Molecules that modulate the Wnt signaling pathway and their uses

Abstract

The present invention provides Wnt pathway substitute molecules and related compositions that can be used in any of a variety of therapeutic methods for treating diseases. In some embodiments, the peptide linker in the Wnt substitute molecule is about 0 to about 100 amino acids, or about 1 to about 100 amino acids. In certain embodiments, the peptide linker is a linear peptide linker. In certain embodiments, the peptide linker comprises one or more glycine and / or serine residues.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims priority to U.S. Provisional Application No. 63 / 462,402, filed Apr. 27, 2023, and U.S. Provisional Application No. 63 / 469,498, filed May 29, 2023, each of which is hereby incorporated by reference in its entirety.

[0002] Statement Regarding the Sequence Listing The accompanying sequence listing xml for this application is in text format and is hereby incorporated by reference in this specification. The name of the xml file containing the sequence listing is SRZN_08_01WO_ST26.xml. This xml file is 75,503 bytes, was created on Apr. 23, 2024, and was submitted electronically via the U.S. Patent Center.

[0003] Background Technical Field The present invention generally relates to multivalent polypeptide molecules that bind to one or more of LRP5 and / or LRP6 and one or more FZD receptors, and related compositions and methods of using them. Such molecules are useful, for example, for modulating the Wnt signaling pathway.

Background Art

[0004] Description of Related Art WNT ( "Wingless - related integration site" or "Wingless and Int - 1" or "Wingless - Int") ligands and their signals play important roles in the development, homeostasis, and regeneration of many essential organs and tissues, including bone, liver, skin, stomach, intestine, kidney, central nervous system, mammary gland, taste buds, ovary, cochlea, and many other tissues (e.g., there is a review by Clevers, Loh, and Nusse, 2014; 346:1248012). Modulation of the Wnt signaling pathway may have potential for treating degenerative diseases and tissue injuries. One of the challenges in modulating Wnt signaling therapeutically is the existence of multiple WNT ligands and WNT receptors, Frizzled 1-10 (FZD1-10), and the fact that many tissues express multiple overlapping FZDs. In addition to FZDs, standard Wnt signaling also involves low-density lipoprotein (LDL) receptor-associated protein 5 (LRP5) or low-density lipoprotein (LDL) receptor-associated protein 6 (LRP6) as co-receptors, which are widely expressed in various tissues. Therefore, there is a clear need in the art for binding sites that specifically bind to one or more FZDs, LRP5, or LRP6 to modulate the Wnt signaling pathway. Furthermore, it is necessary to reduce or eliminate the effector function of these molecules, and in certain cases, to increase or decrease systemic removal of the molecules. The present invention addresses this need. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Clevers, Loh, and Nusse, 2014; 346:1248012 [Overview of the project] [Problems that the invention aims to solve]

[0006] overview Previous studies have determined that multivalent binding to FZD and LRP is important for efficient Wnt signaling induction (e.g., PCT Publication WO2019 / 126398; PCT Publication WO2020 / 010308; Chen et al, Cell Chemical Biology, 27:598, 2020; and Tao et al, eLife, 8:e46134, 2019). These studies explored Fc dimerization-based molecules for multivalent design. These designs resulted in molecules with large molecular weights and often required Fc manipulation for effector-less functionality, FcRn mutations to increase removal, and / or Fc pairing techniques for heterodimerization.

[0007] To address the challenges associated with Fc-based molecules, this disclosure provides polyvalent forms that do not contain Fc. This allows for more rapid removal in the absence of FcRn binding and the absence of effector function. In addition, some of these forms enable single-chain design and compatibility with a wider range of expression systems.

[0008] In various embodiments, the present invention provides polypeptides that modulate the Wnt signaling pathway and their use. [Means for solving the problem]

