Tetravalent FZD and WNT co-receptor binding antibody molecules and uses thereof

Abstract

Provided are tetravalent antibody molecules comprising a FZD receptor binding domain and an LRP5 / 6 coreceptor binding domain at opposite ends of an Fc domain, which activate the Wnt β-catenin signaling pathway, and methods for using the same. [Solution] A tetravalent antibody molecule comprising (a) an Fc domain or a fragment thereof comprising constant heavy chain domain 3 (CH3), (b) a bivalent low-density lipoprotein receptor-related protein 5 (LRP5) binding domain, and (c) a bivalent Frizzled (FZD) binding domain, wherein the LRP5 binding domain is bound to one end of the Fc domain and the FZD binding domain is bound to the other end of the Fc domain, the LRP5 binding domain comprises a diabody that binds to LRP5, and the FZD binding domain comprises two scFvs or two Fabs that bind to FZD4.

Description

[Technical Field]

[0001] This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy created on December 16, 2021 is named 117946_PD606WO_FINAL.txt and is 989,432 bytes in size. [Background technology]

[0002] The Wnt signaling pathway is important for embryonic development and tissue homeostasis in adults. Wnt signaling is initiated when Frizzled (FZD) receptors on the cell surface membrane bind to Wnt ligands. Wnt ligands are secreted growth factors that regulate various cellular processes, such as proliferation, differentiation, survival, and migration.

[0003] In humans, there are 19 Wnt ligands that interact with a network of 10 Frizzled cell surface receptors (FZDs) and one of several coreceptors that guide 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, R. T. transduction. Nat. Rev. Mol. Cell Biol. 10, 468-477 (2009)). FZDs share conserved structural features, including seven hydrophobic transmembrane domains and a cysteine-rich ligand-binding domain. FZDs are known to function in three distinct signaling pathways: the Wnt planar cell polarity (PCP) pathway, the canonical Wnt / β-catenin pathway, and the Wnt / calcium pathway. The presence of Wnt coreceptors is also required to direct the differential engagement of the intracellular signaling cascades listed above. For example, Wnt ligands bind to Frizzled receptors and members of the low-density lipoprotein receptor-related protein 5 and 6 (LRP5 / 6) coreceptor family to activate the Wnt / β-catenin pathway, or bind together with receptor tyrosine kinase (RYK) or protein tyrosine kinase 7 (PTK7) coreceptors, receptor tyrosine kinase-like orphan receptors 1 and 2 (ROR1 / 2), to activate alternative β-catenin-independent signaling pathways.

[0004] Wnt ligands are universally important in controlling the self-renewal of tissue stem cells and regulating many progenitor cell populations, but the hydrophobicity and sensitive tertiary structure of Wnt proteins make their biochemical purification difficult and their use in vitro and in vivo inefficient. Described herein are tetravalent binding antibody molecules that activate the Wnt signaling pathway and methods for their use. Summary of the Invention

[0005] Tetravalent binding antibody molecules that activate the Wnt signaling pathway and methods of use thereof are described herein. The tetravalent binding antibody molecules bind to both a FZD receptor (e.g., Frizzled class receptor 1 (FZD1), Frizzled class receptor 2 (FZD2), Frizzled class receptor 3 (FZD3), Frizzled class receptor 4 (FZD4), Frizzled class receptor 5 (FZD5), Frizzled class receptor 6 (FZD6), Frizzled class receptor 7 (FZD7), Frizzled class receptor 8 (FZD8), Frizzled class receptor 9 (FZD9), or Frizzled class receptor 10 (FZD10)) and a Wnt coreceptor (e.g., LRP5 or LRP6 (LRP5 / 6)), thereby activating the Wnt signaling pathway. In one embodiment, the tetravalent binding antibody molecule binds to both the FZD4 receptor and LRP5 and / or LRP6 and activates the Wnt / β-catenin signaling pathway. The tetravalent binding antibody molecules of the invention are also referred to herein as "FZD agonists," Frizzled and LRP5 / 6 agonists (FLAgs), and in some embodiments, "ANTs."

[0006] A tetravalent binding antibody molecule comprises an Fc domain composed of a CH2 domain and a CH3 domain or a fragment thereof containing the CH3 domain, a first bivalent binding domain that interacts with one or more FZD receptors (e.g., one or more of FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, and FZD10), and a second bivalent binding domain that binds to a WNT coreceptor (e.g., LRP5 or LRP6), wherein the FZD binding domain is attached to one end of the Fc domain and the coreceptor binding domain is attached to the other end of the Fc domain. Thus, the binding domain for the FZD receptor and the binding domain for the WNT coreceptor are not directly linked; rather, they are separated by the Fc domain or a fragment thereof containing the CH3 domain.

[0007] The Fc domain of the FZD agonist can be an Fc domain of an immunoglobulin, with or without effector function. The immunoglobulin can be IgG, for example, IgG1. In one embodiment of the present invention, the tetravalent binding antibody molecule comprises two polypeptides containing Fc regions that dimerize via the inherent ability of the Fc regions in each polypeptide to dimerize or via a knob-in-hole arrangement within the Fc. Thus, the Fc dimer can be a heterodimer or a homodimer. Methods for dimerizing peptides via knobs-in-holes configuration are described in WO 2018 / 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, et al. (1998) Nat. Biotechnol. 16, 677-681, and; Atwell et al., (1997) J. Mol. Biol. 270, 26-35, all of which are incorporated herein by reference.

[0008] In one embodiment, each binding domain of the FZD agonists described herein is bivalent and may each be monospecific, have two binding sites for the same epitope on an FZD receptor, e.g., FZD4, or a Wnt coreceptor, e.g., LRP5 / 6, or may be bispecific, with each binding site binding to a different epitope on an FZD or Wnt coreceptor, e.g., a Wnt1-binding site (domains E1-E2) in the extracellular domain of LRP5 / 6 and a Wnt3-binding site (domains E3-E4) in the LRP5 / 6 coreceptor. In one embodiment, the LRP5 / 6-binding domain binds to the Wnt3A site (domains E3-E4) on LRP5 and the Wnt3A site (domains E3-E4) on LRP6.

[0009] In an embodiment of the invention, the FZD-binding domain linked to the Fc domain of the FZD agonist comprises one or more immunoglobulin heavy chain variable domain (VH) fragments and / or one or more immunoglobulin light chain variable domain (VL) fragments that bind to a FZD, e.g., FZD4. In one embodiment of the invention, the FZD-binding domain comprises a Fab, diabody, or single-chain variable fragment (scFv) single-domain antibody fragment, e.g., a VFv, that binds to the same or different epitopes on a FZD. H H, or a combination thereof.

[0010] In one embodiment of the present invention, the VH and / or VL of the FZD-binding domain binds to FZD4 or FZD5 and comprises the light chain CDRs and heavy chain CDRs of an FZD4 or FZD5-binding antibody of Table 1, Table 2 or Table 6, and / or comprises light chain CDRs and heavy chain CDRs that are 50%, 55%, 60%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the CDRs of an FZD4 antibody of Table 1, Table 2 or Table 6 and still retain binding to the FZD4 or FZD5 receptor. For example, in one embodiment of the present invention, the FZD-binding domain comprises a first heavy chain (CDR-H1), a second heavy chain (CDR-H2), and / or a third heavy chain (CDR-H3) (VHs that bind to FZD include CDR-H1 of SEQ ID NO: 24, SEQ ID NO: 365, or SEQ ID NO: 893, CDR-H2 of SEQ ID NO: 51, SEQ ID NO: 61, SEQ ID NO: 462, or SEQ ID NO: 894, and / or CDR-H3 of SEQ ID NO: 79, SEQ ID NO: 90, SEQ ID NO: 484, or SEQ ID NO: 89). and a first light chain (CDR-L1), a second light chain (CDR-L2) and / or a third light chain (CDR-L3) (the VL that binds to FZD may comprise CDR-L1 of SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 12, CDR-L2 of SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 12 and / or CDR-L3 of SEQ ID NO: 3, SEQ ID NO: 12, SEQ ID NO: 285 or SEQ ID NO: 896).

[0011] In embodiments of the invention, the co-receptor (LRP5 / 6) binding domain linked to the Fc domain of the FZD agonist comprises one or more immunoglobulin heavy chain variable domain (VH) fragments and / or one or more immunoglobulin light chain variable domain (VL) fragments that bind to a Wnt co-receptor, e.g., LRP5 and / or LRP6. For example, in one embodiment of the invention, the LRP-binding domain comprises a first heavy chain (CDR-H1), a second heavy chain (CDR-H2), and / or a third heavy chain (CDR-H3) (VHs that bind to LRP include CDR-H1 of SEQ ID NO:527, SEQ ID NO:528, SEQ ID NO:536, SEQ ID NO:716, or SEQ ID NO:720, CDR-H2 of SEQ ID NO:552, SEQ ID NO:553, SEQ ID NO:566, SEQ ID NO:785, or SEQ ID NO:791, and / or CDR-H3 of SEQ ID NO:584, SEQ ID NO:585, SEQ ID NO:586, SEQ ID NO:603, SEQ ID NO:856, or SEQ ID NO:862). and a first light chain (CDR-L1), a second light chain (CDR-L2) and / or a third light chain (CDR-L3) (the VL that binds to the LRP may comprise CDR-L1 of SEQ ID NO: 1, CDR-L2 of SEQ ID NO: 2, or SEQ ID NO: 491 and / or CDR-L3 of SEQ ID NO: 130, SEQ ID NO: 492, SEQ ID NO: 493, SEQ ID NO: 510, SEQ ID NO: 623 or SEQ ID NO: 665).

[0012] In one embodiment of the present invention, the Wnt coreceptor binding domains are bivalent and may comprise diabodies, or may comprise Fab, single chain variable fragments (scFv), or single domain antibody fragments (VFv) for binding to the same or different epitopes on the coreceptor. HIn embodiments of the invention, the VH and VL of the Wnt coreceptor binding domain comprise the light chain and / or heavy chain CDRs of an LRP5 and / or LRP6 binding antibody of Table 3, Table 4 or Table 6, or comprise light chain and / or heavy chain CDRs that are 50%, 55%, 60%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the CDRs of an LRP5 and / or LRP6 antibody of Table 3, Table 4 or Table 6 and still retain binding to the LRP5 and / or LRP6 coreceptor.

[0013] In one embodiment of the present invention, the Wnt coreceptor binding domain linked to the Fc domain of a FZD agonist described herein comprises a diabody formed by two peptides, each comprising a heavy chain variable domain (VH or VH domain) linked to a light chain variable domain (VL or VL domain), where the VH and VL from one peptide pair with the VL and VH of the other peptide to form the diabody. In this configuration, the binding domain has two binding sites that bind to a Wnt coreceptor, e.g., LRP5 or LRP6. The diabody may monospecifically bind to the same site on the coreceptor, or it may bispecifically bind to two different sites on the coreceptor. By using a knobs-in-holes Fc configuration, peptides comprising a VH and a VL linked to the Fc region may not be identical but still pair to form a bispecific binding domain that can bind to two different sites on a Wnt coreceptor (e.g., LRP5 or LRP6).

[0014] Diabody forming the binding domain, V HThe peptides forming the H, scFv, and Fab can be derived from an antibody, or "source antibody," selected for binding to a desired target. In the case of an FZD-binding domain, the "FZD source antibody" can be an antibody that binds to one or more FZD receptors, e.g., one or more of FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, and FZD10, and antagonizes Wnt signaling or inhibits Wnt binding to a given FZD receptor(s). Alternatively, the FZD source antibody can be an antibody that binds to a FZD receptor(s) without antagonizing Wnt signaling or inhibiting Wnt binding to the FZD receptor. Similarly, in the case of a coreceptor-binding domain, the "coreceptor source antibody" can be an antibody that binds to a Wnt coreceptor, e.g., LRP5 / 6, and antagonizes Wnt signaling or inhibits Wnt binding to the Wnt coreceptor. Alternatively, the co-receptor source antibody can be an antibody that binds to a co-receptor, such as LRP5 / 6, without antagonizing Wnt signaling or inhibiting Wnt binding to the co-receptor.

[0015] In one embodiment of the present invention, the FZD-binding domain of the FZD agonist may specifically bind to a specific FZD, e.g., FZD4, with higher affinity than to other FZDs, i.e., FZD1, FZD2, FZD3, FZD5, FZD6, FZD7, FZD8, FZD9, and FZD10, or may be pan-specific, binding to one or more other members of the FZD receptor family. In one embodiment, the FZD-binding domain specifically binds to a FZD with an affinity that is greater than 10-fold greater than its binding to any other Frizzled family member.

[0016] In one embodiment of the present invention, the FZD agonist binds to FZD4 ("FZD4 agonist"). The FZD4-binding domain of the FZD4 agonist may specifically bind to FZD4 and bind to FZD4 with higher affinity than other FZDs, or may pan-specifically bind to FZD4 and one or more other members of the FZD receptor family, such as Frizzled class receptor 1 (FZD1), Frizzled class receptor 2 (FZD2), Frizzled class receptor 3 (FZD3), Frizzled class receptor 5 (FZD5), Frizzled class receptor 6 (FZD6), Frizzled class receptor 7 (FZD7), Frizzled class receptor 8 (FZD8), Frizzled class receptor 9 (FZD9), or Frizzled class receptor 10 (FZD10). In one embodiment, the FZD-binding domain specifically binds to FZD4 with greater than 10-fold affinity than any other Frizzled family member listed above.

[0017] In one embodiment of the present invention, the FZD agonist binds to FZD5 ("FZD5 agonist"). The FZD5-binding domain of the FZD5 agonist may specifically bind to FZD5, bind to FZD5 with greater affinity than other FZDs, or may be pan-specific, binding to FZD5 and one or more other members of the FZD receptor family, e.g., FZD1, FZD2, FZD3, FZD4, FZD6, FZD7, FZD8, FZD9, or FZD10. In one embodiment, the FZD-binding domain specifically binds to FZD5 with greater than 10-fold greater affinity than any other Frizzled family member listed above.

[0018] In one embodiment of the FZD agonists of the invention, the Wnt coreceptor-binding domain is a monospecific, bivalent LRP5 / 6 coreceptor-binding domain that binds to a single epitope on the LRP5 and / or LRP6 coreceptor, e.g., an epitope on the LRP5 and / or LRP6 coreceptor that binds to Wnt1 (the E1-E2 domains of LRP5 or LRP6) or Wnt3a (the E3-E4 domains of LRP5 or LRP6). In one embodiment of the invention, the coreceptor-binding domain is a bispecific, bivalent LRP5 / 6 binding domain that binds to two epitopes within the extracellular domain of the LRP5 and / or LRP6 coreceptor, e.g., the coreceptor-binding domain interacts with the Wnt1 (E1-E2) and Wnt3 (E3-E4) epitopes on the LRP5 and / or LRP6 coreceptor. In one embodiment of the invention, the coreceptor binding domain is a bispecific, bivalent binding domain that binds to the extracellular domains of LRP5 and LPR6, e.g., the domain interacts with the Wnt1 (E1-E2) epitope of the LRP5 coreceptor and the Wnt1 (E1-E2) epitope of the LRP6 coreceptor LRP5, or the domain interacts with the Wnt3a (E3-E4) epitope of the LRP5 coreceptor and the Wnt3a (E3-E4) epitope of the LRP6 coreceptor, or the domain interacts with the Wnt1 (E1-E2) epitope of the LRP5 coreceptor and the Wnt3a (E3-E4) epitope of the LPR6 coreceptor, or vice versa.

[0019] Various formats of the tetravalent antibody molecules described herein are shown in Figure 6. In a particular format, diabody-Fc-Fab, an LRP5 / 6-binding diabody is linked to the N-terminus of the Fc domain, and two Fabs are linked to the C-terminus of the Fc domain, with the Fabs linked to the CH3 of the Fc domain via the Fab heavy chain (VH) variable domain. Alternatively, the Fabs are linked to the CH3 of the Fc domain via the light chain variable region (VL).

[0020] The present inventors have previously reported multivalent binding molecules comprising an Fc domain, a FZD-binding domain, and a Wnt co-receptor (LRP5 / 6)-binding domain at opposite ends of the Fc domain, e.g., a molecule having a FZD4 diabody linked to one end of the Fc domain and an LRP5 / 6-binding diabody linked to the other end of the Fc domain (see PCT / IB2019 / 051174 inventor Angers et al. and PCT / IB2020 / 055463 inventor Angers et al., both of which are incorporated by reference in their entireties).

[0021] Wnt-β-catenin signaling, particularly via activation of FZD4, has been reported to be important for vasculature development and homeostasis in the adult vasculature. More specifically, it is important for the barrier function of the blood-retinal barrier and the blood-brain barrier (BRB and BBB). Defects in FZD4 signaling can lead to defects in endothelial cell permeability, and genetic mutations in this pathway are known to result in vascular defects (e.g., Norrie disease, FEVR). At the blood-retinal barrier, the extracellular ligand Norrin primarily activates the FZD4-TSPAN12-LRP5 complex to regulate endothelial cell-cell interactions, barrier function, and permeability (Wang et al. (2012) Norrin / Frizzled4 signaling in retinal vascular development and blood-brain barrier plasticity. Cell. 151:1332-1344). At the blood-brain barrier, secreted Wnt7a / b growth factors primarily activate the FZD4-GPR124-LRP6 receptor complex (Chang et al., 2017). GPR124 is essential for blood-brain barrier integrity in central nervous system diseases (Nat. Med. 23:450-460). The FZD4 agonists described herein, for example, those with a diabody-binding domain for LRP5 / 6 and an FZD4-binding domain composed of two Fab fragments that bind to FZD4, with each binding domain at opposite ends of an Fc domain, produce particularly stable and homogenous molecules that unexpectedly activate the Wnt-β-catenin signaling pathway in endothelial cells, leading to increased barrier function and decreased vascular permeability (Figure 11). Essentially, the FZD4 agonists described herein function as Norrin and Wnt7a / b mimicking molecules.

[0022] The present invention also includes methods for using the FZD agonists described herein. Described herein are methods for activating the Wnt signaling pathway, e.g., the Wnt / β-catenin signaling pathway, using tetravalent binding antibody molecules of the present invention that are intended to promote proximity of one or more of an FZD receptor and a Wnt coreceptor, e.g., FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, and FZD10 receptor and an LRP5 and / or LRP6 coreceptor, on a cell, wherein binding of the FZD agonist to both the FZD receptor(s) and the LRP5 and / or LPR6 coreceptor(s) activates the Wnt signaling pathway.

[0023] Blood-retinal barrier (BRB) formation and retinal neovascularization require β-catenin signaling induced by the ligand norlin (NDP [Norry disease protein]), receptor FZD4, co-receptor LRP5, and TSPAN12 (tetraspanin 12). Accordingly, one aspect of the present invention is a method of promoting and / or maintaining retinal vasculature barrier function and angiogenesis by treating ocular tissue, e.g., retinal tissue, with an effective amount of a tetravalent FZD4 agonist of the present invention.

[0024] Also, one aspect of the present invention is a method for promoting, restoring, and / or maintaining BRB and BBB function by treating the BRB or BBB vasculature with an effective amount of a tetravalent FZD4 agonist described herein. A further aspect of the present invention is a method for treating a subject having a disorder or condition characterized by defective retinal or cerebral angiogenesis, characterized by impaired endothelial cell barrier function resulting in vascular leakage, by administering to such subject an effective amount of an FZD4 agonist of the present invention. A further aspect of the present invention is a FZD4 / LRP5 tetravalent-binding antibody molecule or pharmaceutical composition for use in the treatment or prevention of a disorder or condition characterized by defective retinal or cerebral angiogenesis and / or characterized by impaired endothelial cell barrier function and / or vascular leakage. A further aspect of the present invention is a method for treating or preventing a disorder or condition characterized by defective retinal or cerebral angiogenesis and / or impaired endothelial cell barrier function and / or vascular leakage, comprising administering to a subject in need thereof a therapeutically effective amount of a FZD4 / LRP5 tetravalent-binding antibody molecule described herein. A further aspect of the present invention is the use of a FZD4 / LRP5 tetravalent-binding antibody molecule for the manufacture of a medicament for treating or preventing a disorder or condition characterized by defective retinal or cerebral angiogenesis and / or reduced endothelial cell barrier function and / or vascular leakage. Such disorders or conditions include, but are not limited to, ocular disorders, including disorders of the retina or macula. Such disorders of the retina or macula include, but are not limited to, diabetic retinopathy, retinopathy of prematurity, Coats' disease, FEVR, Norrie's disease, macular degeneration, diabetic macular edema, and pediatric vitreoretinopathy. Further disorders or conditions encompassed by embodiments of the present invention include, but are not limited to, Alzheimer's disease, epilepsy, multiple sclerosis, ischemia, and stroke.

[0025] One embodiment of the present invention includes a method of producing vascularized cerebral organoids by promoting barrier function of the vasculature network throughout the organoid, thereby mimicking blood-brain barrier function using an effective amount of a tetravalent FZD4 agonist described herein.

[0026] Also, an embodiment of the present invention is a method of treating a subject suffering from a gastrointestinal disorder, including a subject having inflammation of all or part of the intestine, also known as inflammatory bowel disease, by administering to such a subject an effective amount of a pharmaceutical composition of the present invention, such as a composition comprising an FZD5 agonist. Examples of inflammatory bowel disease include, but are not limited to, Crohn's disease and ulcerative colitis.

[0027] Also, one embodiment of the present invention is a method for differentiating iPS cells or other pluripotent stem cells (PSCs) into various lineages by culturing these cells in the presence of an effective amount of a tetravalent binding antibody molecule of the present invention.

[0028] Methods for making the tetravalent binding antibody molecules of the present invention are also described herein.

[0029] The modular aspect of the present invention allows for mixing and matching of binding domains from FZD-binding antibodies and LRP5 / 6-binding antibodies at opposite ends of an Fc domain to generate tetravalent binding antibody molecules that can engage the FZD-LRP5 / 6 coreceptor complex and selectively activate Wnt signaling. The modularity and effectiveness of the tetravalent binding antibody molecules described herein for activating the Wnt signaling pathway contrasts with prior art Wnt surrogates that consist of monovalent FZD and Wnt coreceptor-binding ligands or FZD and Wnt coreceptor-binding ligands in which the binding ligands are not attached to both ends of an Fc domain. [Brief explanation of the drawings]

[0030] [Figure 1A] Single-point ELISA. [Figure 1B]Single-point ELISA. FZD4-binding antibodies isolated from affinity maturation libraries of known FZD4-binding antibodies 5044 (Figure 1A) and 5027 (Figure 1B) bind to FZD4 sites that compete with their parent antibodies. The reaction was stopped by adding 1 M H3PO4, and absorbance was measured spectrophotometrically at 450 nm in a microtiter plate reader. White = BSA; black and white stripes = Fc; gray = FZD4 + blocking antibody; black = FZD4. [Figure 2] Epitope mapping of FZD4 antibodies. FZD4 and 5027 and 5044 have overlapping epitopes. The pan-FZD binder 5016 is a positive control showing that the antigens are functional, except for "FZD4_Swap10." Both FZD4-specific antibodies 5027 and 5044 were unable to bind to "FZD4 Swap 7," suggesting that these molecules bind to this region of the FZD ECD. [Figure 3A] Size Exclusion Chromatography (SEC). Analysis of FZD4 antibodies compared to trastuzumab. Protein elution was monitored using absorbance at 280 nM. [Figure 3B] ELISA specificity. Measurement of FZD4 antibodies against FZD4 and against FZD1 and FZD10, the two most closely related members of the FZD family to FZD4. The reaction was stopped by adding 1 M H3PO4, and the absorbance was measured spectrophotometrically at 450 nm in a microtiter plate reader. [Figure 4] Phage clone ELISA of synthetic antibodies targeting LRP5. The results demonstrate that the synthetic antibodies bound to LRP5. Absorbance was measured spectrophotometrically at 450 nm in a microtiter plate reader. Gray = BSA; light gray = his-Fc; dark gray = LRP5. [Figure 5A] Phage clone ELISA of synthetic antibodies targeting LRP6. [Figure 5B]Phage clone ELISA of a synthetic antibody targeting LRP6. The results demonstrate that the synthetic antibody bound to LRP6. Absorbance was measured spectrophotometrically at 450 nm in a microtiter plate reader. Black = BSA; Gray = Fc; Light gray = LRP6-Fc. [Figure 6] Modalities of tetravalent antibody molecules include the diabody-Fc-diabody format, which has a FZD-binding monospecific diabody at the N-terminus of the Fc domain and an LPR5 / 6-binding bispecific diabody at the C-terminus of the Fc domain; the diabody-Fc-scFv format, which has an N-terminal LPR5 / 6-binding bispecific diabody and two C-terminal FZD-binding scFvs; the IgG-diabody format, which has two FZD-binding Fabs forming an N-terminal binding domain and a bispecific LRP5 / 6-binding diabody forming a C-terminal binding domain; the IgG-scFv format, which has two FZD-binding Fabs forming an N-terminal binding domain and two LRP5 / 6-binding scFvs forming a C-terminal binding domain; and the diabody-Fc-Fab format, which has a bispecific LRP5 / 6-binding diabody forming an N-terminal binding domain and two FZD-binding Fabs forming a C-terminal binding domain, where the Fabs are linked to the CH3 of the Fc domain via the Fab variable heavy chain region. In another diabody-Fc-Fab format, it is specifically contemplated that the Fab is linked to the CH3 of the Fc domain via the Fab variable light chain region. The various domains of the tetravalent molecule, VL, VH, CH1, CH2, CH3, CL1, and Fc, are linked via linkers, e.g., peptide linkers. The Fc domain is formed by dimerization of the CH2 and CH3 domains of the hole construct Fc region and the knob construct Fc region. The various domains of the tetravalent molecule, VL, VH, CH1, CH2, CH3, CL1, and Fc, are linked via linkers, e.g., peptide linkers. [Figure 7]FZD4 agonists having a diabody-Fc-Fab format. The diabody-Fc-Fab format has an LRP5-binding bispecific diabody that forms a bivalent, bispecific N-terminal LRP5-binding domain, two FZD4-binding Fabs that form bivalent, monospecific C-terminal FZD4-binding domains, and an Fc region with reduced effector function due to amino acid mutations (e.g., N297G (NG) and D265A (DANG) variants). The various domains of the tetravalent molecule, VL, VH, CH1, CH2, CH3, CL1, and Fc, are linked via a linker, e.g., a peptide linker. [Figure 8] FZD4 agonists with a diabody-Fc-Fab format (ANT) bind to FZD4 with high selectivity. Figure 8A shows the apparent selectivity of FZD4 agonists for the recombinant extracellular domains (ECDs) of nine of the ten FZDs, as determined by biolayer interferometry (BLI). Figure 8B demonstrates that the FZD agonists do not recognize common nonspecific antigens. As described in Mouquet et al., FZD agonists were tested at 100 nM for binding to a panel of antigens. Polyreactivity increases the apparent affinity of anti-HIV antibodies through hetero-oligomerization. Nature.2010 Sep;467(7315):591-595.DOI:10.1038 / nature09385,PMC3699875, and Jain T. et al.Biophysical properties of the clinical-stage antibody landscape.Proceedings of the National Academy of Sciences of the United States of America.2017 Jan;114(5):944-949.DOI:10.1073 / pnas.1616408114,PMC5293111. [Figure 9]FZD4 agonists (ANTs) with a diabody-Fc-Fab format (having an LRP-binding bispecific diabody and two FZD4-binding Fabs) are stable and monomeric in solution. Figure 9A shows analytical SEC analysis results of FZD agonists compared to trastuzumab IgG. Figure 9B shows differential scanning fluorimetry results demonstrating that FZD4 agonists in the diabody-Fc-Fab format have a thermal denaturation profile similar to trastuzumab, although the first-generation diabody-Fc-diabody FZD4 modality (CM0199) is less optimal. [Figure 10] FZD4-LRP5-specific FZD4 agonists in the diabody-Fc-Fab format (ANT) stimulate FZD4 expression in a mouse endothelial cell line (bEND3.1), resulting in a concentration-dependent increase in Axin2 (beta-catenin target gene) gene transcription. [Figure 11A] We show that FZD4-LRP5 specific agonists with diabody-fc-diabody format promote endothelial cell barrier function in a mechanism that counteracts VEGF-induced permeability. [Figure 11B]We demonstrate that FZD4-LRP5-specific agonists in the diabody-fc-diabody format promote endothelial cell barrier function in a mechanism that counteracts VEGF-induced permeability. Figure 11A shows immunofluorescence of ZO-1 / CLDN3 and ZO-1 / CLDN5 localization on bEnd.3 cell junctions. bEnd.3 cells were treated with or without 30 nM F4L5.13 (also known as CM0199) and Norrin (NDP) in the presence or absence of VEGF (100 ng / ml) for 1 hour. Starting from the top row and proceeding downwards: NT (untreated) shows no change in permeability; VEGF treatment of bEND3.1 cells results in junctional dissociation, as seen by the loss of plasma membrane staining for CLDN3, CLDN5, and ZO-1; co-treatment of cells with VEGF and the FZD4 agonist CM0199 (F4L5.13) results in a near-complete rescue of the effects of VEGF alone; the last row in Figure 11A shows co-treatment of cells with VEGF and NDP, which also results in a near-complete rescue of the effects of VEGF alone, suggesting that the FZD4 agonists described herein function as Norrin and Wnt7a / b mimicking molecules. Figure 11B shows a transendothelial permeability assay quantifying the passage of FITC-dextran through a monolayer of bEnd3.1 cells. FITC-dextran passage was measured after exposure of bEnd.3 cells to 100 ng / ml VEGF, 30 nM F4L5.13, or both, or after 1 hour of pretreatment with VEGF followed by 1 hour of treatment with F4L5.13. Data are presented as mean ± SD, n = 5 independent experiments. Significance was calculated by one-way ANOVA with Bonferroni's multiple comparison test (*P < 0.05 compared with VEGF treatment). [Figure 12]Single-point ELISA. FZD5 antibody that binds to the extracellular domain of FZD5 at a site overlapping with 2919 identified from an affinity maturation library. The novel FZD5 antibody binds to FZD5 at a site overlapping with 2919 identified from an affinity maturation library. Single-point ELISA was performed in 96-well Maxisorp plates coated with the ECD of human FZD5 protein in the presence or absence of saturating concentrations of 2919 IgG. Absorbance was measured spectrophotometrically at 450 nm in a microtiter plate reader. Black on white stripes = BSA; white on black stripes = Fc; gray = FZD5 + blocking antibody; black = FZD5. [Figure 13] Single-point ELISA demonstrates that novel FZD5 antibodies from the 2928 affinity maturation library selectively bind to FZD5. Novel FZD5 antibodies from the 2928 affinity maturation library selectively bind to FZD5. Single-point ELISA was performed on 96-well Maxisorp plates coated with the ECD of human FZD2, FZD5, or FZD8 proteins. Absorbance was measured spectrophotometrically at 450 nm in a microtiter plate reader. Black on white stripes = Fc; white on black stripes = FZD2; gray = FZD8; black = FZD5. [Figure 14] Luciferase assay. Pan-FZD / LRP6 ANT9 and FZD5-specific / LRP6 ANT59 activate Wnt signaling in cells. TOPFLASH cells were treated overnight with various concentrations of FZD agonists or a non-targeting control molecule (CM0156), and TCF / LEF-driven luciferase expression was measured using a standard luciferase assay. Both molecules can activate FZD-mediated luciferase expression in a concentration-responsive manner. ANT9 can bind to seven of the ten FZD receptor subtypes and produces a higher maximal activation signal than FZD5-specific ANT59. [Figure 15]The original format ANT39 and the reversed format ANT39i are available. The FZD4 agonist ANT39 has a diabody-Fc-Fab format, and the FZD4 agonist ANT39i has an IgG-diabody format (with two FZD-binding Fabs forming an N-terminal binding domain and a bispecific LRP5 / 6-binding diabody forming a C-terminal binding domain) and an Fc domain. The FZD-binding domain of ANT39i contains two Fab fragments attached to the N-terminus of the Fc domain, with each Fab binding to an FZD. The LRP5 / 6 coreceptor-binding domain is attached to the C-terminus of the Fc domain and is composed of a diabody that binds to two different sites on the coreceptor, such as the Wnt1 site (E1-E2) and the Wnt3 site (E3-E4) on LRP5 / 6. The Fab may be specific for a particular FZD, such as FZD4, or may be pan-specific, binding to more than one FZD, such as FZD4 and one or more other FZDs. The Fc region may have weakened effector function due to amino acid mutations, such as the N297G (NG) and D265A (DANG) variants. The various domains of the tetravalent molecule, VL, VH, CH1, CH2, CH3, CL1 and Fc, are connected via linkers, such as peptide linkers. [Figure 16A]

[0013] The present invention discloses an FZD4 agonist, ANT39, having a diabody-Fc-Fab format (having an LRP5-binding bispecific diabody forming a bivalent, bispecific N-terminal LRP5-binding domain and two FZD4-binding Fabs forming a bivalent, monospecific C-terminal FZD4-binding domain), wherein the Fc region has attenuated effector function due to amino acid mutations to the N297G and D265A (DANG) variant or the L234A, L235A, P331S (LALAPS) variant, and the Fc region further comprises the knobs-in-holes heterodimerization variants Merrimack, Merchant, or Merchant S:S (Merrimack CH3 mutations described in WO2018 / 026942A1, Merchant CH3 mutations described in Merchant AM et al. Nature Biotechnology 1998 vol 16 p677-681). Figure 16A discloses SEQ ID NOs: 886, 892, 891, 886, 892, 891, 886, 892, 891, 886, 892, and 891, respectively, in order of appearance. [Figure 16B] Figure 16B shows the FZD4 agonist ANT39i in an IgG-Fc-diabody format (with two Fab fragments (each Fab binds to a FZD) attached to the N-terminus of the Fc domain and an LRP5 / 6 coreceptor binding domain (comprised of diabodies that bind to two different sites on the coreceptor) attached to the C-terminus of the Fc domain) and an Fc region with attenuated effector function due to DANG or LALAPS variants and Merrimack, Merchant, or Merchant S:S heterodimerization variants. Figure 16B discloses SEQ ID NOs: 891, 886, 891, 886, 891, and 886, respectively, in order of appearance. [Figure 17]Thermal stability of ANT39 variants. Figure 17 shows the results of a differential scanning fluorimetry experiment demonstrating that the LALA variant of the FZD4 agonist ANT39 (ANT39 LALA) has improved thermal stability compared to the parent ANT39 (containing a DANG mutation in the Fc). Specifically, the LALA variant exhibited improved thermal stability that more closely resembles the profile of a variant of trastuzumab that contains the same knob / hole Fc mutations as ANT. [Figure 18] FZD4 agonist ANT42 having a diabody-Fc-Fab format. ANT42 is an FZD4 agonist having an LRP5-binding bispecific diabody that forms a bivalent, bispecific N-terminal LRP5-binding domain and two FZD4-binding Fabs that form a bivalent, monospecific C-terminal FZD4-binding domain. The Fc region has attenuated effector function due to amino acid mutations to the N297G and D265A (DANG) variant or the L234A, L235A, P331S (LALAPS) variant, and the Fc region further comprises the knobs-in-holes heterodimerization variants Merrimack, Merchant, or Merchant S:S (the Merrimack CH3 mutation described in WO 2018 / 026942 A1 and the Merchant CH3 mutation described in Merchant AM et al. Nature Biotechnology 1998 vol 16 pp 677-681). The FZD4 agonist ANT42i has an IgG-Fc-diabody format (with two Fab fragments (each Fab binds to a FZD) attached to the N-terminus of the Fc domain and an LRP5 / 6 coreceptor binding domain (composed of diabodies that bind to two different sites on the coreceptor) attached to the C-terminus of the Fc domain) and an Fc region with attenuated effector function due to DANG or LALAPS variants and Merrimack, Merchant, or Merchant S:S heterodimerization variants. Figure 18 discloses SEQ ID NOs: 886, 892, 891, 891, 886, 886, 892, 891, 891, 886, 886, 892, 891, 891, 886, 886, 892, 891, 891, and 886, respectively, in order of appearance. [Figure 19] Antibody modalities tested for FZD agonism. A) Diabody-Fc-diabody, VH, and VL were tested with both FZD-binding and LRP-binding variable domains. B) Diabody-Fc-scFv; C) scFv-Fc-diabody; D) scFv-Fc-scFv; E) IgG-diabody; F) IgG-scFv; G) Diabody-Fc-Fab; H) Diabody-CH3-diabody; I) Fab-diabody. In Figure 19, molecules B-F and H-I contain an N-terminal variable domain that binds LRP and a C-terminal variable domain that binds FZD. In Figure 19, molecule G contains an N-terminal variable domain that binds FZD and a C-terminal variable domain that binds LRP. These antibody formats, marked with an *, were tested using knobs-in-holes Fc. [Figure 20] Multiple antibody structures can induce potent FZD agonism. Pan-FZD and LRP6-targeting paratopes were constructed in various configurations as described in Table 14. Canonical Wnt pathway stimulation by each antibody was measured in a blinded manner by two different scientists in wild-type HEK cells expressing the TOPFLASH reporter. Data are presented as mean ± SD and are representative of four separate experiments. [Figure 21] Expression titers of various FZD agonist modalities. Various FZD agonist modalities were expressed in HEK cells, purified via protein A chromatography, and expression titers were determined based on absorbance at 280 nm. EC50 values ​​for FZD activation were measured in a blinded manner by two different scientists on wild-type HEK cells expressing the TOPFLASH reporter. [Figure 22] Organoid viability assay. Mouse small intestinal organoids were grown in the presence of 1 μM LGK-974 to block endogenous Wnt secretion and treated with PBS, Wnt3a-conditioned medium, or FLAg molecules as indicated. Left: Representative images from n=3 independent experiments. Right: Quantification of organoid viability by CellTiter-Glo luminescence assay. Bars represent the mean + / - standard error from three independent experiments. [Figure 23] Mouse colon histology. Histological appearance of mouse colon after DSS treatment cycles (7 days of 2% DSS, 3 days of 0.5% DSS) with intraperitoneal injection of either control IgG or ANT59 (10 mg / kg) on ​​days 4 and 7. (A) Images captured at 20x magnification showing overall structure. (B) Images captured at 100x magnification showing rescue of mucosal integrity with ANT59 treatment. [Figure 24] (A) Body weight changes in mice across a DSS treatment cycle (7 days of 2% DSS, 3 days of 0.5% DSS) following intraperitoneal injection of either control CM0156, PanFZD agonist, or ANT59 (10 mg / kg) on ​​days 4 and 7. (B) Left: Representative images of excised colons from 6-8 mice per treatment group, with centimeter scale for comparison. Right: Colon length from each treatment group. Bars represent mean colon length + / - SD, and individual data points are shown. *** indicates p<0.0001 by one-way ANOVA; HO indicates normal water (no DSS). [Figure 25] Characterization of FZD5 / LRP6 ANTs. ANTs were expressed in HEK cells and purified via protein A chromatography. Expression titers were determined based on absorbance at 280 nm. Biolayer interferometry was used to determine the apparent affinity (binding activity) of each molecule for recombinant Fc-fused human FZD5 and measure selectivity over other human FZDs. Dose-response curves for activation of the LEF / TCF reporter gene in FZD knockout (1, 2, 4, 5, 7) HEK293 cells overexpressing FZD5. Cells were seeded in 96-well dishes for 24 h and then treated as indicated for 17 h. Reporter activation was assessed using the Dual-Luciferase Reporter Assay System (Promega). Data are presented as mean ± SD for technical duplicates and representative of three independent experiments. DETAILED DESCRIPTION OF THE INVENTION

[0031] Described herein are tetravalent binding antibody molecules comprising an Fc domain with or without effector function, a bivalent FZD-binding domain, and a bivalent LRP-binding domain, each binding domain attached to opposite ends of the Fc domain. In one embodiment, the FZD-binding domain is attached to the carboxy terminus of the Fc domain, and the LRP coreceptor-binding domain is attached to the amino terminus of the Fc domain. Alternatively, the FZD-binding domain is attached to the amino terminus of the Fc domain, and the coreceptor-binding domain is attached to the carboxy terminus of the Fc domain. The binding domains may be attached to the Fc domain directly or via a linker. The FZD-binding domain may bind to one or more FZD receptors, i.e., one or more of FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, and FZD10.

[0032] In one embodiment of the present invention, the FZD-binding domain is bivalent and includes a diabody or scfv that binds to FZD. H a diabody or V that binds to the LRP5 / 6 coreceptor, comprising an H fragment, a Fab fragment, or a combination thereof, and wherein the coreceptor binding domain is bivalent; H In one embodiment of the present invention, the FZD-binding domain is linked to the carboxy terminus of the Fc domain, and includes two scFvs, two Vs, and two Fabs. H A diabody that binds to an H fragment, two Fab fragments, or an FZD, and a coreceptor binding domain attached to the amino terminus of the Fc domain, a diabody that binds to an LRP5 / 6 coreceptor, two V H The FZD-binding domain comprises an H fragment or two scFvs. When attached to the carboxy terminus of the Fc domain, the FZD-binding Fab is linked to the CH3 of the Fc domain via the Fab variable heavy or light region. In other embodiments, the FZD-binding domain is attached to the amino terminus of the Fc domain and consists of two Fabs, and the LRP5 / 6 coreceptor-binding domain is attached to the carboxy terminus of the Fc domain and consists of a diabody or two scFvs that bind to the coreceptor.

[0033] Figure 6 shows a tetravalent antibody molecule of the present invention in a diabody-Fc-scFv format having an LRP5 / 6 coreceptor-binding domain, an Fc domain, and an FZD-binding domain. The diabody-Fc-sFv comprises (i) an Fc domain, (ii) a bispecific diabody that binds to two different sites on a coreceptor, e.g., the Wnt1 (E1-E2) site on LRP5 / 6 and the Wnt3 (E3-E4) site on LRP5 / 6, linked to the N-terminus of the Fc domain, and (iii) an FZD-binding domain comprising two FZD-binding scFv fragments linked to the carboxy terminus of the Fc domain. The scFv may be specific for a particular FZD, e.g., FZD4, or may be pan-specific, binding to more than one FZD, e.g., FZD4 and one or more other FZDs.

[0034] One embodiment of the present invention is a tetravalent antibody molecule in a diabody-Fc-scFv format, having (i) an Fc domain, (ii) an LRP5 / 6 coreceptor-binding domain (the diabody is attached to the amino terminus of the Fc domain) that contains a bispecific diabody that binds to two different sites on the coreceptor, e.g., the Wnt1 (E1-E2) site on LRP5 / 6 and the Wnt3 (E3-E4) site on LRP5 / 6, and (iii) an FZD-binding domain attached to the carboxy terminus of the Fc domain that contains two scFv fragments, each of which binds to a FZD. The scFvs can be specific for a FZD or can be pan-specific, binding to a FZD and one or more other FZDs.