[0009] In one embodiment, the present disclosure is a Wnt substitute molecule, and the Wnt substitute molecule is a) N' AB1 n -X-AB2 m -Y-AB3 p -Z-AB4 q C' polypeptides having the structure; or b) Each N' AB1 n -X-AB2 m -Y-AB3 p -Z-AB4 q C' Dimers of first and second polypeptide monomers having the structure (In the formula, AB1 is an antigen-binding antibody fragment selected from the group consisting of Fab, a single-chain Fv fragment (scFv), a variable heavy chain (VH) and a variable light chain (VL) linked via a linker in any order, and a VH or VL or single-domain antibody (VHH), which binds to one or more FZD receptors or to LRP5 and / or LRP6; AB2 is an antigen-binding antibody fragment selected from the group consisting of a variable heavy chain (VH) and a variable light chain (VL) linked via a linker in either the order Fab, scFv, and a VH or VL or a single-domain antibody (VHH), which binds to one or more FZD receptors or to LRP5 and / or LRP6; AB3 is an antigen-binding antibody fragment selected from the group consisting of a variable heavy chain (VH) and a variable light chain (VL) linked via a linker in either the order Fab, scFv, and a VH or VL or a single-domain antibody (VHH), which binds to one or more FZD receptors or to LRP5 and / or LRP6; AB4 is an antigen-binding antibody fragment selected from the group consisting of a variable heavy chain (VH) and a variable light chain (VL) linked via a linker in either the order Fab, scFv, and a VH or VL or a single-domain antibody (VHH), which binds to one or more FZD receptors or to LRP5 and / or LRP6; X, Y, and Z are either absent or peptide linkers, and if there are no adjacent fragments at both the N-terminus and C-terminus of each of X, Y, or Z, then each of X, Y, and Z is absent; n is 0, 1, or 2; m is 0, 1, or 2; p is 0, 1, or 2; (q is 0, 1, or 2) Includes, Each polypeptide and polypeptide monomer comprises at least two fragments selected from the group consisting of AB1, AB2, AB3, and AB4. The molecule contains one or more antigen-binding antibody fragments that bind to one or more FZD receptors, and one or more antigen-binding fragments that bind to LRP5 and / or LRP6. This provides a WNT substitute molecule that includes [unclear].

[0010] In some embodiments, the peptide linker in the Wnt substitute molecule is approximately 0 to 100 amino acids, or approximately 1 to 100 amino acids. In certain embodiments, the peptide linker is a linear peptide linker. In certain embodiments, the peptide linker comprises one or more glycine and / or serine residues. In certain embodiments, the peptide linker between different AB domains of scFv or between the VH chain and the VL chain is a) Glycine x 4-serine: (G4S) (SEQ ID NO: 59); b) (Glycine x 4-serine) x 3: (G4S)3 (SEQ ID NO: 60); c) ASTKG (SEQ ID NO: 61); d) DKTHT (SEQ ID NO: 62); e) G4S (SEQ ID NO: 59) and ASTKG (SEQ ID NO: 61); and f) G4S (SEQ ID NO: 59) and DKTHT (SEQ ID NO: 60) are selected peptides from this group.

[0011] In some embodiments, the molecule is a dimer, formed by one or more interactions between VH and VL present in the first monomer and VH and VL present in the second monomer. In certain embodiments, the dimer is a homodimer, and in other embodiments, the dimer is a heterodimer. In certain embodiments, the Wnt substitute molecule includes a diabody, which includes intramolecular pairings of VH and VL present in the polypeptide, or intermolecular or intramolecular pairings of VH and VL present in the first monomer and VH and VL present in the second monomer, wherein the VH of the first monomer binds to the VL of the second monomer to form a first antigen-binding domain, and the VL of the first monomer binds to the VH of the second monomer to form a second antigen-binding domain.

[0012] In some embodiments, the molecule is a dimer, and for each polypeptide monomer, AB1 is VHH; AB2 is VH and VL linked via a linker in any order; n=1; m=1; p=0; q=0; X is present; Y and Z are absent, and the VH and VL of the first monomer bond to the VL and VH of the second monomer to form a diabody. In other embodiments, the molecule is a dimer, and for each polypeptide monomer, AB1 is VH and VL linked via a linker in any order; AB2 is VHH, n=1; m=1; p=0; q=0; X is present; Y and Z are absent, and the VH and VL of each monomer bond to the VL and VH of the other monomer to form a diabody. In certain embodiments, VH of the first monomer binds to VL of the second monomer to form a first binding domain, VH of the second monomer binds to VL of the first monomer to form a second binding domain, the dimer is a homodimer, and the molecule is a polyvalent, multispecific binding molecule.

[0013] In some embodiments, the molecule is a heterodimer, where the first monomer VH binds to the second monomer VL to form a first antigen-binding domain, and the first monomer VL binds to the second monomer VH to form a second antigen-binding domain, the first antigen-binding domain binds to a first set of one or more antigens, and the second antigen-binding domain binds to a second set of one or more antigens, the first and second sets being the same or different antigens, or different sets of antigens, which may overlap as needed. In some embodiments, VHH binds to LRP5 and / or LRP6, and VH and VL form a diabody that binds to one or more FZD receptors.