[0035] Figure 6 also shows a tetravalent antibody molecule of the invention in an IgG-diabody format having (i) an Fc domain, (ii) an FZD-binding domain comprising two Fab fragments attached to the N-terminus of the Fc domain (each Fab binds to a FZD), and (iii) an LRP5 / 6 coreceptor-binding domain attached to the C-terminus of the Fc domain, which is composed of a diabody that binds to two different sites on a coreceptor, e.g., the Wnt1 site (E1-E2) and the Wnt3 site (E3-E4) on LRP5 / 6. The Fab may be specific for a particular FZD, e.g., FZD4, or may be pan-specific, binding to more than one FZD, e.g., FZD4 and one or more other FZDs.

[0036] One embodiment of the present invention is a tetravalent binding antibody molecule in an IgG-diabody format, comprising (i) an Fc domain, (ii) an N-terminal binding domain for a FZD, comprising two FZD-binding Fabs, and (ii) a C-terminal binding domain for an LRP5 and / or LRP6 co-receptor, comprising an LRP5 / 6 co-receptor binding diabody. This FZD agonist in the IgG-diabody format is (1) First and second heavy chain monomers, each heavy chain monomer comprising a single polypeptide chain comprising, from N-terminus to C-terminus: (a) a heavy chain variable domain (VH) that binds to FZD; (b) heavy chain constant region domain 1 (CH1 domain); (c) an Fc region (or a fragment thereof) comprising constant heavy chain domain 3 (CH3 domain); (d) a peptide comprising a VH that binds to the LRP5 / 6 coreceptor, a light chain variable domain (VL) that binds to the LRP5 / 6 coreceptor, and (2) Each light chain monomer comprises, from N-terminus to C-terminus, a first and a second light chain monomer, each of which comprises a VL that binds to FZD, a constant light chain domain 1 (CL1 domain).

[0037] The first and second heavy chain monomers dimerize via their Fc regions or fragments thereof. The linker between the VH and VL that binds LRP5 / 6 is long enough to promote pairing of the VH and VL of the first heavy chain monomer with the VL and VH of the second heavy chain monomer, thereby forming an LRP5 / 6 coreceptor-binding diabody. An FZD-binding Fab is formed by pairing each heavy chain monomer with a light chain monomer such that the CH1 of the VH and each heavy chain monomer that binds FZD4 pairs with the CL1 of the VL and light chain monomer that binds FZD4. In this IgG-diabody format, the Fab forms an FZD4-binding domain at the N-terminus of the Fc domain, and the diabody forms a coreceptor-binding domain at the C-terminus of the Fc domain. The Fab may be specific for one FZD, e.g., FZD4 or FZD5, or it may be pan-specific, binding to more than one FZD, e.g., FZD4 and / or FZD5, and optionally more FZDs. The Fc region can dimerize via a knob-in-hole configuration. Methods for dimerizing peptides via a knob-in-hole configuration are described in WO 2018 / 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 et al. (1998) Nat. Biotechnol. 16, 677-681, and Atwell et al. (1997) J. Mol. Biol. 270, 26-35. The Fc region may be Merrimack (knob chain: Q347M, Y349F, T350D, T366W, and L368M; hole chain: S354I, E357L, T366S, L368A, and Y407V), Merchant (knob chain: T366W; hole chain: T336S, L368A, and Y407V), or Merchant S:S (Merchant mutation with an additional S354C variant in the knob chain and Y349C in the hole chain).The Fc regions may also contain mutations that alter their effector functions, for example, the Fc regions may have attenuated effector functions due to amino acid mutations such as DANG variants and LALAPS variants.

[0038] In Figure 6, in the IgG-diabody format, the diabody-forming peptides are linked to the C-terminus of the Fc domain via the VH domain in a VH-VL orientation (N- to C-terminus), whereas in some embodiments, the diabody-forming peptides are linked to the C-terminus of the Fc domain via the VL domain in a VL-VH orientation (N- to C-terminus). Also, the heavy chain is shown as comprising a VH domain and a CH1 domain linked to the N-terminus of the Fc domain, and the light chain is shown as comprising a VL domain and a CL1 domain, forming a Fab; however, in some embodiments (diabody-Fc-Fab in Figures 6 and 7A), the diabody is fused to the N-terminus of the Fc and the Fab is fused to the C-terminus of the Fc. To accomplish this, the CH3 domain of the Fc is fused directly to the heavy chain of the Fab via its VH domain (VH-CH1) or directly to the light chain via its VL domain (VL-CL), with the light and heavy chains still associating to form the Fab.

[0039] Figure 6 shows a tetravalent antibody molecule in a diabody-Fc-Fab configuration, with an LRP5 / 6-binding bispecific bivalent diabody forming the N-terminal binding domain and two FZD-binding Fabs forming the C-terminal binding domain. The Fabs may be specific for a particular FZD, e.g., FZD4, or may be pan-specific, binding to more than one FZD, e.g., FZD4 and one or more other FZDs. See also Figure 7A, which shows a tetravalent antibody molecule in a diabody-Fc-Fab format, with an Fc and an LRP5-binding bispecific bivalent diabody in a knob-in-hole (kiH) configuration forming the N-terminal binding domain and two FZD4-binding Fabs forming the C-terminal binding domain. While Figures 6 and 7A show the Fabs linked (at the C-terminus) to the CH3 of the Fc domain via the Fab variable heavy domain (VH), it is specifically contemplated that in alternative diabody-Fc-Fab formats, the Fabs are linked to the CH3 of the Fc domain via the Fab variable light domain (VL). The various domains of the tetravalent molecule, VL, VH, CH1, CH2, CH3, CL1 and Fc, are connected via linkers, for example peptide linkers.

[0040] Also, one embodiment of the present invention is a tetravalent binding antibody molecule in a diabody-Fc-Fab format, comprising (i) an Fc domain, (ii) an N-terminal binding domain comprising a diabody that binds to a co-receptor, e.g., LRP5 and / or LRP6 co-receptor, and (ii) a C-terminal binding domain comprising two Fabs that bind to one or more FZDs, e.g., FZD4 or FZD5. This FZD agonist in the diabody-Fc-Fab format is (1) First and second heavy chain monomers, each heavy chain monomer comprising a single polypeptide chain comprising, from N-terminus to C-terminus: (a) a peptide comprising a heavy chain variable (VH) domain that binds to the LRP5 / 6 co-receptor and a light chain variable (VL) domain that binds to the LRP5 / 6 co-receptor; (b) an Fc region (or a fragment thereof containing constant heavy chain domain 3 (CH3 domain)); (c) a VH domain that binds to FZD; (d) a CH1 domain, and (2) Each light chain comprises, from N-terminus to C-terminus, first and second light chain monomers that comprise a VL domain that binds to FZD and a constant light chain domain 1 (CL1).

[0041] The first and second heavy chain monomers dimerize via the Fc region or fragment thereof, and a bivalent LRP5 / 6-binding diabody is formed by pairing the LRP5 / 6-binding VH and VL domains of the first heavy chain monomer with the LRP5 / 6-binding VL and VH domains of the second heavy chain monomer. Two FZD-binding Fabs are formed by pairing each heavy chain monomer with a light chain monomer such that the FZD-binding VL and CL1 of the light chain monomer pair with the FZD-binding VH and CH1 of each heavy chain monomer. In this diabody-Fc-Fab format, the diabody forms an LRP5 / 6 coreceptor-binding domain at the amino terminus of the tetravalent molecule, and the two Fabs form FZD-binding domains at the C-terminus of the tetravalent antibody molecule. The Fc regions can dimerize via a knob-in-hole configuration.

[0042] Methods for dimerizing peptides via knob-in-hole configurations are described in WO 2018 / 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, et al. (1998) Nat. Biotechnol. 16, 677-681, and; Atwell et al., (1997) J. Mol. Biol. 270, 26-35. The Fc region may be Merrimack (knob chain: Q347M, Y349F, T350D, T366W, and L368M; hole chain: S354I, E357L, T366S, L368A, and Y407V), Merchant (knob chain: T366W; hole chain: T336S, L368A, and Y407V), or Merchant S:S (Merchant mutation with an additional S354C variant in the knob chain and Y349C in the hole chain). Fc regions may also contain mutations that alter their effector function; for example, Fc regions may have attenuated effector function due to amino acid mutations such as DANG and LALAPS variants.

[0043] In Figures 6 and 7A, the diabody-forming peptides in the diabody-Fc-Fab format are linked to the Fc domain via their VL domains, and thus are in a VH-VL orientation (N- to C-terminus); in some embodiments, the orientation can be switched such that the diabody-forming peptides are linked to the N-terminus of the Fc domain via their VH domains, and thus are in a VL-VH orientation (N- to C-terminus). Also, while the heavy chain in the diabody-Fc-Fab format is shown as comprising a VH domain and a CH1 domain, which pairs with a light chain comprising a VL domain and a CL1 domain to form a Fab, it is contemplated that in some embodiments the variable and constant domains can be switched such that the heavy chain comprises a VL domain and a CL1 domain, and the light chain comprises a VH domain and a CH1 domain, and the heavy and light chains still pair to form a Fab.

[0044] In one embodiment of the present invention, the binding portion of the FZD-binding domain is derived from an antibody or antibody fragment that specifically binds to one FZD, such as FZD4 or FZD5, or that pan-specifically interacts with a particular FZD, such as FZD4 or FZD5, and one or more additional FZD receptors (FZD source antibody), and the coreceptor-binding domain comprises a binding portion derived from an antibody or antibody fragment that binds to LPR5 and / or LRP6 (LRP5 / 6 coreceptor source antibody). In one embodiment of the present invention, the FZD-binding antibody binds to the extracellular cysteine-rich domain (CRD) of a FZD receptor. The FZD-binding antibody can be an antibody that binds to a FZD receptor and antagonizes Wnt signaling or inhibits binding of a Wnt ligand to the FZD receptor. The FZD-binding antibody can be an antibody that binds to a FZD receptor without antagonizing or inhibiting binding of a Wnt ligand to the FZD receptor. The FZD-binding antibody can be an antibody that binds to a FZD and enhances Wnt signaling. The antibody that binds to the LRP5 / 6 coreceptor can be an antibody that binds to the LRP5 / 6 coreceptor and antagonizes Wnt signaling or inhibits binding of a Wnt ligand to the coreceptor, or the antibody that binds to the LRP5 / 6 coreceptor can be an antibody that binds to the coreceptor without antagonizing Wnt or Norrin signaling or inhibiting binding of a Wnt or Norrin ligand to the coreceptor.

[0045] In one embodiment of the present invention, the LRP5 / 6 coreceptor binding domain binds to a single epitope on the coreceptor, e.g., an epitope that binds to the Wnt1 (E1-E2) or Wnt3 (E3-E4) interacting domain of LRP5 / 6. In one embodiment of the present invention, the LRP5 / 6 coreceptor binding domain binds to two epitopes within the coreceptor, e.g., a paratope that binds to the Wnt1 (E1-E2) interacting epitope of LRP5 / 6 and a paratope that binds to the Wnt3 (E3-E4) epitope of LRP5 / 6. In one embodiment of the present invention, the multivalent binding molecule comprises an Fc domain, and the Fc domain is an immunoglobulin Fc domain or a fragment thereof comprising a CH3 domain. In one embodiment of the present invention, the immunoglobulin is IgG. In one embodiment of the present invention, the IgG is IgG1.

[0046] In one embodiment of the present invention, the LRP5 / 6 binding domain comprises a diabody comprising two peptides, each comprising a heavy chain variable domain (VH) that binds LRP5 / 6 linked to a light chain variable domain (VL) that binds LRP5 / 6, wherein the binding domain is formed by pairing the VH and VL from one peptide with the VL and VH of the other peptide, thereby forming the LRP5 / 6 binding domain.

[0047] In tetravalent binding antibody molecules of the invention, both binding domains are bivalent, and one or both of the bivalent binding domains can be bispecific for a respective FZD receptor, e.g., FZD4 or FZD5, or the LRP5 / 6 coreceptor. For example, a binding molecule may contain FZD binding domains that are bivalent and monospecific (each binding site binds to the same epitope), and the LRP5 / 6 binding domain is bivalent and bispecific, binding to two different epitopes (Wnt1 (E1-E2) and Wnt3 (E3-E4) sites on the LRP5 / 6 ectodomain). In one embodiment of the invention, both binding domains are bivalent and bispecific, and each binding domain binds to two different epitopes on its respective target FZD receptor or LRP5 / 6 coreceptor.

[0048] The VH domain and VL domain of the FZD-binding domain of the tetravalent molecule of the invention can comprise three light chain CDRs and three heavy chain CDRs of a FZD source antibody, e.g., FZD4 or FZD5, a binding antibody of Table 1, Table 2 or Table 6, or three light chain CDRs and three heavy chain CDRs that are at least 50%, at least 55%, at least 60%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the CDRs of a FZD source antibody, e.g., a FZD4 antibody of Table 1, Table 2 or Table 6, and still retain binding to the FZD or FZD5 receptor bound by the source antibody.

[0049] The VH and VL domains of the LRP5 / 6 coreceptor binding domain of the tetravalent molecules of the invention can comprise three light chain CDRs and three heavy chain CDRs that are at least 50%, at least 55%, at least 60%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the three light chain CDRs and three heavy chain CDRs of an LRP5 / 6 coreceptor source antibody, e.g., an LRP5 / 6 binding antibody of Table 3, Table 4, or Table 6, or the VH and VL of a Wnt coreceptor source antibody, e.g., an LRP5 / 6 binding antibody of Table 3, Table 4, or Table 6, and still bind to the LRP5 / 6 coreceptor.

[0050] In one embodiment of the invention, the FZD-binding domain of a tetravalent binding molecule of the invention binds to FZD4 (FZD4 agonist), or FZD5 (FZD5 agonist), or FZD4 and / or FZD5 and one or more other FZDs (pan-FZD agonist); and which are at least 50%, at least 55%, at least 60%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to CDR-H1, CDR-H2, and CDR-H3 and CDR-L1, CDR-L2, and CDR-L3 of an antibody of Table 1, Table 2, or Table 6, and which still contain CDRs that bind to FZD4 or FZD5; The LRP5 / 6 binding domain of a FZD4 agonist, a FZD5 agonist, or a pan-FZD agonist is or the CDRs comprise CDR-H1, CDR-H2 and CDR-H3 and CDR-L1, CDR-L2 and CDR-L3 of an antibody of Table 3, Table 4 or Table 6, or the CDRs are at least 50%, at least 55%, at least 60%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to CDR-H1, CDR-H2 and CDR-H3 and CDR-L1, CDR-L2 and CDR-L3 of an antibody of Table 3, Table 4 or Table 6, and still bind to LRP5 or LRP6.

[0051] In one embodiment, the FZD-binding domain of the tetravalent binding antibody molecule does not comprise a diabody, scFv, or Fab comprising the three heavy chain CDRs or three light chain CDRs of FZD4-binding antibody 5044 in combination with a Wnt-coreceptor binding domain comprising a diabody, scFv, or Fab comprising the three heavy chain CDRs and three light chain CDRs of LRP6-binding antibody 2542 and / or antibody 2539. In one embodiment, the tetravalent binding molecule does not comprise a diabody, scFv, or Fab comprising the three heavy chain CDRs and three light chain CDRs of FZD4-binding antibody 5027 in combination with a Wnt-coreceptor binding domain comprising a diabody, scFv, or Fab comprising the three heavy chain CDRs and three light chain CDRs of LRP6-binding antibody 2542 and / or antibody 2539.

[0052] Also, one embodiment of the present invention is a nucleic acid molecule encoding a tetravalent binding molecule described herein. One embodiment of the present invention is a nucleic acid molecule encoding a polypeptide of a tetravalent binding molecule described herein comprising the heavy and light chain CDRs listed in Tables 1, 2, 3, 4, and 6. Another embodiment of the present invention is a nucleic acid molecule encoding a polypeptide of a tetravalent binding molecule of Figures 7A and 7B, e.g., a FZD5 agonist or FZD4 agonist, comprising the CDRs of Table 6. Another embodiment of the present invention is a nucleic acid molecule encoding VH and VL domains comprising the heavy and light chain CDRs listed in Tables 1, 2, 3, 4, and 6, respectively. The nucleic acid molecule may be inserted into a vector and expressed in a suitable host cell, and the tetravalent binding antibody molecule may then be isolated from the cells using methods well known in the art. Accordingly, one aspect of the invention is also expression cassettes and vectors comprising the tetravalent binding molecules described herein, e.g., nucleic acid molecules encoding FZD4 or FZD5 agonist polypeptides, VL and VH domains, Fab domains and diabodies comprising the CDRs set forth in Tables 1, 2, 3, 4, and 6, and Fc domains described herein. One aspect of the invention is host cells expressing these expression cassettes and vectors.

[0053] As used herein, 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 tetravalent binding antibody molecule described herein. A vector capable of expressing a protein upon insertion of a polynucleotide is called an expression vector. A vector can be inserted into a host cell by transformation, transduction, or transfection, thereby allowing the expression of the carried genetic material in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), and P1-derived artificial chromosomes (PACs); phages, such as lambda phage or M13 phage; and animal viruses. Animal viruses may include, but are not limited to, reverse transcriptase viruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex viruses), varicella viruses, baculoviruses, papilloma viruses, and papova viruses (such as SV40). A vector can contain multiple components that control the expression of the tetravalent binding antibody molecules described herein, including, but not limited to, a promoter, e.g., a viral or eukaryotic promoter, e.g., a CMV promoter, a signal peptide, e.g., the TRYP2 signal peptide, a transcription initiation factor, an enhancer, a selection element, and a reporter gene. Additionally, a vector can also contain replication origin(s).

[0054] As used herein, the term "host cell" refers to cells into which expression cassettes and vectors can be introduced, including, but not limited to, prokaryotic cells such as E. 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, for example, fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, or HEK293 cells.

[0055] One embodiment of the present invention is a pharmaceutical composition comprising the FZD agonist described herein or a nucleic acid molecule, expression cassette and vector encoding the FZD agonist, and a pharmaceutically acceptable carrier, diluent or excipient.The pharmaceutical composition may further comprise an additional drug, such as a second therapeutic antibody, such as an anti-VEGF antibody (aflibercept, ranibizumab and bevacizumab), a growth factor such as VEGF, or an agent that activates the Wnt pathway, such as the small molecule CHIR99021, Norrin or R-Spondin, or a nucleic acid molecule, expression cassette and vector encoding the agent.The pharmaceutical composition may consist essentially of or consist of the FZD agonist described herein or a nucleic acid molecule, expression cassette or vector encoding the FZD agonist, and a pharmaceutically acceptable diluent, carrier or excipient. Suitable carriers, diluents, and excipients, as well as their formulations, are described in Remington: The Science and Practice of Pharmacy (19th ed.) 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 render the formulation isotonic. Pharmaceutically acceptable carriers include, but are not limited to, saline, Ringer's solution, and dextrose solution. The pH of the solution can be, for example, about 5 to about 8, about 5 to 7.5, or about 6 to 7. Further carriers include sustained-release preparations, such as semipermeable matrices of solid hydrophobic polymers containing the agonist, which matrices are in the form of shaped articles, e.g., films, liposomes, or microparticles. It will be apparent to those skilled in the art that certain carriers may be more preferable depending, for example, on the route of administration and the concentration of the FZD agonist being administered.

[0056] The present invention also includes methods for using the FZD agonists described herein. One embodiment of the present invention is a method for activating the Wnt signaling pathway in a cell, comprising contacting a cell having a FZD receptor and an LRP5 / 6 co-receptor with a tetravalent binding antibody molecule of the present invention that binds to a FZD, such as FZD4 and LRP5 / 6, in an amount effective to activate Wnt signaling. The Norrin-FZD4 pathway has been reported to play a role in retinal neovascularization (see Wang et al. Cell. 2012;151(6):1332-1344; Braunger BM, Tamm ER. Adv Exp Med Biol. 2012;723:679-683; Ohlmann A, Tamm ER. Prog Retin Eye Res. 2012;31(3):243-257; and Ye et al. Trends Mol Med. 2010;16(9):417-425). Signal transduction via the Norrin-FZD4 pathway is necessary for the development and maintenance of the retinal vasculature. Mutations affecting genes in this pathway can result in several vitreoretinopathies, such as Norrie disease, familial exudative vitreoretinopathy (FEVR), and pseudoglioma and osteoporosis syndrome. Furthermore, retinopathy of prematurity (ROP) is associated with mutations in this Norrin-FZD4 pathway, and Wnt pathway mutations have been reported in Coats disease and persistent fetal vasculature (PFV). FZD4 signaling activated by the Norrin and / or WNT7A / B pathways is also involved in the development and homeostasis of the CNS blood-brain barrier. Genetic ablation of Norrin, FZD4, LRP5, LRP6, and the co-receptor tetraspanin-12 (Tspan-12) results in defective angiogenesis and barrier disruption in retinal and / or small cerebral vessels (Cho et al. (2017) Neuron 95, 1056-1073; Zhou et al., (2014) J Clin Invest 124:3825-3846). Thus, a functional Wnt signaling system plays an important fundamental role in the development of sufficient vascular and neural networks in the eye and retina to support vision, and in the CNS to support BBB development and homeostasis.