[0014] In some embodiments, the molecule is a single polypeptide, where AB1 is VHH; AB2 is VH and VL linked via a linker in any order; AB3 is VH and VL linked via a linker in any order; n=1; m=1; p=1; q=0; X and Y are present, Z is absent, and the VH and VL of AB2 are bonded to the VL and VL of AB3 to form a diamond body. In certain embodiments, the VH and VL of AB2 are identical to the VH and VL of AB3. In other embodiments, the VH and VL of AB2 are different from the VH and VL of AB3. In certain embodiments, the VH of AB2 binds to the VL of AB3 to form a first antigen-binding domain, and the VL of AB2 binds to the VH of AB3 to form a second antigen-binding domain, the first antigen-binding domain binds to a first set of one or more antigens, and the second antigen-binding domain binds to a second set of one or more antigens, the first and second sets being the same or different antigens, or different sets of antigens, which may overlap as needed. In some embodiments, in this molecule, for each of AB2 and AB3, the VH is on the amino-terminal side relative to the VL. In other embodiments, in this molecule, for each of AB2 and AB3, the VL is on the amino-terminal side relative to the VH. In some embodiments, the VHH of the molecule binds to LRP5 and / or LRP6, and the diabody binds to one or more FZD receptors.

[0015] In some embodiments of the Wnt substitute molecule, AB1 is VHH; AB2 is scFv; AB3 is scFv; n=1; m=1; p=1; q=0; X and Y are present, and Z is absent. In some embodiments, the first and second scFv are identical. In other embodiments, the first and second scFv are not identical, and the Wnt substitute molecule is a trivalent, triple-specific polypeptide. In certain embodiments, AB1 binds to LRP5 and / or LRP6, and AB2 and AB3 each independently bind to one or more FZD receptors.

[0016] In some embodiments of the Wnt substitute molecule, AB1 is VHH; AB2 is VH and VL connected via a linker in either order; AB3 consists of VH and VL connected via a linker in any order; AB4 consists of VHH; n=1; m=1; p=1; q=1; X, Y, and Z are present; and VH and VL of AB2 bind to VL and VH of AB3 to form a diabody. In certain embodiments, VH and VL of AB2 are identical to VH and VL of AB3. In other embodiments, VH and VL of AB2 are different from VH and VL of AB3. In certain embodiments, VH of AB2 binds to VL of AB3 to form a first antigen-binding domain, and VL of AB2 binds to VH of AB3 to form a second antigen-binding domain, the first antigen-binding domain binds to a first set of one or more antigens, and the second antigen-binding domain binds to a second set of one or more antigens, the first and second sets may be the same or different antigens, or different, possibly overlapping sets of antigens. In some embodiments, for each of AB2 and AB3, VH is located on the amino-terminal side relative to VL. In certain embodiments, for each of AB2 and AB3, VL is located on the amino-terminal side relative to VH. In some embodiments, VHH of AB1 is identical to VHH of AB4. In some embodiments, VHH of AB1 is different from VHH of AB4, and the Wnt substitute molecule is a tetravalent tetraspecific binding molecule. In some embodiments, AB1 and AB4 bind to LRP5 and / or LRP6, and the diabody binds to one or more FZD receptors.

[0017] In some embodiments of the Wnt substitute molecule, AB1 is VHH; AB2 is scFv; AB3 is scFv; AB4 is VHH; n=1; m=1; p=1; q=1; X, Y, and Z exist. In certain embodiments, the scFv of AB2 is identical to the scFv of AB3, and the Wnt substitute molecule is a tetravalent bispecific binding molecule. In some embodiments, the scFv of AB2 is not identical to the scFv of AB3, the VHH of AB1 is identical to the VHH of AB4, and the Wnt substitute molecule is a tetravalent triple specific binding molecule. In some embodiments, the VHH of AB1 is not identical to the VHH of AB4, the scFv of AB2 is identical to the scFv of AB3, and the Wnt substitute molecule is a tetravalent triple specific binding molecule. In some embodiments, AB1 and AB4 independently bind to LRP5 and / or LRP6, and AB2 and AB3 independently bind to one or more FZD receptors.

[0018] In some embodiments of the Wnt substitute molecule, AB1 is VHH; AB2 is scFv; AB3 is VHH; AB4 is scFv; n=1; m=1; p=1; q=1; X, Y, and Z are present. In certain embodiments, AB2 and AB4 are identical, and the molecule is a tetravalent bispecific binding molecule. In some embodiments, AB1 and AB3 are not identical, and the molecule is a tetravalent tripspecific binding molecule. In certain embodiments, AB2 and AB4 are not identical, AB1 and AB3 are identical, and the molecule is a tetravalent tripspecific binding molecule. In some embodiments, AB2 and AB4 are not identical, AB1 and AB3 are not identical, and the molecule is a tetravalent quadruspecific binding molecule. In some embodiments, AB1 and AB3 independently bind to LRP5 and / or LRP6, and AB2 and AB4 independently bind to one or more FZD receptors.

[0019] In some embodiments of the Wnt substitute molecule, AB1 is scFv; AB2 is VHH; AB3 is scFv; n=1; m=1; p=1; q=0; X and Y are present, and Z is absent. In certain embodiments, AB1 and AB3 are identical, and the molecule is a trivalent, bispecific molecule. In other embodiments, AB1 and AB3 are not identical, and the molecule is a trivalent, triplicate molecule. In some embodiments, AB1 and AB3 each independently bind to one or more FZD receptors, and AB2 binds to LRP5 and / or LRP6.

[0020] In certain embodiments of the Wnt substitute molecule, AB1 is VHH; AB2 is VHH; AB3 is VHH; n=1; m=1; p=1; q=0; X and Y are present, and Z is absent. In some embodiments, AB1 binds to LRP5 and / or LRP6; AB2 binds to one or more FZD receptors; and AB3 binds to one or more FZD receptors, and in some cases where AB2 and AB3 are identical, the molecule is a trivalent bispecific binding molecule. In some embodiments, AB2 and AB3 are not identical, and the molecule is a trivalent triplespecific binding molecule. In other embodiments, AB2 and AB3 each independently bind to the same or different FZD receptors, or to different, optionally overlapping sets of FZD receptors.

[0021] In some embodiments of the Wnt substitute molecule, AB1 is VHH; AB2 is VHH; AB3 is VHH; AB4 is VHH; n=1; m=1; p=1; q=1; X, Y, and Z are present. In certain embodiments, the molecule is a tetravalent bispecific binding molecule having two VHHs that bind to the same FZD receptor and two VHHs that each bind to LRP5 and / or LRP6. In some embodiments, the molecule is a tetravalent tripspecific binding molecule having two VHHs that bind to different FZD receptors, or to different, optionally overlapping sets of FZD receptors, or to different epitopes on the same FZD receptor, and two VHHs that bind to the same LRP5 and / or LRP6. In further embodiments, the molecule is a tetravalent, triple-specific binding molecule having two identical VHHs that bind to one or more FZD receptors and two different VHHs that bind to different LRP5 and / or LRP6, or to different epitopes of the same LRP5 and / or LRP6. In particular embodiments, the molecule is a tetravalent, quadruple-specific binding molecule having two VHHs that each bind to a different FZD receptor or a different but potentially overlapping combination of FZD receptors and two VHHs that each bind to a different LRP5 and / or LRP6.

[0022] In a further embodiment, one of the Wnt substitute molecules modulates the Wnt signaling pathway in cells, optionally mammalian cells, e.g., human cells. In a particular embodiment, the Wnt substitute molecule increases signaling by the Wnt signaling pathway in cells. In a particular embodiment, the Wnt signaling pathway is either a standard Wnt signaling pathway or a non-standard Wnt signaling pathway.

[0023] In some embodiments, the Wnt substitute molecule is a polypeptide shown in any one of SEQ ID NOs: 1 to 56 or a polypeptide having at least 90% or 95% identity with an internal domain of any one of SEQ ID NOs: 1 to 56.

[0024] In one relevant embodiment, the Disclosure provides an isolated polynucleotide encoding a polypeptide sequence comprising one or more FZD-binding domains and / or one or more LRP5 / 6-binding domains of a Wnt substitute polypeptide. In some embodiments, the polynucleotide encodes a Wnt substitute molecule, or a polypeptide monomer of a Wnt substitute molecule. In certain embodiments, the Disclosure provides an expression vector comprising the isolated polynucleotide. In further embodiments, the Disclosure provides an isolated host cell, which is a prokaryotic or eukaryotic cell, comprising the expression vector.