[0057] One aspect of the present invention is a method for promoting and / or maintaining retinal vasculature by treating ocular tissue, e.g., retinal tissue, with an effective amount of a pharmaceutical composition comprising a tetravalent antibody molecule of the present invention, e.g., a tetravalent antibody molecule that binds FZD4 and LRP5 / 6, an FZD4 agonist, and having the structure shown in Figure 6, via topical or systemic administration. Also, one aspect of the present invention is a method for promoting and / or maintaining BBB vasculature by treating a subject in need thereof with an effective amount of a pharmaceutical composition of the present invention, e.g., a composition comprising an FZD4 agonist having the structure shown in Figure 6. The BBB is initiated during development, and its integrity remains essential for homeostasis and neuroprotection throughout life. Subjects in need thereof include those with neurological conditions associated with BBB dysfunction, e.g., neurodegenerative diseases such as Alzheimer's disease, as well as epilepsy, multiple sclerosis, and stroke.

[0058] A further aspect of the present invention is a method of treating a subject having a disorder characterized by vascular leakage, particularly retinal vascular leakage and / or endothelial cell leakage, as well as disorders characterized by impaired retinal or brain endothelial cell barrier function or impaired BBB or BRB, such as diabetic retinopathy, retinopathy of prematurity, Coats disease, FEVR, Norrie disease, macular degeneration, diabetic macular edema, and pediatric vitreoretinopathy, by administering an effective amount of a pharmaceutical composition of the present invention, e.g., a composition comprising a FZD4 agonist having the structure shown in Figure 6. An effective amount of such a composition is, for example, an amount sufficient to increase or restore endothelial cell barrier function, thereby reducing vascular leakage in such a subject. The subject may be a fetus. The FZD4 agonists of the present invention, particularly those in the diabody-Fc-Fab format, which contain two Fab fragments forming an FZD4-binding domain at the carboxy terminus of an Fc receptor and an LRP5 and / or LRP6-binding domain composed of a diabody at the amino terminus of the Fc domain, activate FZD4 and β-catenin signaling in endothelial cells, promote barrier function, thereby reducing endothelial cell permeability, and thereby significantly enhance angiogenesis, as shown, for example, in Figure 6. In particular, treatment of endothelial cells in vivo, ex vivo, or in vitro with these FZD4 agonists, preferably those in the diabody-Fc-Fab format, enhances the development and maintenance of retinal vasculature and / or BRB and BBB more effectively than other molecules without this structure.

[0059] A further aspect of the present invention is a method for treating a subject with inflammation of all or part of the intestine, also known as inflammatory bowel disease, by administering to such a subject an effective amount of a pharmaceutical composition of the present invention, e.g., a composition comprising an FZD5 agonist. Examples of inflammatory bowel disease include, but are not limited to, Crohn's disease and ulcerative colitis. An effective amount of such a composition is an amount sufficient to reduce, ameliorate, eliminate, or treat the inflammation. Subjects in need thereof include subjects with inflammation of the mucosa of the gastrointestinal tract. The methods disclosed herein can be performed to reduce inflammation (e.g., inflammation associated with IBD or inflammation in tissue affected by IBD (e.g., gastrointestinal tissue (e.g., small intestine, large intestine, or colon))), activate WNT signaling, or reduce any of the histological symptoms of IBD (e.g., those disclosed herein).

[0060] The FZD agonists of the present invention can be administered systemically or locally, for example, by injection (e.g., subcutaneously, intravenously, intraperitoneally, intrathecally, intraocularly, intravitreously, etc.), implantation, topical administration, or oral administration. Depending on the route of administration, the FZD agonist may be coated with a material to protect the agonist from conditions that may inactivate the agonist. The tetravalent binding antibody molecules described herein can be dissolved or suspended in a pharmaceutically acceptable, preferably aqueous, carrier. Furthermore, compositions containing FZD agonists can contain excipients such as buffers, binders, blasting agents, diluents, flavoring agents, lubricants, etc. An extensive list of excipients that can be used in such compositions can be found, for example, in A. Kibbe, Handbook of Pharmaceutical Excipients (Kibbe, 2000). The tetravalent binding antibody molecules can also be administered together with immune stimulants such as cytokines.

[0061] One embodiment of the present invention comprises the method of using tetravalent antibody molecule as described herein to induce the cerebral organoid with the vascular network that shows barrier function.It is assumed that the tetravalent binding antibody molecule as described herein that activates FZD4 signal transduction can promote the barrier function in the endothelial cells that are cultured with cerebral organoid, thereby promoting angiogenesis.

[0062] One embodiment of the present invention includes a method for directed differentiation of multipotent or pluripotent stem cells (PSCs) or induced pluripotent stem cells (iPSCs), comprising culturing the cells under conditions suitable for directed differentiation, wherein the culture conditions further comprise an effective amount of a tetravalent binding antibody molecule described herein. Studies in mouse and human PSCs have identified specific approaches to the addition of growth factors, including Wnt, that can induce PSC differentiation 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 the FZD agonists described herein, e.g., FZD4 agonists, can be used in amounts sufficient to result in activation of the Wnt signaling pathway to direct the differentiation of PSCs into specific mesodermal lineages, such as cardiomyocytes (citing Yoon et al. FZD4 Marks Lateral Plate Mesoderm and Signals with NORRIN to Increase Cardiomyocyte Induction from Pluripotent Stem Cell-Derived Cardiac Progenitors. Stem Cell Reports. 2018 Jan;10(1):87-100. DOI:10.1016 / j.stemcr.2017.11.008. PMID:29249665).

[0063] One embodiment of the present invention is a method for enhancing tissue regeneration in a subject in need thereof by activating Wnt signaling in such a subject by administering to the subject an effective amount of a FZD agonist described herein.

[0064] One embodiment of the present invention includes a method for promoting endothelial cell barrier function in ocular tissue, such as retinal tissue, in a subject in need thereof by administering an effective amount of a tetravalent binding molecule of the invention, an FZD4 agonist, that binds to FZD4 and LRP5 / 6. In certain embodiments, the FZD4 agonist of the invention that binds to FZD4 and the binding domain that binds to LRP5 or / and LRP6 have the diabody-Fc-Fab structure shown in Figures 6 and 7. In one embodiment of the present invention, the FZD4 agonist for enhancing retinal angiogenesis comprises the light chain CDRs, i.e., CDR-L1, CDR-L2, and CDR-L3, and heavy chain CDRs, i.e., CDR-H1, CDR-H2, and CDR-H3, of the FZD4-binding antibodies shown in Tables 1, 2, and 6, and the LRP5 / 6-binding antibodies shown in Tables 3, 4, and 6.

[0065] As used herein, a subject can be any animal (e.g., a mammal), including, but not limited to, a human, a non-human primate, a horse, a cow, a dog, a cat, a rodent, etc. The subject can be a fetus. Typically, the subject is a human.

[0066] Effective dosages and schedules for administering the FZD agonists described herein and the nucleic acids encoding them can be determined empirically, and making such determinations is within the skill of one of ordinary skill in the art. Those skilled in the art will understand that the dosage of such FZD agonists to be administered will vary depending, for example, on the subject receiving the antibody, the route of administration, the specific type of FZD agonist used, and other drugs administered. Guidance for selecting appropriate doses for FZD agonists can be found in 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 administration of the composition is sufficient to produce the desired effect, for example, promoting endothelial cell barrier function, vascular homeostasis, or enhancing Wnt signaling. The dosage should not be so large as to cause adverse side effects, such as undesirable cross-reactions or anaphylactic reactions. Generally, the dosage will vary depending on the patient's age, condition, sex, and severity of the disease or disorder, and can be determined by one skilled in the art. The dosage can be adjusted by an individual physician in the event of any contraindications. Dosages can vary and can be administered in one or multiple doses daily for one or several days. While individual needs vary, determining the optimal range of effective amounts of the vector is within the skill of one of ordinary skill in the art.

[0067] Also, one aspect of the present invention is a method of making the tetravalent binding antibody molecules described herein. The amino acid sequences of FZD receptors, e.g., FZD4 and Wnt coreceptor LRP5 / 6, and nucleotide sequences encoding FZD receptors and Wnt coreceptor LRP5 / 6, as well as antibodies and antibody libraries that bind to FZDs, e.g., FZD4 or Wnt coreceptor LRP5 / 6, are readily available or can be generated using methods well known in the art (see, e.g., U.S. Patent Application Publication No. 2015 / 0232554, inventor Gurney, et al.). (See, for example, U.S. Patent Application Publication No. 2016 / 0194394, inventors Sidhu et al., and U.S. Patent Application Publication No. 20190040144, inventors Pan et al.; U.S. Patent Application Publication No. 2017 / 0166636, inventors Wu et al.; U.S. Patent Application Publication No. 2016 / 0208018, inventors Chen et al.; U.S. Patent Application Publication No. 2016 / 0053022, inventors Macheda et al.; U.S. Patent Application Publication No. 2015 / 031293, inventors Damelin et al.) Additionally, various methods for generating and screening such phage display libraries for antibodies and antibody fragments, scFv, Fab, VL, and VH, having desired binding properties are known in the art.Such methods are reviewed, for example, by Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001), and further described, for example, by 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. 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), all of which are incorporated herein by reference. In certain phage display methods, repertoires of VH and VL genes can be separately cloned by polymerase chain reaction (PCR) and randomly recombined into phage libraries, which can then be screened for antigen-binding phage (described in Winter et al., Ann. Rev. Immunol., 12:433-455 (1994)). Phage typically display antibody fragments as either single-chain Fv (scFv) fragments or Fab fragments. Libraries from immunized sources provide high-affinity antibodies to immunogens without the need to construct hybridomas. Alternatively, naive repertoires can be cloned (e.g., from humans) without immunization to provide a single source of antibodies against a wide range of non-self and self antigens, as described by Griffiths et al., EMBO J, 12:725-734 (1993).Finally, naive libraries can also be generated synthetically by cloning unrearranged V gene segments from stem cells and using PCR primers containing random sequences to encode the hypervariable CDR3 regions and achieve rearrangement in vitro, as described by Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example, U.S. Patent No. 5,750,373, and U.S. Patent Application Publication Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360, all of which are incorporated herein by reference. Antibodies or antibody fragments isolated from human antibody libraries are considered human antibodies or human antibody fragments herein.

[0068] In one embodiment of the present invention, a tetravalent binding antibody molecule in a diabody-Fc-scFv format comprising an LRP5 / 6 coreceptor binding domain comprising an LRP5 / 6-binding diabody and a FZD-binding domain comprising two FZD-binding scFvs is (a) selecting an Fc domain having a C-terminus and an N-terminus; (b) identifying an antibody that binds to a FZD receptor (a "FZD source antibody"); and (c) identifying antibodies that bind to the LRP5 / 6 coreceptor ("coreceptor source antibodies" or "LRP5 / 6 source antibodies"); (d) generating a nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide monomer comprising: (i) a peptide comprising a VL domain linked to a VH domain, wherein the domain comprises the heavy and / or light chain CDRs of the antibody of step b that binds to a FZD receptor, or comprises the heavy and / or light chain CDRs from the antibody of step b that still binds to a FZD; (ii) linked to the Fc domain of step a, (iii) comprising a VL domain linked to a VH domain comprising the light and / or heavy chain CDRs of the antibody of step c, or linked to a peptide comprising CDRs derived from the antibody of step c and still binding to the LRP5 / 6 co-receptor; (e) expressing the nucleic acid molecule of step d to produce polypeptide monomers and then dimerizing the polypeptides; The VH and VL domains that bind to the FZD of each monomer form an scFv that binds to the FZD, and the VH and VL domains that bind to the LRP5 / 6 coreceptor of one monomer bind to the VL and VH domains that bind to the Wnt coreceptor of another monomer to form an LRP5 / 6 coreceptor-binding diabody; The polypeptide monomers dimerize via the Fc region to form a tetravalent antibody molecule comprising an Fc domain, an FZD-binding domain composed of two FZD-binding scFvs, and an LRP5 / 6 coreceptor-binding domain composed of a diabody; The FZD-binding domain and the LRP5 / 6 coreceptor-binding domain are at opposite ends of the Fc domain. If the FZD-binding domain and the LRP-binding domain are at opposite ends of the Fc domain, it is contemplated that a peptide comprising a VL domain and a VH domain that binds to an FZD or an LRP can be linked to either the N-terminus or C-terminus of the Fc domain via the VL domain or the VH domain. The FZD can be one or more of FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, and FZD10.

[0069] In one embodiment of the present invention, a tetravalent binding antibody molecule has two FZD-binding Fabs, e.g., FZD4-binding Fabs, linked to one end of an Fc domain, and two LRP5 / 6-binding scFvs or LRP5 / 6-binding diabodies linked to the other end of the Fc domain, and is generated by: (a) identifying the light chain complementarity determining regions (CDR-L1, CDR-L2, and CDR-L3) and / or heavy chain complementarity determining regions (CDR-H1, CDR-H2, and CDR-H3) of an antibody ("FZD source antibody") that binds to a FZD, e.g., FZD4 or FZD5; and (b) identifying the CDR-L1, CDR-L2, and CDR-L3 and / or CDR-H1, CDR-H2, and CDR-H3 of one or more antibodies that bind to LRP5 or LRP6 ("LRP5 / 6 source antibodies"); (c) generating a nucleic acid molecule encoding a "heavy chain" polypeptide comprising: (i) a peptide comprising an immunoglobulin constant heavy chain region 1 (CH1 domain) linked to a VH domain comprising CDR-H1, H2, and H3 of the antibody of step a), or a peptide comprising CDR-H1, CDR-H2, and CDR-H3 derived from the antibody of step a), which still binds to FZD4, (ii) linked to an Fc region; (iii) a VL domain comprising CDR-L1, CDR-L2 and CDR-L3 of the antibody of step b) linked to a VH domain comprising CDR-H1, CDR-H2 and CDR-H3 of the antibody of step b), or a peptide comprising CDR-H1, CDR-H2 and CDR-H3 from the antibody of step b) that binds to LRP5 or LRP6; (d) in step a), generating a nucleic acid molecule comprising a nucleic acid sequence encoding a "light chain" polypeptide comprising an immunoglobulin constant light chain region 1 (CL1) linked to a VL domain, wherein the VL domain comprises the antibody FZD light chain CDR-L1, CDR-L2 and CDR-L3; (e) expressing the nucleic acid molecules of (c) and (d) to produce heavy chain polypeptides and light chain polypeptides; wherein the two heavy chain polypeptides dimerize via their Fc regions, and the CH1 domains of the VH and heavy chain polypeptides that bind to FZD pair with the CL1 domains of the VL and light chain polypeptides that bind to FZD to form two FZD Fabs; The VH and VL that bind to LRP5 / 6 of each heavy chain polypeptide pair to form an LRP5 / 6-binding scFv, or the VH and VL that bind to LRP5 / 6 of one heavy chain polypeptide of a dimeric pair and the VL and VH that bind to LRP5 / 6 of the other heavy chain polypeptide of the dimer, form a diabody, thereby forming a tetravalent antibody molecule comprising an Fc domain, two FZD Fabs linked to either the N-terminus or C-terminus of the Fc domain, and two LRP5 / 6-binding scFvs or LRP5 / 6-binding diabodies linked to the other end of the Fc domain.

[0070] The FZD source antibody may be an antibody that specifically binds to one FZD, such as FZD4, or a pan-specific antibody that binds to an FZD, such as FZD4 or FZD5, and one or more other FZD receptors, and antagonizes Wnt signaling or inhibits Wnt binding to the receptor. Alternatively, the FZD source antibody may be an antibody that specifically binds to one FZD, such as FZD4 or FZD5, or a pan-specific antibody that binds to one FZD, such as FZD4 or FZD5, and one or more other FZD receptors, and does not antagonize Wnt signaling or inhibit Wnt binding to the receptor. The LRP source antibody may be an antibody that specifically binds to LRP5 / 6, or an antibody that pan-specifically binds to LRP5 / 6 and one or more Wnt coreceptors, and antagonizes Wnt signaling or inhibits Wnt binding to the coreceptor. Alternatively, the LRP5 / 6 source antibody may be an antibody that binds to the LRP5 / 6 coreceptor or that binds pan-specifically to LRP5 / 6 and one or more Wnt coreceptors, and does not antagonize Wnt signaling or inhibit Wnt binding to the LRP5 / 6 coreceptor.

[0071] The FZD source antibody can be an antibody fragment, such as a Fab, VL, or VH, that binds to a FZD receptor. The light and heavy chain CDRs, VH, and / or VL in the FZD-binding domain of the FZD agonist can be identical to the CDRs, VH, and / or VL of the FZD source antibody, or can be at least 50%, at least 55%, at least 60%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the CDRs, VH, or VL of the source antibody and still retain binding to a FZD receptor. The CDRs, VH and / or VL in the FZD-binding domain of the FZD agonist may be identical to the CDRs, VH and / or VL of an FZD4-binding antibody or FZD5-binding antibody of Table 1, Table 2 or Table 6, or may be at least 50%, at least 55%, at least 60%, 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the CDRs, VH or VL of an FZD4-binding antibody or FZD5-binding antibody of Table 1, Table 2 or Table 6 and still retain binding to the FZD receptor.

[0072] Similarly, the Wnt coreceptor source antibody can be an antibody fragment, such as a Fab, VL, or VH, that binds to an LRP coreceptor, e.g., LRP5 / 6. The light chain and heavy chain CDRs, VH, and / or VL in the Wnt coreceptor-binding domain of the FZD4 agonist can be identical to the CDRs, VH, and / or VL of the Wnt coreceptor source antibody, or can be at least at least 50%, at least 55%, at least 60%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the CDRs, VH, or VL of the source antibody and still retain binding to an LRP coreceptor. The light and heavy chain CDRs, VH and / or VL in the LRP5 / 6 binding domain of the FZD agonist may be identical to the light and heavy chain CDRs, VH and / or VL of an LRP-binding antibody of Table 3, Table 4 or Table 6, or may be at least at least 50%, at least 55%, at least 60%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the light and heavy chain CDRs, VH and / or VL of an LRP-binding antibody of Table 3, Table 4 or Table 6 and still retain binding to an LRP co-receptor.

[0073] In one embodiment of the present invention, the two polypeptides of a tetravalent binding antibody molecule dimerize via a knobs-in-hole arrangement of their Fc sequences. Tetravalent binding antibody molecules of the present invention can be generated by dimerizing two polypeptides in a "knobs-in-hole" arrangement. The knobs-in-hole arrangement enhances the modularity of the present invention by facilitating the association of peptides containing binding moieties that bind to different epitopes on the FZD receptor or LRP5 / 6 coreceptor or epitopes on different members of the FZD receptor or coreceptor family (see, e.g., Figure 6). Methods for engineering Fc molecules via knob-into-hole designs are well known in the art, see, e.g., WO 2018 / 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, et al. (1998) Nat. Biotechnol. 16, 677-681, and; Atwell et al., (1997) J. Mol. Biol. 270, 26-35.

[0074] Without wishing to be bound by theory, it is contemplated that the tetravalent binding antibody molecule of the present invention promotes the interaction between a FZD receptor and an LRP5 / 6 coreceptor on a cell by promoting proximity and a stabilized conformation of the receptor proteins that is favorable for activating the Wnt signaling pathway. Another embodiment of the present invention is a method for promoting the interaction between a FZD receptor and an LRP5 / 6 coreceptor on a cell, thereby activating the Wnt signaling pathway in the cell, comprising: (a) selecting an Fc domain or a fragment thereof comprising a CH3 domain having a C-terminus and an N-terminus; (b) linking a first bivalent binding domain that binds to the FZD receptor to one end of the Fc domain and a second bivalent binding domain that binds to the Wnt coreceptor to the other end of the Fc domain, thereby forming a tetravalent binding antibody molecule; and (c) contacting the tetravalent binding antibody molecule with cells expressing the FZD receptor and the Wnt coreceptor under conditions in which both the FZD receptor and the coreceptor bind to the tetravalent binding antibody molecule, thereby activating the Wnt signaling pathway. The Wnt coreceptor-binding domain and the FZD-binding domain are bivalent and each have a VL domain and / or a VH domain or a V H The Wnt co-receptor binding domain comprises an H domain, and one or both of the binding domains can be monospecific. In one embodiment of the present invention, one or both of the Wnt co-receptor binding domain and the FZD binding domain are bispecific. In one embodiment of the present invention, the Wnt co-receptor binding domain is bivalent and bispecific. The FZD binding domain can be an scFV that binds to FZD, a V that binds to FZD, or both of the FZD binding domains can be monospecific. H The Wnt coreceptor binding domain may comprise a scFV that binds to the LRP5 / 6 coreceptor, a V that binds to the LRP5 / 6 coreceptor, or a Fab that binds to the FZD, or a combination thereof, or a diabody that binds to the FZD. H The FZD-binding domain may comprise two FZD-binding Fabs, an LRP5 / 6 co-receptor-binding Fab, or a combination thereof, or a diabody that binds to the LRP5 / 6 co-receptor. In one embodiment of the present invention, the FZD-binding domain comprises two FZD-binding Fabs, and the Wnt co-receptor-binding domain comprises a bispecific, bivalent diabody that binds to LRP5 / 6 on two different epitopes.

[0075] The tetravalent binding antibody molecule of the present invention initiates the Wnt signaling pathway stimulated by the FZD-coreceptor complex, for example, the β-catenin pathway stimulated by the FZD-LRP5 / 6 complex. Wnt ligands function by promoting the clustering of coreceptors and FZD receptors. Without wishing to be bound by theory, it is contemplated that the FZD agonists described herein bind to both the FZD receptor and its LRP5 / 6 coreceptor, thereby forming a complex that mimics the binding of Wnt molecules to the FZD receptor and LRP5 / 6 coreceptor, which activates the Wnt signaling pathway, i.e., the Wnt β-catenin pathway.

[0076] An embodiment of the present invention is a method for activating the Wnt signaling pathway, comprising contacting a cell expressing a FZD receptor and its LRP5 / 6 co-receptor with an effective amount of a FZD agonist of the present invention comprising a FZD-binding domain and an LRP5 / 6 co-receptor-binding domain.

[0077] The FZD agonists of the present invention may be produced 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 the molecules may be synthetically produced, for example, using a commercially available synthesis device, such as an automated synthesizer from Applied Biosystems, Inc., Beckman, etc. By using a synthesizer, naturally occurring amino acids may be substituted with unnatural amino acids. The particular order and mode of preparation are determined by convenience, economy, required purity, etc. If desired, various groups can be introduced into the peptide during synthesis or expression, allowing for binding to other molecules or surfaces.

[0078] The binding domain of the FZD agonist can be linked to the Fc domain via a linker. In some embodiments, adjacent VH and VL domains can be linked to each other via a peptide linker. In some embodiments, adjacent constant and variable domains are linked via a peptide linker. The linker can be, for example, a polypeptide linker or a non-peptide linker. In some embodiments, the constant and variable domains of the FZD agonist are linked to the Fc domain via a peptide linker. Suitable linkers, such as XTEN linkers (see WO2013120683, inventors Schellenberger et al.), are well known in the art. In some embodiments, the peptide linker comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 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, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 20, 21, In some embodiments, the peptide linker comprises 0, 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, 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 peptide linker is 1 to 100, 5 to 75, 1 to 50, 5 to 50, 1 to 30, 1 to 25, 5 to 25, 5 to 20, 5 to 15, 5 to 10, 1 to 10, or 1 to 5 amino acids in length. The modular aspects of the invention allow for the mixing and matching of binding domains derived from antibodies that bind to FZD receptors or antibodies that bind to LRP5 / 6 coreceptors at opposite ends of the Fc domain to generate tetravalent binding antibody molecules that can engage the FZD receptor-LRP5 / 6 coreceptor complex and activate Wnt signaling.

[0079] The Fc domain of the FZD agonist, with or without a linker, has a length and flexibility that allows the tetravalent binding antibody molecule of the present invention to bind to both the FZD receptor and its LRP5 / 6 coreceptor, thereby stabilizing a receptor conformation compatible with activation of the downstream Wnt signaling pathway. In one embodiment of the present invention, the Fc domain or fragment thereof, including the CH3 domain, with or without a linker, is greater than 100 amino acids up to 300 Å, greater than 125 amino acids up to 375 Å, greater than 150 amino acids up to 450 Å, greater than 175 amino acids up to 525 Å, or greater than 300 amino acids up to 900 Å. Preferably, the Fc domain is about 200 to about 300 amino acids in length.

[0080] As used in this specification and 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 "the peptide" includes a reference to one or more peptides and equivalents thereof, such as polypeptides known to those skilled in the art.

[0081] An "affinity matured" antibody or "antibody maturation" refers to an antibody with one or more changes in one or more hypervariable regions (HVRs) compared to a parent or source antibody that does not possess such changes, which changes result in an improvement in the affinity of the antibody for antigen or other desired property of the molecule.

[0082] By "comprising," it is meant that the listed elements are required for the composition / method / kit, but that other elements may be included to form the composition / method / kit, etc., within the scope of the claim. For example, a composition comprising a tetravalently bound antibody molecule is a composition that may include other elements in addition to the tetravalently bound antibody molecule, such as a functional moiety, such as a polypeptide, small molecule, or nucleic acid, attached, e.g., covalently bound, to the tetravalently bound antibody molecule; an agent that promotes the stability of the tetravalently bound antibody molecule composition; an agent that promotes the solubility of the tetravalently bound antibody molecule composition; an adjuvant; etc. (excluding elements encompassed by any negative qualification, as would be readily understood in the art).

[0083] "Consisting essentially of" refers to a limitation of the scope of the described composition or method to particular materials or steps that do not materially affect the basic and novel feature(s) of the invention. For example, a tetravalent binding antibody molecule "consisting essentially of" a disclosed sequence has the amino acid sequence of the disclosed sequence plus or minus about 5 amino acid residues at the boundary of the sequence based on the sequence from which it is derived, e.g., about 5, 4, 3, 2, or about 1 residue less than the recited boundary amino acid residue, or about 1, 2, 3, 4, or 5 residues more than the recited boundary amino acid residue.

[0084] "Consisting of" means excluding from the composition, method, or kit any element, step, or ingredient not specified in the claim. For example, a tetravalent binding antibody molecule "consisting of" a disclosed sequence consists only of the disclosed amino acid sequence.

[0085] Where a range of values ​​is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limit of that range is also specifically disclosed, unless the context clearly dictates otherwise. Every smaller range between any stated or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded, and every range in which either or both limits are included in the smaller range, or neither, is also encompassed within the invention, subject to any specifically excluded limit in the stated range. When a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0086] The basic antibody structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" chain (approximately 25 kDa) and one "heavy" chain (approximately 50-70 kDa). The amino-terminal portion of each chain contains a variable region of about 100-110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector functions, such as Fc receptor binding and activation of antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Methods for dimerizing peptides via knob-in-hole configurations are described in WO 2018 / 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, et al. (1998) Nat. Biotechnol. 16, 677-681, and; Atwell et al., (1997) J. Mol. Biol. 270, 26-35. The Fc region may be Merrimack (knob chain: Q347M, Y349F, T350D, T366W, and L368M; hole chain: S354I, E357L, T366S, L368A, and Y407V), Merchant (knob chain: T366W; hole chain: T336S, L368A, and Y407V), or Merchant S:S (Merchant mutation with an additional S354C variant in the knob chain and Y349C in the hole chain). Fc regions may also contain mutations that alter their effector function; for example, Fc regions may have attenuated effector function due to amino acid mutations such as DANG and LALAPS variants.Methods for reducing antibody effector function are well known in the art, including, for example, amino acid substitutions in the Fc region, such as N297G and D265A, the N297G (DANG) variant, the L234A, L235A, P331S (LALAPS), the LALAPS Merchant, the LALAPS Merchant SS (Merchant AM et al Nature Biotechnol 1998 vol 16 p677-681) variant, or the L234A, L235A, P329G (LALA-PG) substitution. See, for example, Lo et al. "Effector Attenuating Substitutions that Maintain Antibody Stability and Reduce Toxicity in Mice." The Journal of Biological Chemistry Vol. 292, No. 9, pp. 3900-3908, March 3, 2017 (incorporated herein by reference). Generally, antibody molecules obtained from humans relate to one of the classes IgG, IgM, IgA, IgE, and IgD, which differ from each other depending on the nature of the heavy chain present in the molecule. Particular classes also have subclasses, such as IgG1, IgG2, etc. Furthermore, in humans, the light chain may be a kappa chain or a lambda chain.

[0087] Three highly divergent stretches within each of the heavy chain variable domain (VH or VH domain) and the light chain variable domain (VL or VL domain), called complementarity-determining regions (CDRs), are sandwiched between more conserved flanking stretches known as "framework regions" or "FRs." Accordingly, the term "FR" refers to the amino acid sequences naturally found between and adjacent to the CDRs in immunoglobulins. A 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, a 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 of the VL domain (CDR-L1, CDR-L2, and CDR-L3) and the three CDRs of the VH domain (CDR-H1, CDR-H2, and CDR-H3) are arranged relative to each other in three-dimensional space to form an antigen-binding site within the antibody variable region. The surface of the antigen-binding site is complementary to the three-dimensional surface of a bound antigen. The amino acid sequences of the VL and VH domains can be numbered according to the Kabat numbering system (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md.) or the INTERNATIONAL IMMUNOGENETICS INFORMATION SYSTEM (IMGT numbering system; Lefranc et al., 2003, Development and Comparative Immunology 27:55-77), both of which are incorporated herein by reference, and the CDRs and FRs therein can be identified / defined.Those skilled in the art will have the knowledge to number the amino acid residues of the VL domain and VH domain and identify the CDRs and FRs therein according to commonly used numbering systems such as the IMGT numbering system or the Kabat numbering system.

[0088] The term "antibody" as referred to herein includes whole antibodies and any antigen-binding fragment (i.e., "antigen-binding portion") or single chain thereof. A "whole antibody" or full length refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen-binding portion thereof. Each heavy chain consists of a heavy chain variable region or domain (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region or domain (abbreviated herein as VL) and a light chain constant region. The light chain constant region is composed of one domain, CL or CL1. The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0089] 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, portions, 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) a Fab fragment (a monovalent fragment consisting of the VL, VH, CL1, and CH1 domains); (ii) a F(ab')2 fragment (a bivalent fragment comprising two F(ab)' fragments linked by a disulfide bridge at the hinge region); (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a dAb fragment consisting of the VH domain (Ward et al. (1989) Nature 241:544-546); and (vi) an isolated complementarity-determining region (CDR). Furthermore, although the two domains of an Fv fragment, VL and VH, are encoded by separate genes, they can be linked by a synthetic linker that allows them to be produced using recombinant methods as a single continuous chain in which the VL and VH regions pair to form a monovalent molecule (known as a single-chain Fv (scFv); see, e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain antibodies are also intended to be encompassed by the term "antigen-binding portion" of an antibody. Other forms of single-chain antibodies, such as diabodies, are also encompassed (see, e.g., Holliger et al. (1993) PNAS. USA 90:6444-6448).

[0090] As used herein, a "diabody," or sometimes referred to herein as "Dia," is a dimeric antibody fragment. In each polypeptide of a diabody, a heavy chain variable domain (VH) is linked to a light chain variable domain (VL). However, unlike single-chain Fv fragments, the linker between the VL and VH is too short for intramolecular pairing; therefore, each antigen-binding site is formed by pairing the VH and VL of one polypeptide with the VH and VL of another polypeptide. Thus, diabodies 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 Dubel eds., Antibody Engineering (2001) Springer-Verlag. New York. 790 pp. (ISBN 3-540-41354-5), incorporated herein by reference.

[0091] As used herein, an "effective amount" of a pharmaceutical agent, e.g., a tetravalent binding antibody molecule or pharmaceutical composition comprising the molecule, refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired result. In some embodiments, a therapeutically effective amount is an amount that reduces the incidence and / or severity of, stabilizes one or more characteristics of, and / or delays the onset of, one or more symptoms of a disease, disorder, and / or condition. In some embodiments, the amount of FZD agonist administered to a subject ranges from about 0.001 mg / kg to 10 mg / kg, 0.5 mg / kg to about 10 mg / kg, or about 0.5 mg / kg to about 1 mg / kg of the subject's body weight. For example, in some embodiments, an FZD4 agonist may be applied to the eye in an amount of, e.g., about 0.02 to 1.5 mg, about 0.05 to 1.0 mg, or about 0.1 to 0.5 mg per eye.

[0092] As used herein, the term "epitope" includes any protein determinant capable of specific binding to an immunoglobulin or fragment thereof, or a T-cell receptor. The term "epitope" includes any protein determinant capable of specific binding to an immunoglobulin or a T-cell receptor. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics. An antibody is said to specifically bind an antigen if the dissociation constant is ≦10 μM; e.g., ≦100 nM, preferably ≦10 nM, and more preferably ≦1 nM.

[0093] The constant region of an immunoglobulin molecule is also called the fragment crystallizable region, "Fc region," or "Fc domain." The Fc domain is composed of two identical protein fragments derived from the second and third constant domains of the two heavy chains of an antibody, and the Fc domain of IgG has highly conserved N-glycosylation sites. Glycosylation of the Fc fragment is essential for Fc receptor-mediated activity. In one embodiment of the present invention, the Fc domain of a tetravalent binding antibody molecule is engineered so as not to target cells that bind the tetravalent binding antibody molecule for ADCC or CDC-dependent killing. In one embodiment of the present invention, the Fc domain of a tetravalent binding antibody molecule is a peptide dimer in a knobs-in-holes configuration. The peptide dimer may be a heterodimer.

[0094] The terms "individual," "subject," "host," and "patient" are used interchangeably herein and refer to any mammalian subject, particularly humans, for whom diagnosis, treatment, or therapy is desired.

[0095] The terms "LRP," "LRP protein," and "LRP receptor" are used herein to refer to members of the low-density lipoprotein receptor-related protein family. These receptors are single-pass transmembrane proteins that bind and internalize ligands during receptor-mediated endocytosis. The LRP proteins LRP5 (e.g., LRP5:NP_002326.2) and LRP6 (e.g., LRP6:NP_002327.2) are included in the Wnt receptor complex, which is required for activation of the Wnt-β-catenin signaling pathway. For information on human / mouse LRP5 and LRP6, see https: / / www.uniprot.org / uniprot / O75197, https: / / www.uniprot.org / uniprot / Q91VN0, https: / / www.uniprot.org / uniprot / O75581, and https: / / www.uniprot.org / uniprot / O88572.

[0096] As used herein, the term "polypeptide fragment" refers to a polypeptide that has an amino-terminal and / or carboxy-terminal deletion, but where the remaining amino acid sequence is identical to the corresponding positions of a naturally occurring sequence as deduced, e.g., from a full-length cDNA sequence.

[0097] As used herein, the term "paratope" includes the antigen-binding site within the variable region of an antibody that binds to an epitope.

[0098] "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding. 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.).

[0099] A "single domain antibody" (sdAb) or "nanobody" is an antibody fragment consisting of a single monomeric variable antibody domain. As used herein, "VHH" or "VHH fragment" refers to a human VH that has been engineered to be independent of the light chain (Nilvebrant et al. Curr Pharm Des. (2016) 22(43):6527-6537; Barthelemy et al., Journal of Biological Chemistry (2007) 283:3639-3654).

[0100] The terms "treatment," "treating," and the like are used herein to generally mean obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, in that a disease or its symptoms are completely or partially prevented, and / or therapeutic, in that a disease and / or adverse effects resulting from the disease are partially or completely cured. As used herein, "treatment" encompasses any treatment of a disease in a mammal, including (a) preventing the disease from occurring in a subject who may be predisposed to, but has not yet been diagnosed with, the disease; (b) inhibiting the disease, i.e., slowing or halting its development; or (c) relieving the disease, i.e., causing regression of the disease. A therapeutic agent may be administered before, during, or after the onset of a disease or injury. Treatment of an ongoing disease is particularly interesting if the treatment stabilizes or alleviates undesirable clinical symptoms in the patient. Such treatment desirably occurs before complete loss of function in the affected tissue. The target therapy may be administered during, or in some cases after, the symptomatic phase of the disease.

[0101] The ability of the tetravalent binding antibody molecules of the present invention to activate Wnt signaling can be confirmed by several assays. The tetravalent binding antibody molecules of the present invention typically initiate a reaction or activity similar to or identical to that initiated by the natural ligand of the FZD receptor. The tetravalent binding antibody molecules of the present invention activate the Wnt signaling pathway, for example, the canonical Wnt-β-catenin signaling pathway. As used herein, the term "activation" refers to a measurable increase in the intracellular level of the Wnt signaling pathway, for example, the Wnt-β-catenin signaling pathway, compared to the level in the absence of the FZD agonist of the present invention.

[0102] Various methods for measuring the level of Wnt-β-catenin activation are known in the art. These include, but are not limited to, assays measuring Wnt-β-catenin target gene expression; LEF / TCF reporter gene expression (e.g., TopFLASH, superTopFLASH, pBAR); β-catenin stabilization; LRP5 / 6 phosphorylation; disheveled phosphorylation; and axin translocation from the cytoplasm to the plasma membrane and its binding to LRP5 / 6. The canonical Wnt-β-catenin signaling pathway ultimately leads to changes in gene expression via the transcription factors TCF1, TCF7L1, TCF7L2, and LEF1. The transcriptional response to Wnt activation has been characterized in several cells and tissues. Therefore, global transcriptional profiling by methods well known in the art can be used to evaluate Wnt-β-catenin signaling activation.

[0103] Changes in Wnt-responsive gene expression are generally mediated by TCF and LEF transcription factors. The TCF reporter assay assesses changes in transcription of TCF / LEF-regulated genes to determine the level of Wnt-β-catenin signaling. The TCF reporter assay was first described by Korinek, V. et al., 1997. This method, also known as TOP / FOP, involves using three copies of the optimal TCF motif CCTTTGATC or three copies of the mutant motif CCTTTGGCC upstream of a minimal c-Fos promoter (pTOPFLASH, pFOPFLASH) driving luciferase expression to determine the transactivation activity of endogenous β-catenin / TCF. A higher ratio of these two reporter activities (TOP / FOP) indicates higher β-catenin / TCF activity. A newer, more sensitive version of this reporter, called pBAR, contains 12 repeats of the TCF motif (Biechele and Moon, Methods Mol Biol. 2008;468:99-110, PMID:19099249).

[0104] General methods in molecular and cellular biochemistry can be found in standard textbooks such as Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., CSH Laboratory Press 2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); Nonviral Vectors for Gene Therapy (Wagner et al. eds., Academic Press 1999); Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998).

[0105] Unless otherwise defined, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. Furthermore, unless the context otherwise requires, singular terms shall include the plural and plural terms shall include the singular. Generally, the nomenclature utilized in connection with, and techniques of, cell and tissue culture, molecular biology, and protein and oligo- or polynucleotide chemistry and hybridization described herein are those 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 performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures are generally carried out according to conventional methods well known in the art and as described in the various general and more specific references cited and discussed throughout this specification. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)). The nomenclature utilized in connection with, and the laboratory procedures and techniques of analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation and delivery, and treatment of patients.

[0106] example Example 1 Identification and characterization of FZD4- or FZD5-binding Fab-phages. A. FZD4 antibodies from the affinity maturation library of FZD4-binding antibodies 5027 and 5044; FZD5 antibodies from the affinity maturation library of FZD5-binding antibodies 2919 and 2928. Affinity maturation libraries of known FZD4-binding antibodies 5027 and 5044 and known FZD5-binding antibodies 2919 and 2928 were prepared using routine methods essentially as described in U.S. Publication No. 2016 / 0194394, inventors Sidhu et al. (see also Persson et al. J. Mol. Biol., 2013 Feb 22;425(4):803-11 https: / / pubmed.ncbi.nlm.nih.gov / 23219464 / , both of which are incorporated herein by reference in their entireties).

[0107] The six CDRs of the heavy chain (CDR-H1, CDR-H2, and CDR-H3) and light chain (CDR-L1, CDR-L2, and CDR-L3) of antibodies 5044, 5027, 2919, and 2928 isolated from the affinity maturation library are shown in Tables 1 and 2.

[0108] Single-point ELISA was performed in 96-well Maxisorp plates coated with the extracellular domain (ECD) of human FZD4 protein in the presence or absence of a saturating concentration of 5027 diabody-Fc (a diabody containing the VL and VH of 5027 linked to the Fc domain). The plates were incubated with monoclonal Fab phage, followed by incubation with horseradish peroxidase (HRP)-conjugated anti-M13 antibody. The wells were then washed eight times and incubated with 3,3,'5,5'-tetramethylbenzidine / H2O2 peroxidase (TMB) substrate for 5-10 minutes. The reaction was stopped by adding 1 M H3PO4, and absorbance was measured spectrophotometrically at 450 nm in a microtiter plate reader. The results of the assay are shown in Figures 1 and 2 and demonstrate that the newly identified FZD4 antibody binds to FZD4 at a site that overlaps with the site recognized by antibody 5027. The FZD4 binding antibodies 5027 and 5044 are described in US Provisional Patent Application No. 62 / 885,781, which is incorporated herein by reference.

[0109] B. Epitope mapping of the lead FZD4 antibody. ELISA assays were performed in 384-well Maxisorp plates coated with wild-type FZD4 ECD (FZD4) or mutant FZD4 proteins (FZD_swap1-18) in which segments of the FZD4 ECD were replaced with the corresponding region from FZD5. The plates were incubated with 10 nM IgGs known to specifically bind FZD4, i.e., 5044 and 5027, or pan-specific IgG, i.e., 5016 (which binds to FZD4, FZD5, and other FZD receptors), followed by incubation with horseradish peroxidase (HRP)-conjugated anti-kappa light chain antibody. Phosphate-buffered saline (PBS) and IgG 4275, which does not bind to FZD4 or FZD5, were used as controls. The wells were washed six times and then incubated with 3,3,'5,5'-tetramethylbenzidine / H2O2 peroxidase (TMB) substrate for 3-5 minutes. The reaction was stopped by adding 1M H3PO4, and absorbance was measured spectrophotometrically at 450 nm in a microtiter plate reader (see Figure 2). The pan-FZD binder 5016 is a positive control demonstrating that the antigens are functional, except for "FZD4_Swap10." Both FZD4-specific antibodies 5027 and 5044 were unable to bind to "FZD4 Swap 7," suggesting that these molecules bind to this region of the FZD ECD.