[0025] In one relevant embodiment, the Disclosure provides a pharmaceutical composition comprising a physiologically acceptable additive, diluent or carrier, as disclosed herein, and a Wnt substitute polypeptide, a polynucleotide encoding the Wnt substitute polypeptide as a peptide monomer, an expression vector comprising the polynucleotide, or a host cell comprising the expression vector. In a particular embodiment, the pharmaceutical composition comprises a physiologically acceptable additive, diluent or carrier, and a therapeutically effective amount of a Wnt substitute polypeptide, a polynucleotide, a host cell, or an expression vector.

[0026] In related embodiments, the present disclosure provides a method for stimulating the Wnt signaling pathway in cells, comprising contacting the cells with a Wnt substitute polypeptide, a polynucleotide encoding a Wnt substitute polypeptide, or an expression vector.

[0027] In certain embodiments, the disclosure provides a method for treating a subject having a disease or disorder related to reduced Wnt signaling, comprising administering an effective amount of a pharmaceutical composition to the subject, wherein the Wnt substitute polypeptide is an agonist of the Wnt signaling pathway.In certain embodiments, the disease or disorder is fracture, stress fracture, vertebral compression fracture, osteoporosis, osteoporotic fracture, non-union fracture, delayed union fracture, spinal fusion, preoperative optimization for spinal surgery, osteonecrosis, osseointegration of implants or orthopedic devices, osteogenesis imperfecta, bone grafting, tendon repair, tendon-osseointegration, tooth growth and regeneration, salivary gland disorders, maxillofacial surgery, dental implants, periodontal disease, maxillofacial reconstruction, osteonecrosis of the jaw, hip or femoral head, avascular necrosis, alopecia, hearing loss, vestibular dysfunction, macular degeneration, age-related macular degeneration (AMD), vitreoretinopathy, retinopathy, diabetic retinopathy, retinal degenerative diseases, Fuchs dystrophy, corneal diseases, dry Lacrimal gland disorders including Eyes syndrome and Sjögren's syndrome, stroke, traumatic brain injury, Alzheimer's disease, multiple sclerosis, muscular dystrophy, muscle atrophy due to sarcopenia or cachexia, diseases affecting the blood-brain barrier (BBB), spinal cord injury, spinal cord diseases, oral mucositis, short bowel syndrome, inflammatory bowel disease (IBD) including but not limited to Crohn's disease and ulcerative colitis, metabolic syndrome, diabetes mellitus, dyslipidemia, pancreatitis, exocrine pancreatic insufficiency, impaired wound healing, diabetic impaired wound healing, diabetic foot ulcers, pressure ulcers, Venous ulcers, epidermolysis bullosa, cutaneous dysplasia, myocardial infarction, coronary artery disease, heart failure, hematopoietic cell damage, immunodeficiency, graft-versus-host disease, acute kidney injury, chronic kidney disease, chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis, acute liver failure of any cause, drug-induced acute liver failure, alcoholic liver disease, chronic liver failure of any cause, cirrhosis, hepatic fibrosis of any cause, portal hypertension, chronic liver dysfunction of any cause, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD) (fat The group consists of the following: liver, alcoholic hepatitis, hepatitis C virus-induced liver disease (HCV), hepatitis B virus-induced liver disease (HBV), other viral hepatitis (e.g., hepatitis A virus-induced liver disease (HAV) and hepatitis D virus-induced liver disease (HDV)), primary biliary cirrhosis, autoimmune hepatitis, liver surgery, liver injury, liver transplantation, "slightly under-grafted" syndrome in liver surgery and transplantation, congenital liver disease and impairment, genetic disorders, degeneration, aging, drugs and any other liver impairment or defect resulting from injury. [Brief explanation of the drawing]

[0028] [Figure 1] Figures 1A–1D show various forms of homodimerized polypeptide monomers containing one VHH LRP-binding domain as well as VH and VL FZD-binding domains, separated by a short 5-amino acid G4S linker (SEQ ID NO: 59), for preventing single-chain Fv(scFv) formation and generating a polyvalent, multispecific binding molecule, the FZD-binding diabody.