[0110] C. Characterization of FZD4 IgG. FZD4-binding full-length IgG was expressed by transient transfection in the Expi293 cell culture system (essentially as described in Tao et al., Tailored tetravalent antibodies potently and specifically activate Wnt / Frizzled pathways in cells, organoids and mice. Elife. 2019 Aug 27;8:e46134. doi:10.7554 / eLife.46134; PMID:31452509.) and purified via Protein A affinity chromatography. Briefly, approximately 2.5 × 10 cells were transfected in Expi293 expression medium (Gibco) in baffled cell culture flasks. 6 Cells were grown to a density of 1000 cells / ml and transfected with the appropriate vector using FectoPRO transfection reagent (Polyplus-transfection) using the standard manufacturer's protocol (ThermoFisher). Expression proceeded for 5 days at 37°C and 8% CO2 with shaking at 125 rpm. After expression, cells were removed by centrifugation, and protein was purified from the conditioned medium using rProtein A Sepharose (GE Healthcare). Purified protein was buffer-exchanged into either PBS or a formulated stabilizing buffer (36.8 mM citric acid, 63.2 mM Na2HPO4, 10% trehalose, 0.2 M L-arginine, 0.01% Tween-80, pH 6.0) for storage. Protein concentration was determined by absorbance at 280 nm, and purity was confirmed by SDS-PAGE analysis.

[0111] Expression titers were determined as mg of purified protein per liter of mammalian cell culture. Size exclusion chromatography (SEC) results in Table A below are defined as: "-": evidence of multiple peaks on the SEC trace, <50% monomeric species; "+": >50% monomeric species, delayed retention time (>14 min); "++": >90% of the major peak at / approximate the expected retention time of monomeric IgG. Standard retention times were determined by comparison with trastuzumab. [Table 1]

[0112] Trac IDs correspond to the antibody numbers in Tables 1 and 2.

[0113] D. Size exclusion chromatography analysis and ELISA specificity determination of FZD4 IgG. Twenty micrograms of FZD4-binding IgG was separated on an AdvanceBio SEC, 300 Å, 2.7 μm, 4.6 × 300 mm column in a mobile phase of PBS using an Agilent Bio-Inert HPLC. Protein elution was monitored using absorbance at 280 nM. The results are shown in Figure 3A.

[0114] ELISA specificity of FZD4 antibodies was determined for FZD1 and FZD10, the two FZD family members most closely related to FZD4. ELISA assays were performed in 384-well Maxisorp plates coated with 1 μg / ml of FZD ECD wild-type or mutant protein, and excess binding sites were blocked with 0.5% BSA. The plates were incubated with 10 nM FZD4-binding IgG, followed by horseradish peroxidase (HRP)-conjugated anti-kappa light chain antibody. After washing the wells six times, the plates were incubated with 3,3,'5,5'-tetramethylbenzidine / H2O2 peroxidase (TMB) substrate for 3–5 min. The reaction was stopped by adding 1 M H3PO4, and absorbance was measured spectrophotometrically at 450 nm in a microtiter plate reader. The results are shown in Figure 3B.

[0115] E. Identification of CDRs of FZD4- or FZD5-binding antibodies. The amino acid sequences of the CDRs of FZD4-binding immunoglobulins and FZD5-binding immunoglobulins are shown in Tables 1 and 2. The CDRs were identified according to the INTERNATIONAL IMMUNOGENETICS INFORMATION SYSTEM (IMGT numbering system; Lefranc et al., 2003, Development and Comparative Immunology 27:55-77) and annotated as described in Persson et al. J Mol Biol. 2013 Feb 22;425(4):803-11, both of which are incorporated herein by reference. [Table 2-1] [Table 2-2] [Table 3-1] [Table 3-2] [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5] [Table 4-6]

[0116] Example 2: Identification and characterization of LRP-binding synthetic antibodies A. Phage clone ELISA of synthetic antibodies targeting LRP5 and LRP6. Single-point ELISA was performed on 96-well Maxisorp plates coated with the ECD of mouse LRP5-his protein or human Fc and blocked with BSA (0.5%). The plates were loaded with monoclonal Fab-phages or VH-phages and titers >10. 9 The wells were incubated with phage / ml, followed by incubation with horseradish peroxidase (HRP)-conjugated anti-M13 antibody. After washing the wells eight times, they were incubated with 3,3,'5,5'-tetramethylbenzidine / H2O2 peroxidase (TMB) substrate for 5-10 minutes. The reaction was stopped by adding 1 M H3PO4, and absorbance was measured spectrophotometrically at 450 nm in a microtiter plate reader. The results are shown in Figure 4. The results demonstrate that the synthetic antibodies bound to LRP5. LRP5-binding antibodies 2459, 2460, and 8716 are described in U.S. Provisional Patent Application No. 62 / 886,913, which is incorporated herein by reference.

[0117] Single-point ELISA was performed on 96-well Maxisorp plates coated with the ECD of human LRP6-Fc protein chimera. The plates were incubated with monoclonal Fab phage or VH-phage at a titer of >10 phage / ml, followed by incubation with horseradish peroxidase (HRP)-conjugated anti-M13 antibody. After washing the wells eight times, the plates were incubated with 3,3,'5,5'-tetramethylbenzidine / H2O2 peroxidase (TMB) substrate for 5-10 minutes. The reaction was stopped by adding 1 M H3PO4, and absorbance was measured spectrophotometrically at 450 nm in a microtiter plate reader. The results are shown in Figures 5A and 5B. The results demonstrate that the synthetic antibodies bound to LRP6. LRP6-binding antibodies 2539, 2540, and 2542 are described in U.S. Provisional Patent Application No. 62 / 886,918, which is incorporated herein by reference.

[0118] B. Identification of CDRs of synthetic antibodies targeting LRP5 and LRP6. The CDRs of the LRP5- and LRP6-binding immunoglobulins listed in Tables 3 and 4 were identified according to the INTERNATIONAL IMMUNOGENETICS INFORMATION SYSTEM (IMGT numbering system; Lefranc et al., 2003, Development and Comparative Immunology 27:55-77) and annotated as described in Persson et al. J Mol Biol. 2013 Feb 22;425(4):803-11, both of which are incorporated herein by reference. [Table 5-1] [Table 5-2] [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4] [Table 6-5]

[0119] Example 3: Tetravalent binding antibody molecule format A. We generated various formats of tetravalent antibody molecules containing pan-FZD and LRP5 / 6 antibody fragments, e.g., scFv, diabody, and Fab, flanking the Fc domain (see Table 5 and Figure 6) and assayed their Wnt agonist activity. DNA fragments encoding antibody variable domains were amplified by PCR from phagemid DNA templates or constructed by chemical synthesis (Twist Biosciences). The DNA fragments were cloned into a mammalian expression vector (pSCSTa). The bispecific diabody and IgG contained an optimized version of the "knobs-in-holes" heterodimeric Fc (Ridgway et al., Protein Eng. 9, 617-621 (1996)). The diabody domains were arranged in a VH-VL orientation, with the variable domains separated by a short GGGGS linker (SEQ ID NO: 886), which facilitates intermolecular association between the VH and VL domains and thus diabody formation. To generate diabody fusion constructs, diabody chains were fused to human IgG1 Fc. The diabody-fc-diabody protein was constructed as VH-x-VL-y-[human IgG1 Fc]-z-VH-x-VL, where the linkers were x = GGGGS (SEQ ID NO: 886), y = LEDKTHTKVEPKSS (SEQ ID NO: 887), and z = SGSETPGTSESATPESGGG (SEQ ID NO: 888). In this format, the human IgG1 Fc or knobs-in-holes IgG1 Fc fragment spanned positions 234 to 478 (Kabat numbering). For scFv-Fc fusions, the variable domains were arranged in a VL-VH orientation and linked by a long GTTAASGSSGGSSSGA linker (SEQ ID NO: 889), which promotes intramolecular association between the VH and VL domains and thus scFv formation. Variants with the Fab domain fused to the C-terminus of the Fc were generated via chemical synthesis (Twist Biosciences). For all constructs, the entire coding region was cloned in frame with the secretory signal peptide into a mammalian expression vector.

[0120] These various tetravalent binding antibody molecules, including pan-specific FZD and LRP5 / 6 antibody fragments, were tested in the TOPFLASH assay to monitor β-catenin-mediated gene reporter activity. Proteins were compared to the natural ligand Wnt3a. Assays were performed by plating TOPFLASH cells at approximately 70% confluency in 96-well tissue culture-treated plates. Agonists were diluted in DMEM to final assay concentrations ranging from 0.046 nM to 100 nM, and cells were treated overnight at 37°C under 5% CO2. Luciferase expression was quantified in 96-well black plates using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer's instructions. Briefly, HEK293T cells were transduced with a lentivirus encoding the pBARls reporter (Biechele and Moon in Wnt Signaling: Pathway Methods and Mammalian Models, E. Vincan, 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. Before transfection or stimulation, 1-2 x 10 cells were transduced in 120 μl. 3Cells were seeded into each well of a 96-well plate for 24 hours. The next day, FZD agonist or Ab protein was added. After 15–20 hours of stimulation, cells were lysed and luminescence was measured using an Envision plate reader (PerkinElmer) according to the dual-luciferase protocol (Promega). For FZD4 agonist assays, FZD4 cDNA was transfected for 6 hours, followed by the addition of FZD agonist. For Wnt inhibition assays, Wnt1 was introduced by cDNA transfection, or WNT3A protein was applied 6 hours before the addition of Ab protein. All assays were repeated at least three times. The results are shown in Table 5. As shown in Table 5, each tetravalent format activates FZD signaling to different degrees when clustering FZD4 and LRP5. These formats were also evaluated for stability, uniformity, and yield production from Expi293 (Figures 3 and 9). From these analyses, the diabody-Fc-Fab format offers the best balance of activity, expression, and stability. Finally, we applied the same modality configuration to FZD5 and LRP6 and observed potent agonist activity. The results in Table 5 show that various tetravalent modalities induce WNT agonism, and that engagement of two LRP5 / 6 epitopes results in higher WNT signaling activity (maximum) than engagement of a single LRP5 / 6 epitope. [Table 7-1] [Table 7-2]

[0121] B. Diabody-Fc-Fab Format FZD4 Agonists Bispecific LRP5-binding diabodies and FZD agonists with FZD4-binding domains containing FZD4-binding Fabs (FZD4 agonists), FZD5-binding domains containing FZD5-binding Fabs (FZD5 agonists), or FZD-binding domains that bind to multiple FZDs (pan-FZD agonists) were generated using the knobs-in-holes system. Briefly, constructs were generated by chemical synthesis (Twist Biosciences) or standard molecular biology techniques in a mammalian expression vector (pSCSTa). Diabody constructs were configured in a VH-VL format with a short (GGGGS (SEQ ID NO: 886)) linker connecting the VH and VL to promote intermolecular pairing. For bispecific diabody configuration, the variable domains of paratopes A and B, respectively, were arranged as VH(A)-VL(B) on the hole Fc chain and as VH(B)-VL(A) on the knob Fc chain to facilitate proper paratope formation. The diabody was fused to the N-terminus of an optimized knobs-in-holes heterodimeric Fc (Ridgway et al., Protein Eng. 9, 617-621 (1996)) via a GGGGSGGGGSEPKSS linker (SEQ ID NO: 890). The Fc region also contains effector-null mutations D278A and N314G (Kabat numbering), corresponding to D655A / N297G (EU numbering). The Fab domain was fused to the C-terminus of the heterodimeric Fc via a GGGSGGGSGGGSGGGSTG linker (SEQ ID NO: 891). Directly fused to this linker was the N-terminus of the Fab VH domain, followed by the CH1, terminating at T238 (Kabat numbering). This Fab is paired with a standard kappa light chain cloned as described above. For all constructs, the entire coding region was cloned in frame with the secretory signal peptide into a mammalian expression vector.

[0122] Furthermore, the diabody-fc-Fab format is constructed as VH-x-VL-y-[human IgG1 Fc]-z-VH, with linkers x = GGGGS (SEQ ID NO: 886), y = GGGGSGGGGSEPKSSDKTHT (SEQ ID NO: 892), and z = GGGSGGGSGGGSGGGSGSTG (SEQ ID NO: 891). The diabody domains are arranged in a VH-VL orientation, with the variable domains separated by a short GGGGS linker (SEQ ID NO: 886), which promotes intermolecular association between the VH and VL domains and thus diabody formation. Furthermore, the Fc region can exhibit attenuated effector function by amino acid mutations to the N297G and D265A (DANG) variant or the L234A, L235A, P331S (LALAPS) variant, and the Fc region further comprises the knobs-in-holes heterodimerization variants Merrimack, Merchant, or Merchant S:S.

[0123] Figure 7 shows a diagram of a diabody-Fc-Fab format FZD4 agonist with an LRP5-binding domain composed of a diabody that is bivalent and bispecific for LRP5, and an FZD4-binding domain composed of two FZD4-binding Fab fragments formed by the VL and CL1 of the light chain construct paired with the VH and CH1 of the heavy chain hole and heavy chain knob constructs, respectively. Table 12 shows the amino acid sequences of the heavy and light chains of the FZD4 agonist ANT (diabody-Fc-Fab format): heavy chain knob construct (ANT16 knob), heavy chain hole construct (ANT hole), and light chain construct. The light and heavy chain variable CDRs are in bold, underlined, and italicized.

[0124] FIG. 16A shows diabody-Fc-Fab format FZD4 agonists with Fc regions with attenuated effector function due to amino acid mutations, e.g., N297G(NG) and D265A, (DANG) variants, and / or LALAPS variants, and further comprising knobs-in-holes heterodimerization variants Merrimack, Merchant, or Merchant S:S.

[0125] B. IgG-diabody format FZD4 agonist A FZD agonist with two FZD-binding Fabs forming the N-terminal binding domain, a bispecific LRP5 / 6-binding diabody forming the C-terminal binding domain, and an Fc domain was generated using knobs-in-holes. The IgG-diabody protein was constructed as VH-[human IgG1 Fc]-y-VH-x-VL (linker: x = GGGGS (SEQ ID NO: 886) and y = GGGSGGGSGGGSGGGSGSTG (SEQ ID NO: 891)).

[0126] Figure 15 shows a diagram of an IgG-diabody format FZD4 agonist with an FZD-binding domain comprising two Fab fragments (each Fab binds to an FZD) attached to the N-terminus of the Fc domain. The LRP5 / 6 coreceptor-binding domain is attached to the C-terminus of the Fc domain and is composed of a diabody that binds to two different sites on the coreceptor, e.g., the Wnt1 site (E1-E2) and the Wnt3 site (E3-E4) on LRP5 / 6. The Fab may be specific for a particular FZD, e.g., FZD4, or may be pan-specific, binding to more than one FZD, e.g., FZD4 and one or more other FZDs.

[0127] Figure 16B shows IgG-diabody FZD4 agonists with Fc regions that have attenuated effector function due to amino acid mutations, e.g., N297G(NG) and D265A, (DANG) variants, and / or LALAPS variants, and further include the knob-in-hole heterodimerization variants Merrimack, Merchant, or Merchant S. Table 13 shows the amino acid sequences of the heavy and light chains of the FZD4 agonists, ANT39 (diabody-Fc-Fab format) and ANT39wi (IgG-diabody format): heavy chain knob constructs (ANT39 and ANT39i-knob), heavy chain hole constructs (ANT39 and ANT39i-hole), and light chain constructs. Table 13 also includes the amino acid sequences of the heavy and light chains of the FZD4 agonists, ANT39 and ANT39i variants DANG, LALAPS, LALAPS Merchant, and LALAPS Merchant SS. The light and heavy chain variable CDRs are in bold, underlined and italic. [Table 8] [Table 9] [Table 10] [Table 11]

[0128] C. FZD agonists are highly specific for FZD4, bind with high specificity, and are stable in solution. Using biolayer interferometry (BLI), we found that the FZD4 agonists described herein are highly specific for FZD4 over other FZD receptors. Recombinant FZD ECD proteins were immobilized on BLI sensors. A diabody-Fc-Fab format FZD4 agonist, with an LRP5-binding domain composed of a diabody that is bivalent and bispecific for LRP5 and an FZD4-binding domain composed of two FZD4-binding Fab fragments, was tested for binding to the ECD protein at a concentration of 100 nM in a buffer of PBS + 0.05% Tween-20 and 1% BSA. The results are shown in Figure 8A. Assay controls included CM0199, a diabody-Fc-diabody format FZD agonist that recognizes FZD4 and LRP5, and immunoglobulin 4275, an IgG that does not bind to FZDs or LRPs.

[0129] FZD4 agonists also failed to recognize common nonspecific antigens. FZD4 agonists were tested at 100 nM for binding to a panel of antigens essentially as described in Monquet et al., "Polyreactivity increases the apparent affinity of anti-HIV antibodies by heteroligation," Nature 2010 Sep 30;467(7315):591-5 (PMC3699875), and Jain et al., "Biophysical properties of the clinical-stage antibody landscape," Proc Natl Acad Sci 2017 Jan 31;114(5):944-949 (PMC5293111). Assay controls included CM0199, a diabody-Fc-diabody format FZD agonist that recognizes FZD4 and LRP5, and immunoglobulin 6606, an IgG that is particularly prone to nonspecific binding in this assay. Results are shown in Figure 8B.

[0130] FZD4 agonists containing binding domains for FZD4 and LRP5 bind with high affinity to both FZD4 and LRP5. The apparent affinity of FZD4 agonists for the recombinant ECD of FZD4 was determined by biolayer interferometry essentially as described in Elife. 2019 Aug 27;8:e46134. Briefly, BLI assays were performed using an Octet HTX instrument (ForteBio). To measure antigen binding, FZD-Fc proteins were captured on an AHQ BLI sensor (18-5001, ForteBio) 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). FZD-coated or control (Fc-coated) sensors were transferred to 100-0.1 nM tetravalent FZD agonist in assay buffer (PBS, 1% BSA, 0.05% Tween 20), and association was monitored for 300 s. The sensors were then transferred to assay buffer, and dissociation was monitored for an additional 300 s. The shaking speed was 1000 rpm, and the temperature was 25°C. The results are shown in Table 7. [Table 12]

[0131] The FZD4 agonist was also analyzed by SEC in comparison to trastuzumab IgG. The results are presented in Figure 9A and demonstrate that the agonist in the diabody-Fc-Fab format is stable and homogeneous in solution.

[0132] FZD4 agonists are also stable in solution. Purified FZD4 agonists, ANT16, ANT18, ANT20, ANT21, and ANT36, were resuspended at 1 mg / ml (except for ANT18, which was resuspended at 0.34 mg / ml) in 10 mM histidine, 140 mM NaCl, 0.9% sucrose, pH 6, and stored at either 4°C or 40°C for 6 days. Samples were removed at various time points, centrifuged to remove precipitated protein, and the residual protein concentration was measured. The results are shown in Tables 8 and 9. [Table 13] [Table 14]

[0133] On day 6, the amount of FZD4-specific binding sites remaining in the samples was quantified using BLI. Differential scanning fluorimetry analysis showed that FZD4 agonists with a diabody-Fc-Fab format, which has an LRP-binding diabody at the N-terminus of the Fc domain and two FZD4-binding Fabs at the C-terminus of the Fc domain, had a thermal denaturation profile similar to that of trastuzumab. IgG generally exhibits two peaks in the thermal stability assay, the first corresponding to CH2 and the second corresponding to the Fab domain and CH3 (see Figure 9B).

[0134] FZD4 agonists were also assayed for induction of the beta-catenin target gene AXIN2 in a mouse endothelial cell line (bEND3.1) and were shown to induce transcription in a concentration-dependent manner. These results are shown in Figure 10. [Table 15]

[0135] Example 4. FZD4 agonists were assayed for their ability to counteract the effects on cell junction dissociation and increased permeability mediated by VEGF, a cytokine released during tissue hypoxia. VEGF treatment of bEND3.1 cells results in junction dissociation, as seen by the loss of plasma membrane staining for CLDN3, CLDN5, and ZO-1. Co-treatment of cells with VEGF and an FZD4 agonist results in a near-complete rescue of this effect (Figure 11). This reduction in cell-cell junction stability mediated by VEGF treatment leads to increased endothelial cell permeability, as monitored in a transendothelial permeability assay measuring passage of 40 kDa FITC-dextran across confluent endothelial monolayers of bEnd.3 cells grown on transwell filters. Co-treatment of cells with VEGF and an FZD4 agonist completely rescues the VEGF-mediated increase in cell permeability. These results demonstrate that FZD4 agonists promote endothelial cell barrier function through a VEGF-independent mechanism.

[0136] A) Immunofluorescence of ZO-1 (green) / CLDN3 (red) and ZO-1 (green) / CLDN5 (red) localization at bEnd.3 cell junctions. bEnd.3 cells were treated with 30 nM F4L5.13 (also known as CM0199) and Norrin for 1 hour, or not, in the presence or absence of VEGF (100 ng / ml). DAPI (blue) stains nuclei. B) Transendothelial permeability was determined by measuring the passage of FITC-dextran through bEnd.3 monolayers. VEGF (100 ng / ml) and F4L5.13 (30 nM) were used alone or simultaneously, or pretreated with VEGF for 1 hour followed by F4L5.13 treatment for 1 hour. FITC-dextran passage was measured after bEnd.3 treatment. Error bars indicate SEM, n = 5. Results are shown in Figure 11.

[0137] Example 5. Novel FZD5 antibodies bind to FZD5 at a site overlapping with 2919 identified from an affinity maturation library. Single-point ELISA was performed in 96-well Maxisorp plates coated with the ECD of human FZD5 protein in the presence or absence of saturating concentrations of 2919 IgG. The plates were incubated with monoclonal Fab phage, followed by incubation with horseradish peroxidase (HRP)-conjugated anti-M13 antibody. The wells were then washed eight times and incubated with 3,3,'5,5'-tetramethylbenzidine / H2O2 peroxidase (TMB) substrate for 5-10 minutes. The reaction was stopped by adding 1 M H3PO4, and the absorbance was measured spectrophotometrically at 450 nm in a microtiter plate reader. The results are shown in Figure 12.