[0029] [Figure 2] Figure 2 shows a single-chain polypeptide comprising one VHH that binds to LRP5 and / or LRP6, and two VH and VL FZD binding fragments, each separated by a longer 15-amino acid 3×G4S linker (SEQ ID NO: 60), which is separated by a short 5-amino acid G4S linker (SEQ ID NO: 59) to prevent scFv formation, and each of which binds to one or more FZD receptors upon intramolecular diabody formation, where the intramolecular interaction between VH1 / VL2 and VH2 / VL1 results in a bispecific polypeptide. G4S (SEQ ID NO: 59) represents a single G4S linker (SEQ ID NO: 59), and 3×G4S (SEQ ID NO: 60) represents a (G4S)3 linker (SEQ ID NO: 60).

[0030] [Figure 3] Figure 3 shows a single-chain polypeptide comprising one VHH that binds to LRP5 and / or LRP6, and two scFvs, each of which binds to one or more FZD receptors, with each of the two scFvs, VH1 / VL1 and VH2 / VL2, being specific to one or more FZD receptors. To facilitate scFv formation, the linker between the VH / VL pairs is (G4S)3 (SEQ ID NO: 60), and the linker between the scFvs is G4S (SEQ ID NO: 60).

[0031] [Figure 4]Figure 4 shows two isomers of the single-chain polypeptide of Figure 3, having a longer linker (G4S)3 (SEQ ID NO: 60) between VL1 and VH2. This polypeptide can form isoforms having two consecutive scFv, as shown in isomer forms a and b, respectively, or form diabodies through intramolecular interactions.

[0032] [Figure 5] Figure 5 shows a single-chain polypeptide comprising two identical or different VHHs that bind to LRP5 and / or LRP6, and two VH and VL FZD binding fragments separated by a shorter G4S linker (SEQ ID NO: 59) to prevent scFv formation, and each separated by a longer linker (G4S)3 (SEQ ID NO: 60) that binds to one or more FZD receptors upon intramolecular diabody formation, where the intramolecular interactions between VH1 / VL2 and VH2 / VL1 of these two result in a multispecific polypeptide.

[0033] [Figure 6] Figure 6 shows two isomers (isomer a and isomer b) of the polypeptide of Figure 5, having a (G4S)3 linker (SEQ ID NO: 60) between VH1 and VL1 and between VH2 and VL2. This polypeptide can form isoforms having two consecutive scFv, as shown in isomer forms a and b, respectively, or form diabodies through intramolecular interactions.

[0034] [Figure 7]Figure 7A shows a tetravalent polypeptide in which a first VHH that binds to LRP5 and / or LRP6 is linked to a first scFv that binds to one or more FZD receptors, and this is linked to a second identical or different VHH that binds to LRP and / or LRP6, which is linked to a second identical or different scFv that binds to one or more FZD receptors. Figure 7B shows a trivalent polypeptide in which the VHH that binds to LRP5 and / or LRP6 is located between two identical or different scFvs, each bound to one or more FZD receptors.

[0035] [Figure 8] Figure 8 shows a monomer comprising one VHH that binds to LRP5 and / or LRP6, and two identical or different VHHs, each binding to one or more FZD receptors. The VHH that binds to LRP5 and / or LRP6, as well as the two VHHs, each binding to one or more FZD receptors, may each be AB1, AB2, or AB3.

[0036] [Figure 9] Figure 9 shows a monomer comprising two identical or different VHHs that bind to LRP5 and / or LRP6, and two identical or different VHHs that each bind to one or more FZD receptors. The two VHHs that bind to LRP5 and / or LRP6, and the two VHHs that each bind to one or more FZD receptors, may each be AB1, AB2, AB3, or AB4.

[0037] [Figure 10]Figures 10A–10D show exemplary DNA constructs encoding the various polypeptides shown in Figure 1A (Figure 10A), Figure 2 (Figure 10B), Figure 3 (Figure 10C), and Figure 4 (Figure 10D). All other polypeptides shown in Figures 1–9 are generated using DNA constructs having similar combinations in which a single kappa-SP (signal sequence peptide) is combined with VHH, VH1 / VL1, VH2 / VL2, and the linker regions between them.

[0038] [Figure 11] Figures 11A–11F show various forms of Fab, each having two added identical or different VHHs that bind to LRP5 and / or LRP6, each binding to one or more FZD receptors.

[0039] [Figure 12] Figures 12A–12D show various forms of FV-Fab having identical or different VH / VL pairings, binding to one or more FZD receptors, and having one additional VHH that binds to LRP5 and / or LRP6.