[0138] Example 6. Novel FZD5 antibodies from a 2928 affinity maturation library selectively bind to FZD5. Single-point ELISA was performed on 96-well Maxisorp plates coated with the ECD of human FZD2, FZD5, or FZD8 proteins. The plates were incubated with monoclonal Fab phage, followed by incubation with horseradish peroxidase (HRP)-conjugated anti-M13 antibody. The wells were then washed eight times and incubated with 3,3,'5,5'-tetramethylbenzidine / H2O2 peroxidase (TMB) substrate for 5–10 minutes. The reaction was stopped by adding 1 M H3PO4, and the absorbance was measured spectrophotometrically at 450 nm in a microtiter plate reader. The results are shown in Figure 13.

[0139] Example 7. Pan-FZD / LRP6 ANT9 and FZD5-specific / LRP6 ANT9 activate Wnt signaling in cells. TOPFLASH HEK293 cells were treated overnight with various concentrations of FZD agonists or a non-targeting control molecule (CM0156), and TCF / LEF-driven luciferase expression was measured using a standard luciferase assay. Both molecules can activate FZD-mediated luciferase expression in a concentration-responsive manner. ANT9 can bind to seven of the ten FZD receptor subtypes and produces a higher maximal activation signal than the FZD5-specific ANT59.

[0140] In vivo experiments DSS-induced colitis model In Figure 24, C57 / BL6 mice were given 2% DSS in drinking water for 7 days, followed by 0.5% DSS for an additional 3 days to induce colitis. Control FLAg, Pan-FLAg, and ANT59 were administered by intraperitoneal injection at a dose of 10 mg / kg on days 4 and 7. Mice were weighed daily. On day 10, mice were euthanized, and tissues were collected for colon length and histology.

[0141] histology For histological analysis, harvested tissues were fixed in 4% paraformaldehyde and embedded in paraffin. 5 μm sections were stained with hematoxylin and eosin (H&E). Images were captured using a Nikon Eclipse microscope (Figure 23).

[0142] Organoid culture and viability measurement Small intestinal crypts were harvested from 8-week-old female C57BL / 6 mice and cultured as previously described (O'Rourke et al., 2016). Organoid cultures were passaged, embedded in 25 μl of growth factor-reduced Matrigel (Corning, 356231), and seeded in triplicate into 48-well plates. Organoid cultures were treated with DMSO, 1 μM LGK974, 1 μM LGK974 + 50% WNT3A-conditioned medium, 1 μM LGK974 + 30 nM Pan-FLAg, 1 μM LGK974 + 30 nM FZD2-FLAg, 1 μM LGK974 + 30 nM FZD4-FLAg, 1 μM LGK974 + 30 nM FZD5-FLAg, or 1 μM LGK974 + 30 nM FZD7-FLAg. Treatments were prepared in 250 μl of complete medium and added to each well on the day of passage, with changes every 2–3 days. At the endpoint (7 days), 150 μl of Cell Titer-Glo3D (Promega) was added to each well's 150 μl medium. Organoids were lysed on a rocking platform at room temperature for 30 minutes. Luminescence readings were measured in duplicate for 20 μl of lysate from each well of the Envision multilabel plate reader. The average luminescence readings for each condition were normalized to the control condition to calculate relative survival rates (FIG. 22).

[0143] Example 8. Transient Expression of Eight ANT39 Variants. A series of eight ANT39 variants (Figures 16A and 16B) was transiently expressed in CHO cells using a standard manufacturing lipid-based protocol (ThermoFisher). Briefly, cells were grown in growth medium to a density of approximately 2.0 x 10 cells / ml and transfected with the relevant DNA using the appropriate transfection reagent. For each variant, two alternative input plasmid ratios were tested: either 1:1:2 or 2:1:3 (knob heavy chain:hole heavy chain:light chain). Conditioned medium was collected after 7 days, purified by Protein A Sepharose, and titered. [Table 16]

[0144] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention. Various substitutions, alterations, and modifications can be made to the invention without departing from the spirit and scope of the invention. Other aspects, advantages, and modifications are within the scope of the invention.

[0145] The contents of all references, issued patents, and published patent applications cited throughout this application are incorporated herein by reference, and the appropriate components, processes, and methods of these patents, applications, and other documents may be selected for the present invention and its embodiments.

[0146] Throughout the following specification and claims, unless the context otherwise requires, the word "comprise" and variations such as "comprises" and "comprising" will be understood to imply the inclusion of a stated integer, step, group of integers or group of steps, but not the exclusion of other integers, steps, groups of integers or group of steps.

[0147] For clarity and to inform the public of the use of this specification, 、 , ...and <n> At least one of the following" or "< / n> 、 , ...or <n> At least one of the following" or "< / n> 、 、... <n> , or at least one of a combination thereof" or "< / n> 、 , ...and / or <n>The phrase "a" or "an" is defined by applicant in the broadest sense and supersedes any other implied definition above or below to mean one or more elements selected from the group including A, B, ..., and N, unless expressly stated otherwise by applicant. In other words, the phrase means any combination of one or more of the elements A, B, ..., or N, including any one element only, or one element in combination with one or more other elements, which combination may also include additional, unlisted elements. Unless otherwise indicated or suggested otherwise by context, as used herein, "a" or "an" means "at least one" or "one or more."

[0148] [Table 17] [Table 18] [Table 19] [Table 20] [Table 21] [Table 22-1] [Table 22-2] [Table 23] [Table 24]

[0149] Table 13 Diabody-Fc-Fab and IgG-diabody amino acid sequences of the "heavy chain" hole, "heavy chain" knob, and "light chain" constructs of FZD4 agonists, ANT39 and ANT42, ANT39 and ANT42, and ANT39i and ANT42i, with Fc domain amino acid mutations DANG, LALAPS, LALAPS and Merchant, or LALAPS and Merchant SS. Mature sequences do not include the 5' leader peptide. Chain V region CDRs are underlined, italicized, and bolded; Fc null mutations are italicized; CH3 heterodimerization mutations are underlined and italicized; CH3 cys disulfide bridge is bolded; and linker is underlined.

[0150]

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[0151] Table 25

[0152] Table 26 Table 27 Table 28 Table 29 [Table 30] [Table 31] [Sequence List Free Text]

[0153] Sequence Listing 1-896 <223> Writing artificial sequences: synthetic peptides Sequence list 897~984 <223> Description of artificial sequences: synthetic polypeptides Sequence Listing 985 <223> Writing artificial sequences: synthetic peptides Sequence list 986~1029 <223> Description of artificial sequences: synthetic polypeptides< / n>

Claims

1. (a) an Fc domain or fragment thereof comprising constant heavy chain domain 3 (CH3); (b) a bivalent low-density lipoprotein receptor-related protein 5 (LRP5) binding domain; (c) a bivalent Frizzled (FZD) binding domain; and 1. A tetravalent binding antibody molecule comprising: The LRP5 binding domain is attached to one end of the Fc domain, and the FZD binding domain is attached to the other end of the Fc domain; The LRP5 binding domain comprises a diabody that binds to LRP5, and the FZD binding domain comprises two scFvs or two Fabs that bind to FZD4; The above tetravalent binding antibody molecule.

2. (a) the LRP5 binding domain diabody is linked to the N-terminus of the Fc domain; and (b) the FZD4-binding domain is linked to the C-terminus of the Fc domain; The tetravalent binding antibody molecule of claim 1.

3. (a) the LRP5 binding domain diabody is linked to the N-terminus of the Fc domain via the VL or VH of the diabody; (b) the FZD-binding domain comprises two FZD-binding Fabs fused to the C-terminus of an Fc, each Fab being linked to the Fc domain via a heavy or light chain variable domain (VH or VL) of the Fab linked to the CH3 domain of the Fc domain; The tetravalent binding antibody molecule of claim 2.

4. A tetravalent binding antibody molecule comprising an N-terminal LRP5-binding diabody and a C-terminal domain comprising two FZD4-binding scFvs, wherein the tetravalent binding antibody molecule is A dimer of a first and a second monomer, each monomer comprising, from N-terminus to C-terminus: (a) a first peptide comprising a first heavy chain variable domain (VH) and a first light chain variable domain (VL) that binds to LRP5; (b) an Fc region or fragment thereof comprising constant heavy chain domain 3 (CH3); (c) a second peptide comprising a second VL and a second VH that binds to FZD4; and the dimer comprising a single-chain polypeptide comprising: first and second light chain monomers, each light chain monomer comprising, from N-terminus to C-terminus, a VL that binds to FZD4 and is linked to a constant light chain domain 1 (CL1 domain); Including, The first and second monomers dimerize via the Fc region or fragment thereof, and the first VH and VL of each monomer pair with the first VH and VL of the other monomer to form a diabody that binds to LRP5, and the second VL and VH of each monomer pair to form an scFv that binds to FZD4; The LRP5-binding diabody forms the N-terminal LRP5-binding domain of the tetravalent binding antibody molecule, and the two FZD4-binding scFvs form the C-terminal FZD4-binding domain of the tetravalent binding antibody molecule; The above tetravalent binding antibody molecule.

5. The tetravalent binding antibody molecule of claim 1, wherein the FZD binding domain is attached to the N-terminus of the Fc domain and the LRP5 binding domain is attached to the C-terminus of the Fc domain.

6. (a) the FZD-binding domain comprises two Fabs that bind to FZD4, each Fab being linked to the N-terminus of the Fc domain via a heavy or light chain variable domain (VH or VL) of the Fab linked to a CH2 domain of the Fc domain; (b) The LRP5 binding domain comprises a diabody or two scFvs that bind to LRP5, and the diabody or two scFvs are linked to the C-terminus of the Fc domain via the VL or VH of the diabody or scFv linked to CH3 of the Fc domain; The tetravalent binding antibody molecule of claim 5.

7. A tetravalent binding antibody molecule comprising an Fc domain or fragment thereof comprising constant heavy chain domain 3 (CH3), an N-terminal FZD4 binding domain comprising two FZD4-binding Fabs, and a C-terminal LRP5 binding domain comprising an LRP5-binding diabody; (a) first and second heavy chain monomers, each heavy chain monomer comprising, from N-terminus to C-terminus: Linked to a light chain variable domain (VL) that binds to the LRP5 co-receptor, (i) a heavy chain variable domain (VH) that binds to FZD4; (ii) heavy chain constant region domain 1 (CH1 domain); (iii) the CH2 domain of the Fc region; (iv) a peptide comprising a VH that binds to the LRP5 co-receptor; the heavy chain monomer comprising a single polypeptide chain comprising: (b) first and second light chain monomers, each light chain monomer comprising, from N-terminus to C-terminus, a VL that binds to FZD4 and is linked to a constant light chain domain 1 (CL1 domain); Including, the first and second heavy chain monomers dimerize via their Fc regions or fragments thereof; The linker between the VH and VL that binds to LRP5 is of a length that promotes pairing of the VH and VL of the first heavy chain monomer with the VL and VH of the second heavy chain monomer to form an LRP5 coreceptor-binding diabody, and the FZD-binding Fab is formed by pairing each heavy chain monomer with a light chain monomer such that the VH that binds to FZD4 and CH1 of each heavy chain monomer pairs with the VL that binds to FZD4 and CL1 of the light chain monomer. A tetravalent binding antibody molecule in which the Fab forms an FZD4 binding domain at the N-terminus of the Fc domain and the diabody forms an LRP5 coreceptor binding domain at the C-terminus of the Fc domain.

8. The tetravalent binding antibody molecule of any one of claims 1 to 7, wherein the LRP5-binding diabody is a bispecific, bivalent LRP5-binding domain that binds to two epitopes within the LRP5 co-receptor extracellular domain.

9. The tetravalent binding antibody molecule of claim 8, wherein the LPR5 binding domain interacts with Wnt1 and Wnt3 epitopes of the LRP5 coreceptor.

10. The tetravalent binding antibody molecule of any one of claims 1 to 9, wherein the FZD-binding domain is monospecific.

11. The LRP5 binding domain diabody binds to LRP5 and comprises the heavy chain complementarity determining regions CDR-H1, CDR-H2 and CDR-H3 and the light chain complementarity determining regions CDR-L1, CDR-L2 and CDR-L3 of the antibody shown in Table 3 or Table 6A; A tetravalent binding antibody molecule according to any one of claims 1 to 10.

12. The tetravalent binding antibody molecule of any one of claims 1 to 11, wherein the Fc domain or fragment thereof dimerizes via a knob-in-hole arrangement of the Fc region or fragment thereof.

13. The tetravalent binding antibody molecule of claim 12, wherein the Fc region of the first heavy chain monomer comprises the mutations T366S, L368A and Y407V, and the Fc region of the second heavy chain monomer comprises the mutation T366W, according to EU numbering.

14. The tetravalent binding antibody molecule of claim 13, wherein the Fc region of the first heavy chain monomer further comprises the mutations S354I and E357L, and the Fc region of the second heavy chain monomer further comprises the mutations Q347M, Y349F, T350D, and L368M, according to EU numbering.

15. The tetravalent linked antibody molecule of claim 13, wherein an additional disulfide bond is introduced between the Fc region of the first heavy chain monomer and the second heavy chain monomer, and preferably, the first heavy chain monomer comprises the mutation Y349C and the second heavy chain monomer comprises the mutation S354C, according to EU numbering.

16. A tetravalent binding antibody molecule according to any one of claims 1 to 15, wherein the Fc domain lacks one or more effector functions.

17. The tetravalent binding antibody molecule of claim 16, wherein the Fc region comprises mutations that alter their effector functions by the amino acid mutations N297G (NG) and / or D265A (DA) variants according to EU numbering.

18. The tetravalent binding antibody molecule of claim 16, wherein the Fc region comprises mutations that alter their effector functions by amino acid mutations L234A, L235A and / or P331S according to EU numbering.

19. The tetravalent binding antibody molecule of claim 18, wherein the Fc region comprises mutations that alter their effector functions by amino acid mutations L234A and L235A (LALA).

20. The tetravalent binding antibody molecule of claim 19, wherein the Fc region comprises mutations that alter their effector functions by the amino acid mutations L234A, L235A and P331S (LALAPS).

21. A tetravalent binding antibody molecule according to any one of claims 1 to 20, wherein the LRP5 binding domain and the FZD binding domain are each linked to the Fc domain by a linker.

22. The tetravalent binding antibody molecule of claim 21, wherein the linker comprises 1 to 100, 1 to 50, 1 to 30, 1 to 25, 1 to 10, 1 to 6 amino acids, 1 to 5 amino acids, or 2 to 4 amino acids.

23. 23. The tetravalent binding antibody molecule of claim 21 or 22, wherein the diabody forming the LRP5 co-receptor binding domain is fused to the Fc domain via a GGGGSGGGGGSEPKSSDKTHT (SEQ ID NO: 892) linker.

24. The tetravalent binding antibody molecule of any one of claims 21 to 23, wherein the FZD4-binding Fab is fused to the Fc region via a GGGSGGGSGGGSGGGSGGGSGSTG (SEQ ID NO: 891) linker.

25. A tetravalent binding antibody molecule according to any one of claims 1 to 24, wherein the VH that binds the LRP5 co-receptor is linked to the VL that binds the LRP5 co-receptor via a short GGGGS (SEQ ID NO: 886) linker.

26. The tetravalent binding antibody molecule of any one of claims 1 to 25, wherein the FZD-binding domain comprises two Fabs that bind to FZD4.

27. The tetravalent binding antibody molecule of claim 26, wherein the FZD-binding Fab comprises light chain complementarity determining regions CDR-L1, CDR-L2, and CDR-L3 and heavy chain CDRs CDR-H1, CDR-H2, and CDR-H3 of an antibody set forth in a sequence listed in Table 1, Table 2, or Table 6.

28. (a) a dimer of first and second heavy chain monomers, each monomer comprising a single polypeptide chain comprising, from N-terminus to C-terminus: (1) A peptide comprising a heavy chain variable domain (VH) that binds to LRP5 and a light chain variable domain (VL) that binds to LRP5; (2) an Fc region or a fragment thereof comprising CH3; (3) a VH that binds to FZD4, and (4) constant heavy chain domain 1 (CH1); where: (5) The VH that binds to LRP5 comprises the heavy chain CDRs (CDR-H1, CDR-H2 and CDR-H3) of the antibody shown in the sequence listed in Table 3 or Table 6, and (b) the VL that binds to LRP5 comprises the light chain CDRs (CDR-L1, CDR-L2 and CDR-L3) of an antibody shown in the sequence listed in Table 3 or Table 6; (c) the VH that binds to FZD4 comprises the heavy chain CDRs (CDR-H1, CDR-H2, and CDR-H3) of an antibody shown in the sequence listed in Table 1, Table 2, or Table 6; and (d) a third and fourth light chain monomer, each comprising a VL that binds to FZD4 and a constant light chain domain 1 (CL1), wherein the VL that binds to FZD4 comprises the light chain CDRs (CDR-L1, CDR-L2, CDR-L3) of an antibody shown in a sequence listed in Table 1, Table 2, or Table 6; Including, The first and second heavy chain monomers dimerize via their Fc regions, and the VL and VH that bind to LRP5 of the first monomer pair with the VH and VL that bind to LRP5 of the second monomer to form a bivalent diabody that binds to LRP5; The CL1 and VL that bind to FZD4 of the third and fourth light chain monomers are paired with the CH1 and VH that bind to FZD4 of the first and second heavy chain monomers to form two Fabs that bind to FZD4, the diabody forming an N-terminal bivalent LRP5 binding domain, and the two Fabs forming a C-terminal bivalent FZD4 binding domain. A tetravalent binding antibody molecule according to any one of claims 1 to 4 and 8 to 27.

29. (a) a dimer of first and second heavy chain monomers, each monomer comprising a single polypeptide chain comprising, from N-terminus to C-terminus: (1) a VH that binds to FZD4; (2) an Fc region or a fragment thereof comprising CH3; (3) A peptide comprising a VH that binds to LRP5 and a VL that binds to LRP5; and (4) constant heavy chain domain 1 (CH1); where: (5) The VH that binds to LRP5 contains the heavy chain CDRs (CDR-H1, CDR-H2, and CDR-H3) of the antibody shown in the sequence listed in Table 3 or Table 6, and (b) The VL that binds to LRP5 contains the light chain CDRs (CDR-L1, CDR-L2, and CDR-L3) of the antibody shown in the sequence set forth in Table 3 or Table 6; (c) the VH that binds to FZD4 comprises the heavy chain CDRs (CDR-H1, CDR-H2, and CDR-H3) of an antibody shown in the sequence listed in Table 1, Table 2, or Table 6; and (d) a third and fourth light chain monomer, each comprising, from N-terminus to C-terminus, a VL that binds to FZD4 and a constant light chain domain 1 (CL1), wherein the FZ4-binding VL comprises the light chain CDRs (CDR-L1, CDR-L2, and CDR-L3) of an antibody set forth in a sequence listed in Table 1, Table 2, or Table 6; Including, The first and second heavy chain monomers dimerize via their Fc regions, and the VL and VH that bind to LRP5 of the first monomer pair with the VH and VL that bind to LRP5 of the second monomer to form a bivalent diabody that binds to LRP5; The CL1 and VL that bind to FZD4 of the third and fourth light chain monomers are paired with the CH1 and VH that bind to FZD4 of the first and second heavy chain monomers to form two Fabs that bind to FZD4, the diabody forming a C-terminal bivalent LRP5 binding domain, and the two Fabs forming an N-terminal bivalent FZD4 binding domain. A tetravalent binding antibody molecule according to any one of claims 5 to 7.

30. The LRP5-binding diabody is bispecific; The CDR of the VH that binds to LRP5 of the first heavy chain monomer and the CDR of the VH that binds to LRP5 of the second heavy chain monomer are not identical; and The CDR of the VL that binds to LRP5 of the first heavy chain monomer is not identical to the CDR of the VL that binds to LRP5 of the second heavy chain monomer; A tetravalent binding antibody molecule according to claim 28 or 29.

31. In the first heavy chain monomer: (a) CDR-H1 and CDR-H2 of the VH that binds to LRP5 comprise FSSSSI (SEQ ID NO: 528) and SISSSYGYTY (SEQ ID NO: 553), respectively, or CDR-H1 and CDR-H2 of the VH that binds to LRP5 comprise LSYYYM (SEQ ID NO: 527) and SIYSSYGYTY (SEQ ID NO: 552), respectively; and (b) CDR-L2 and CDR-L3 of the VL that binds to LRP5 comprise SASDLYS (SEQ ID NO: 491) and YAGAGLI (SEQ ID NO: 510), respectively, or CDR-L2 and CDR-L3 of the VL that binds to LRP5 comprise SASSLYS (SEQ ID NO: 2) and SSYSLI (SEQ ID NO: 130), respectively; and In the second heavy chain monomer: (c) CDR-H1 and CDR-H2 of the VH that binds to LRP5 comprise FTAYAM (SEQ ID NO: 536) and SIYPSGGYTA (SEQ ID NO: 566), respectively, or CDR-H1 and CDR-H2 of the VH that binds to LRP5 comprise FSSSSI (SEQ ID NO: 528) and SISSSYGYTY (SEQ ID NO: 553), respectively; and (d) CDR-L2 and CDR-L3 of the VL that binds to LRP5 contain SASSLYS (SEQ ID NO: 2) and YWAYYSPI, respectively, or CDR-L2 and CDR-L3 of the VL that binds to LRP5 contain SASSLYS (SEQ ID NO: 2) and ASYAPI, respectively; The tetravalent binding antibody molecule of claim 30.