[0040] [Figure 13] Figures 13A–13D show various forms of Fab-Fv, each binding to one or more FZD receptors, possessing identical or different VH / VL pairings, and having one additional VHH that binds to LRP5 and / or LRP6.

[0041] [Figure 14] Figures 14A-14H show various forms containing two identical or different VHHs, each having a VHH that binds to one or more FZD receptors, and in which a VHH that binds to LRP5 and / or LRP6 is attached to the constant region of one of the VHHs that binds to one or more FZD receptors.

[0042] [Figure 15]Figures 15A–15F show various forms, including two identical or different VHHs that bind to LRP 5 and / or LRP 6, and two identical or different VHHs that are added from the constant region in a cis or trans configuration, each binding to one or more FZD receptors.

[0043] [Figure 16] Figures 16A–16D show various forms of the molecule, each containing scFv and Fab that bind to one or more FZD receptors, and having VHH attached to the constant region that binds to LRP5 and / or LRP6.

[0044] [Figure 17] Figures 17A–17D show various forms of the molecule, each containing a Fab that binds to LRP5 and / or LRP6, and each having two identical or different attached scFvs that bind to one or more FZD receptors.

[0045] [Figure 18] Figures 18A–18F show various forms of the molecule, each containing two constant regions and having two ScFvs attached to each constant region, with each scFv being attached in the cis form from one of the constant regions to bind to LRP 5 and / or LRP 6, or to one or more FZD receptors. The scFvs that bind to FZD may be the same or different, and the scFvs that bind to LRP may be the same or different.

[0046] [Figure 19] Figures 19A–19D show various forms of the molecule, each containing two identical or different VHHs, each bound to LRP5 and / or LRP6, and two identical or different scFvs, which are added in trans form from the constant region, each bound to one or more FZD receptors.

[0047] [Figure 20]Figures 20A–20B show various molecular forms, each containing two identical or different VHHs, each bound to LRP5 and / or LRP6, and two identical or different scFvs, each attached from the constant region in the cis form and bound to one or more FZD receptors.

[0048] [Figure 21] Figures 21A–21L show various forms of the molecule, each comprising one VHH that binds to LRP5 and / or LRP6, and two identical or different scFvs, each binding to one or more FZD receptors, with the VHH and scFvs added from the constant region.

[0049] [Figure 22] Figures 22A–22F show various molecular forms in which one VHH binds to LRP5 and / or LRP6, and two identical or different scFv each bind to one or more FZD receptors, with each scFv attached to a constant region and each VHH attached to an scFv.

[0050] [Figure 23] Figure 23A shows the SEC profiles of the three shown molecules, which are diabodies illustrated in Figure 1A. All are of the same form but originate from different parental anti-Fzd antibodies. Figure 23B shows two of the diabodies from 23A having various VH-VL orientations (top, illustrated in Figure 1B) or C-terminal VHH (bottom, illustrated in Figure 1C). Figure 23C shows two examples of single-chain VHH-tandem scFv polypeptides (illustrated in Figure 3).

[0051] [Figure 24-1] Figures 24A, 24B, and 24C show the purified polypeptides analyzed by non-reducing (NR) and reducing (R) SDS-PAGE gels. Figure 24A shows the fractions shown in SEC from the upper panel of Figures 23A and 23C (representative of Figures 1 and 2); Figure 24C shows the gels for polypeptides representative of Figures 1A-1D, 2, 4, and 5. [Figure 24-2] Same as above.

[0052] [Figure 25] Figure 25 shows the Super Top Flash Wnt activity reporter assay (STF activity) of the indicated molecules in the Huh7 cell line, compared to their parental IgG-based molecules. At log[protein]nM=0, the lines from top to bottom correspond to: Plate 1 R2M3-26(SZP10048+10277); Plate 3 R2M3-26(SZP10048+10277); Plate 2 R2M3-26(SZP10048+10277); VHH26-tandem-R2M13-scFv(SZP31560) with a 15-mer linker; VHH26-tandem-R2M13-scFv(SZP31559) with a 5-mer linker; R2M13-26(SZP12246+06075); VHH26-R2M13-diabody(SZP31557); and VHH26-tandem-R2M13-diabody(SZP31558).

[0053] [Figure 26] Figure 26 shows the STF activity of the indicated molecules in the Huh7 cell line, compared to their parental IgG-based molecules.