32. The tetravalent binding antibody molecule of any one of claims 28 to 31, wherein in the first and second heavy chain monomers, CDR-H1 and CDR-H2 of the VH that binds to FZD4 comprise LSSYSM (SEQ ID NO: 24) and YISSYYGYTY (SEQ ID NO: 51), respectively, or CDR-H1 and CDR-H2 of the VH that binds to FZD4 comprise LSSYSM (SEQ ID NO: 24) and YISSYDSITD (SEQ ID NO: 61), respectively.

33. A tetravalent binding antibody molecule according to any one of claims 28 to 32, wherein in the third and fourth light chain monomers, CDR-L1 and CDR-L2 of the VL that binds to FZD4 comprise SVSSA (SEQ ID NO: 1) and SASSLYS (SEQ ID NO: 2), respectively, and CDR-L3 of the VL that binds to FZD4 comprises WYYAPI (SEQ ID NO: 3) or WYNAPI (SEQ ID NO: 12).

34. comprising a bivalent, bispecific LRP5 binding domain; (a) in the first heavy chain monomer The VH that binds to LRP-5 comprises CDR-H1 of SEQ ID NO: 528, CDR-H2 of SEQ ID NO: 553, and CDR-H3 of SEQ ID NO: 586 of the ANT16-hole in Table 6A, and the VL that binds to LRP-5 comprises CDR-L1 of SEQ ID NO: 1, CDR-L2 of SEQ ID NO: 491, and CDR-L3 of SEQ ID NO: 510 of the ANT16-hole in Table 6A; Or The VH that binds to LRP-5 comprises CDR-H1 of SEQ ID NO: 527, CDR-H2 of SEQ ID NO: 552, and CDR-H3 of SEQ ID NO: 584 of the ANT18-hole in Table 6A, and the VL that binds to LRP-5 comprises CDR-L1 of SEQ ID NO: 1, CDR-L2 of SEQ ID NO: 2, and CDR-L3 of SEQ ID NO: 130 of the ANT18-hole in Table 6A; Or The VH that binds to LRP-5 comprises CDR-H1 of SEQ ID NO: 527, CDR-H2 of SEQ ID NO: 552, and CDR-H3 of SEQ ID NO: 584 of the ANT20-hole in Table 6A, and the VL that binds to LRP-5 comprises CDR-L1 of SEQ ID NO: 1, CDR-L2 of SEQ ID NO: 491, and CDR-L3 of SEQ ID NO: 510 of the ANT20-hole in Table 6A; Or The VH that binds to LRP-5 comprises CDR-H1 of SEQ ID NO: 528, CDR-H2 of SEQ ID NO: 553, and CDR-H3 of SEQ ID NO: 586 of the ANT21-hole in Table 6A, and the VL that binds to LRP-5 comprises CDR-L1 of SEQ ID NO: 1, CDR-L2 of SEQ ID NO: 2, and CDR-L3 of SEQ ID NO: 130 of the ANT21-hole in Table 6A; Or The VH that binds to LRP-5 comprises CDR-H1 of SEQ ID NO: 527, CDR-H2 of SEQ ID NO: 552, and CDR-H3 of SEQ ID NO: 584 of the ANT36-hole in Table 6A, and the VL that binds to LRP-5 comprises CDR-L1 of SEQ ID NO: 1, CDR-L2 of SEQ ID NO: 2, and CDR-L3 of SEQ ID NO: 130 of the ANT36-hole in Table 6A; Or The VH that binds to LRP-5 comprises CDR-H1 of SEQ ID NO: 528, CDR-H2 of SEQ ID NO: 553, and CDR-H3 of SEQ ID NO: 586 of the ANT39-hole described in Table 6A, and the VL that binds to LRP-5 comprises CDR-L1 of SEQ ID NO: 1, CDR-L2 of SEQ ID NO: 491, and CDR-L3 of SEQ ID NO: 510 of the ANT39-hole described in Table 6A; and the VH that binds to FZD4 comprises the FZD4 VH CDRs CDR-H1 of SEQ ID NO: 24, CDR-H2 of SEQ ID NO: 51, and CDR-H3 of SEQ ID NO: 79 of the ANT16 hole listed in Table 6B; or the VH that binds FZD4 comprises the FZD4 VH CDRs CDR-H1 of SEQ ID NO: 24, CDR-H2 of SEQ ID NO: 51, and CDR-H3 of SEQ ID NO: 79 of ANT18-hole in Table 6B; or the VH that binds FZD4 comprises the FZD4 VH CDRs CDR-H1 of SEQ ID NO: 24, CDR-H2 of SEQ ID NO: 51, and CDR-H3 of SEQ ID NO: 79 of ANT20-hole in Table 6B; or the VH that binds FZD4 comprises the FZD4 VH CDRs CDR-H1 of SEQ ID NO: 24, CDR-H2 of SEQ ID NO: 51, and CDR-H3 of SEQ ID NO: 79 of ANT21-hole in Table 6B; or the VH that binds FZD4 comprises the FZD4 VH CDRs CDR-H1 of SEQ ID NO: 24, CDR-H2 of SEQ ID NO: 61, and CDR-H3 of SEQ ID NO: 90 of ANT36-hole in Table 6B; or the VH that binds to FZD4 comprises the FZD4 VH CDRs CDR-H1 of SEQ ID NO: 24, CDR-H2 of SEQ ID NO: 61, and CDR-H3 of SEQ ID NO: 90 of ANT39-hole in Table 6B; FZD CDR-L1 and CDR-L2 are SVSSA (SEQ ID NO: 1) and SASSLYS (SEQ ID NO: 2), respectively; and (b) in the second monomer, The VH that binds to LRP5 comprises CDR-H1 of SEQ ID NO: 536, CDR-H2 of SEQ ID NO: 566, and CDR-H3 of SEQ ID NO: 603 of the ANT16-knob in Table 6A, and the VL that binds to LRP5 comprises CDR-L1 of SEQ ID NO: 1, CDR-L2 of SEQ ID NO: 2, and CDR-L3 of SEQ ID NO: 493 of the ANT16-knob in Table 6A; Or The VH that binds to LRP5 comprises CDR-H1 of SEQ ID NO: 528, CDR-H2 of SEQ ID NO: 553, and CDR-H3 of SEQ ID NO: 585 of the ANT18-knob in Table 6A, and the VL that binds to LRP5 comprises CDR-L1 of SEQ ID NO: 1, CDR-L2 of SEQ ID NO: 2, and CDR-L3 of SEQ ID NO: 492 of the ANT18-knob in Table 6A; Or The VH that binds to LRP5 comprises CDR-H1 of SEQ ID NO: 536, CDR-H2 of SEQ ID NO: 566, and CDR-H3 of SEQ ID NO: 603 of the ANT20-knob in Table 6A, and the VL that binds to LRP5 comprises CDR-L1 of SEQ ID NO: 1, CDR-L2 of SEQ ID NO: 2, and CDR-L3 of SEQ ID NO: 492 of the ANT20-knob in Table 6A; Or The VH that binds to LRP5 comprises CDR-H1 of SEQ ID NO: 528, CDR-H2 of SEQ ID NO: 553, and CDR-H3 of SEQ ID NO: 585 of the ANT21-knob in Table 6A, and the VL that binds to LRP5 comprises CDR-L1 of SEQ ID NO: 1, CDR-L2 of SEQ ID NO: 2, and CDR-L3 of SEQ ID NO: 493 of the ANT21-knob in Table 6A; Or The VH that binds to LRP5 comprises CDR-H1 of SEQ ID NO: 528, CDR-H2 of SEQ ID NO: 553, and CDR-H3 of SEQ ID NO: 585 of the ANT36-knob in Table 6A, and the VL that binds to LRP5 comprises CDR-L1 of SEQ ID NO: 1, CDR-L2 of SEQ ID NO: 2, and CDR-L3 of SEQ ID NO: 492 of the ANT36-knob in Table 6A; Or The VH that binds to LRP5 comprises CDR-H1 of SEQ ID NO: 536, CDR-H2 of SEQ ID NO: 566, and CDR-H3 of SEQ ID NO: 603 of the ANT39-knob in Table 6A, and the VL that binds to LRP5 comprises CDR-L1 of SEQ ID NO: 1, CDR-L2 of SEQ ID NO: 2, and CDR-L3 of SEQ ID NO: 493 of the ANT39-knob in Table 6A; and a VH that binds to FZD4, FZD4 Fab CDR-H1 of SEQ ID NO: 24, CDR-H2 of SEQ ID NO: 51 and CDR-H3 of SEQ ID NO: 79 of the ANT16 knobs listed in Table 6B; or FZD4 Fab CDR-H1 of SEQ ID NO: 24, CDR-H2 of SEQ ID NO: 51 and CDR-H3 of SEQ ID NO: 79 of the ANT18 knobs listed in Table 6B; or FZD4 Fab CDR-H1 of SEQ ID NO: 24, CDR-H2 of SEQ ID NO: 51 and CDR-H3 of SEQ ID NO: 79 of the ANT20 knobs listed in Table 6B; or FZD4 Fab CDR-H1 of SEQ ID NO: 24, CDR-H2 of SEQ ID NO: 51 and CDR-H3 of SEQ ID NO: 79 of the ANT21 knobs listed in Table 6B; or FZD4 Fab CDR-H1 of SEQ ID NO: 24, CDR-H2 of SEQ ID NO: 61 and CDR-H3 of SEQ ID NO: 90 of the ANT36 knobs listed in Table 6B; or the FZD4 Fab CDR-H1 of SEQ ID NO: 24, the CDR-H2 of SEQ ID NO: 61, and the CDR-H3 of SEQ ID NO: 90 of the ANT39 knobs listed in Table 6B; and, (c) in each of the third and fourth monomers, the VL comprises the FZD4 Fab CDR-L1, CDR-L2 and CDR-L3 of SEQ ID NO: 3 of the ANT16-knob as listed in Table 6B; or the VL comprises FZD4 Fab CDR-L1, CDR-L2 and CDR-L3 of SEQ ID NO: 3 of the ANT18-knob as listed in Table 6B; or the VL comprises the FZD4 Fab CDR-L1, CDR-L2 and CDR-L3 of SEQ ID NO: 3 of the ANT20-knob as listed in Table 6B; or the VL comprises the FZD4 Fab CDR-L1, CDR-L2 and CDR-L3 of SEQ ID NO: 3 of the ANT21-knob as listed in Table 6B; or the VL comprises FZD4 Fab CDR-L1, CDR-L2 and CDR-L3 of SEQ ID NO: 12 of ANT36-knob as listed in Table 6B; or VL comprises FZD4 Fab CDR-L1, CDR-L2 and CDR-L3 of SEQ ID NO: 12 of ANT39-knob as described in Table 6B; FZD4 Fab CDR-L1 comprises SVSSA (SEQ ID NO: 1) and CDR-L2 comprises SASSLYS (SEQ ID NO: 2); A tetravalent binding antibody molecule described in any one of claims 28, 29, 30, 32 and 33.

35. The tetravalent binding antibody molecule of any one of claims 1 to 34, wherein the tetravalent binding antibody molecule does not comprise a FZD binding domain comprising the CDRs of the FZD binding antibody 5044 in combination with a Wnt co-receptor binding domain comprising the CDRs of the LRP6 binding antibody 2542 and / or antibody 2539. The tetravalent binding antibody molecule of any one of claims 1 to 34.

36. The tetravalent binding antibody molecule is (a) a first heavy chain comprising the amino acid sequence of the hole heavy chain construct of SEQ ID NO: 898, a second heavy chain comprising the amino acid sequence of the knob heavy chain construct of SEQ ID NO: 897, and a light chain comprising the amino acid sequence of the light chain construct of SEQ ID NO: 899, wherein the amino acid sequences of the CDRs are the amino acid sequences of the CDRs of ANT16; or (b) a first heavy chain comprising the amino acid sequence of the hole heavy chain construct of SEQ ID NO: 901, a second heavy chain comprising the amino acid sequence of the knob heavy chain construct of SEQ ID NO: 900, and a light chain comprising the amino acid sequence of the light chain construct of SEQ ID NO: 902, wherein the amino acid sequences of the CDRs are the amino acid sequences of the CDRs of ANT18; or (c) a first heavy chain comprising the amino acid sequence of the hole heavy chain construct of SEQ ID NO: 904, a second heavy chain comprising the amino acid sequence of the knob heavy chain construct of SEQ ID NO: 903, and a light chain comprising the amino acid sequence of the light chain construct of SEQ ID NO: 902, wherein the amino acid sequences of the CDRs are the amino acid sequences of the CDRs of ANT20; or (d) a first heavy chain comprising the amino acid sequence of the hole heavy chain construct of SEQ ID NO: 906, a second heavy chain comprising the amino acid sequence of the knob heavy chain construct of SEQ ID NO: 905, and a light chain comprising the amino acid sequence of the light chain construct of SEQ ID NO: 902, wherein the amino acid sequences of the CDRs are the amino acid sequences of the CDRs of ANT21; or (e) a first heavy chain comprising the amino acid sequence of the hole heavy chain construct of SEQ ID NO: 908, a second heavy chain comprising the amino acid sequence of the knob heavy chain construct of SEQ ID NO: 907, and a light chain comprising the amino acid sequence of the light chain construct of SEQ ID NO: 909, wherein the amino acid sequences of the CDRs are the amino acid sequences of the CDRs of ANT39; or (f) a first heavy chain comprising the amino acid sequence of a hole heavy chain construct selected from the group consisting of SEQ ID NO:921; SEQ ID NO:922; SEQ ID NO:923; SEQ ID NO:924; SEQ ID NO:925; SEQ ID NO:926; SEQ ID NO:927; and SEQ ID NO:928; a second heavy chain comprising the amino acid sequence of a knob heavy chain construct selected from the group consisting of SEQ ID NO:929; SEQ ID NO:930; SEQ ID NO:931; SEQ ID NO:932; SEQ ID NO:933; SEQ ID NO:934; SEQ ID NO:935; and SEQ ID NO:936; and a light chain comprising the amino acid sequence of a light chain construct selected from the group consisting of SEQ ID NO:909 and SEQ ID NO:

952.

36. The tetravalent binding antibody molecule of any one of claims 1 to 35, comprising:

37. The tetravalent binding antibody molecule is (a) a first heavy chain comprising the amino acid sequence of a hole heavy chain construct of SEQ ID NO:921 or SEQ ID NO:925, a second heavy chain comprising the amino acid sequence of a knob heavy chain construct of SEQ ID NO:929 or SEQ ID NO:933, and a light chain comprising the amino acid sequence of a light chain construct of SEQ ID NO:909 or SEQ ID NO:952; or (b) a first heavy chain comprising the amino acid sequence of a hole heavy chain construct of SEQ ID NO:922 or SEQ ID NO:926, a second heavy chain comprising the amino acid sequence of a knob heavy chain construct of SEQ ID NO:930 or SEQ ID NO:934, and a light chain comprising the amino acid sequence of a light chain construct of SEQ ID NO:909 or SEQ ID NO:952; or (c) a first heavy chain comprising the amino acid sequence of a hole heavy chain construct of SEQ ID NO:923 or SEQ ID NO:927, a second heavy chain comprising the amino acid sequence of a knob heavy chain construct of SEQ ID NO:931 or SEQ ID NO:935, and a light chain comprising the amino acid sequence of a light chain construct of SEQ ID NO:909 or SEQ ID NO:952; or (d) a first heavy chain comprising the amino acid sequence of a hole heavy chain construct of SEQ ID NO:924 or SEQ ID NO:928, a second heavy chain comprising the amino acid sequence of a knob heavy chain construct of SEQ ID NO:932 or SEQ ID NO:936, and a light chain comprising the amino acid sequence of a light chain construct of SEQ ID NO:909 or SEQ ID NO:952; or (e) a first heavy chain comprising the amino acid sequence of a hole heavy chain construct of SEQ ID NO:908 or SEQ ID NO:940, a second heavy chain comprising the amino acid sequence of a knob heavy chain construct of SEQ ID NO:944 or SEQ ID NO:948, and a light chain comprising the amino acid sequence of a light chain construct of SEQ ID NO:909 or SEQ ID NO:952; or (f) a first heavy chain comprising the amino acid sequence of a hole heavy chain construct of SEQ ID NO:937 or SEQ ID NO:941, a second heavy chain comprising the amino acid sequence of a knob heavy chain construct of SEQ ID NO:945 or SEQ ID NO:949, and a light chain comprising the amino acid sequence of a light chain construct of SEQ ID NO:909 or SEQ ID NO:952; or (g) a first heavy chain comprising the amino acid sequence of a hole heavy chain construct of SEQ ID NO:938 or SEQ ID NO:942, a second heavy chain comprising the amino acid sequence of a knob heavy chain construct of SEQ ID NO:946 or SEQ ID NO:950, and a light chain comprising the amino acid sequence of a light chain construct of SEQ ID NO:909 or SEQ ID NO:952; or (h) a first heavy chain comprising the amino acid sequence of a hole heavy chain construct of SEQ ID NO:939 or SEQ ID NO:943, a second heavy chain comprising the amino acid sequence of a knob heavy chain construct of SEQ ID NO:947 or SEQ ID NO:951, and a light chain comprising the amino acid sequence of a light chain construct of SEQ ID NO:909 or SEQ ID NO:

952.

36. The tetravalent binding antibody molecule of any one of claims 1 to 35, comprising:

38. A pharmaceutical composition comprising the tetravalent binding antibody molecule of any one of claims 1 to 37 and a pharmaceutically acceptable carrier.

39. A method for promoting endothelial cell barrier function in a tissue, comprising administering to the tissue an effective amount of a tetravalent binding antibody molecule according to any one of claims 1 to 37.

40. 40. The method of claim 39, wherein the tissue is brain, kidney, or eye tissue.

41. The method of claim 40, wherein the tetravalent binding antibody molecule is administered to the ocular tissue by intravitreal injection.

42. A method for increasing retinal or brain endothelial cell barrier function, reducing endothelial cell permeability, and enhancing or restoring blood-retinal and blood-brain barrier maintenance in a subject in need thereof, comprising contacting endothelial cells comprising the FZD4 receptor and LRP5 with an effective amount of a tetravalent binding antibody molecule described in any one of claims 1 to 37 in a subject in need thereof.

43. 43. The method of claim 42, wherein the tetravalent binding antibody molecule is administered to a subject in need thereof by injection, topically, or orally.

44. 43. The method of claim 42, wherein the tetravalent binding antibody molecule is administered subcutaneously, intravenously, intraperitoneally, intrathecally, intravitreally, or intraocularly.

45. A tetravalent binding antibody molecule according to any one of claims 1 to 37 or a pharmaceutical composition according to claim 38 for use as a medicament.

46. A tetravalent binding antibody molecule or pharmaceutical composition as described in claim 45 for use in the treatment or prevention of disorders or conditions characterized by defective retinal or cerebral vascularization and / or characterized by impaired endothelial cell barrier function and / or vascular leakage.

47. 39. A method for treating or preventing a disorder or condition characterized by defective retinal or cerebral angiogenesis and / or reduced endothelial cell barrier function and / or vascular leakage, comprising administering to a person in need thereof a therapeutically effective amount of a tetravalent binding antibody molecule according to any one of claims 1 to 37 or a pharmaceutical composition according to claim 38.

48. Use of a tetravalent conjugated antibody molecule according to any one of claims 1 to 37 or a pharmaceutical composition according to claim 38 for the manufacture of a medicament for the treatment or prevention of a disorder or condition characterized by defective retinal or cerebral angiogenesis and / or reduced endothelial cell barrier function and / or vascular leakage.

49. 49. The tetravalent binding antibody molecule or pharmaceutical composition for use, method, or use according to any one of claims 46 to 48, wherein the disorder is selected from diabetic retinopathy, retinopathy of prematurity, Coats' disease, FEVR, Norrie's disease, macular degeneration, diabetic macular edema, pediatric vitreoretinopathy, Alzheimer's disease, epilepsy, multiple sclerosis, stroke, and ischemia.

50. 40. A tetravalent binding antibody molecule according to any one of claims 1 to 37 or a pharmaceutical composition according to claim 37 for use in the treatment or prevention of an ocular disorder, such as a retinal or macular disorder selected from diabetic retinopathy, retinopathy of prematurity, Coats' disease, FEVR, Norrie's disease, macular degeneration, diabetic macular edema and pediatric vitreoretinopathy, or in the treatment or prevention of a disorder selected from Alzheimer's disease, epilepsy, multiple sclerosis, stroke and ischemia.

51. 39. A method for treating or preventing an ocular disorder, such as a retinal or macular disorder selected from diabetic retinopathy, retinopathy of prematurity, Coats' disease, FEVR, Norrie's disease, macular degeneration, diabetic macular edema, and pediatric vitreoretinopathy, or a disorder selected from Alzheimer's disease, epilepsy, multiple sclerosis, stroke, and ischemia, comprising administering to a person in need thereof a therapeutically effective amount of a tetravalent binding antibody molecule according to any one of claims 1 to 37 or a pharmaceutical composition according to claim 38.

52. 39. Use of a tetravalent conjugated antibody molecule according to any one of claims 1 to 36 or a pharmaceutical composition according to claim 38 for the manufacture of a medicament for the treatment or prevention of an ocular disorder, such as a retinal or macular disorder selected from diabetic retinopathy, retinopathy of prematurity, Coats' disease, FEVR, Norrie's disease, macular degeneration, diabetic macular edema and pediatric vitreoretinopathy, or in the treatment or prevention of a disorder selected from Alzheimer's disease, epilepsy, multiple sclerosis, stroke and ischemia.

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