[0054] [Figure 27] Figure 27 shows the Tm / Tagg graph for the molecule shown; representative melting and aggregation curves for the polypeptide VHH-diabody form and VHH-tandem scFv form are shown. Black circles represent melting curves; black triangles represent aggregation curves.

[0055] [Figure 28] Figure 28 shows the in vitro efficacy response in organoids using control tetravalent bispecific VHH-IgG. The EC50s of the selected Wnt substitute polypeptides described herein are shown in Table 4. [Modes for carrying out the invention]

[0056] Detailed Description The present disclosure relates to Wnt surrogate molecules that bind to one or more FZD receptors and one or more LRP5 receptors or LRP6 receptors and modulate the downstream Wnt signaling pathway. In certain embodiments, the Wnt surrogate molecule activates the Wnt signaling pathway or increases signaling through the Wnt signaling pathway. In certain embodiments, the Wnt surrogate molecule (also referred to as a “Wnt mimetic” or “Wnt surrogate polypeptide”) is a) N’ AB1 n -X-AB2 m -Y-AB3 p -Z-AB4 q C’ a polypeptide having the structure of; or b) each of which is N’ AB1 n -X-AB2 m -Y-AB3 p -Z-AB4 q C’ a dimer of the first and second polypeptide monomers having the structure of (wherein AB1 is an antigen-binding antibody fragment that binds to one or more FZD receptors or binds to LRP5 and / or LRP6, selected from the group consisting of Fab, single-chain Fv fragment (scFv), variable heavy chain (VH) and variable light chain (VL) connected via a linker in either order, and VH or VL or single domain antibody (VHH); AB2 is an antigen-binding antibody fragment that binds to one or more FZD receptors or binds to LRP5 and / or LRP6, selected from the group consisting of Fab, scFv, variable heavy chain (VH) and variable light chain (VL) connected via a linker in either order, and VH or VL or single domain antibody (VHH); AB3 is an antigen-binding antibody fragment selected from the group consisting of a variable heavy chain (VH) and a variable light chain (VL) linked via a linker in either the order Fab, scFv, and a VH or VL or a single-domain antibody (VHH), which binds to one or more FZD receptors or to LRP5 and / or LRP6; AB4 is an antigen-binding antibody fragment selected from the group consisting of a variable heavy chain (VH) and a variable light chain (VL) linked via a linker in either the order Fab, scFv, and a VH or VL or a single-domain antibody (VHH), which binds to one or more FZD receptors or to LRP5 and / or LRP6; X, Y, and Z are either absent or peptide linkers, and if there are no fragments adjacent to both the N-terminus and C-terminus of each of X, Y, or Z, then each of X, Y, and Z is absent; n is 0, 1, or 2; m is 0, 1, or 2; p is 0, 1, or 2; (q is 0, 1, or 2) Includes, Each polypeptide comprises at least two fragments selected from the group consisting of AB1, AB2, AB3, and AB4, and The molecule comprises one or more antigen-binding antibody fragments that bind to one or more FZD receptors, and one or more antigen-binding fragments that bind to LRP5 and / or LRP6. In certain embodiments, each polypeptide of the Wnt substitute molecule comprises at least three fragments selected from the group consisting of AB1, AB2, AB3, and AB4. In certain embodiments, each polypeptide of the Wnt substitute molecule comprises at least four fragments selected from the group consisting of AB1, AB2, AB3, and AB4.

[0057] Certain embodiments include specific structural forms or arrangements of FZD-binding fragments or regions and LRP5 / 6-binding fragments or regions of Wnt surrogate molecules that are advantageous in enhancing downstream Wnt pathway signaling and associated biological effects.

[0058] Embodiments of the present invention relate to the use of Wnt substitute molecules for the diagnosis, evaluation, and treatment of diseases and disorders related to the Wnt signaling pathway. In certain embodiments, the Wnt substitute molecules are used to modulate the Wnt signaling pathway in cells or tissues. In certain embodiments, the Wnt substitute molecules are used in the treatment or prevention of diseases and disorders related to abnormal or deregulated (e.g., reduced) Wnt signaling, that is, by modulating, for example, increasing, Wnt signaling, therapeutic benefits may be obtained.

[0059] Unless otherwise expressly stated, the implementation of this invention will utilize conventional methods of virology, immunology, microbiology, molecular biology, and recombinant DNA technology, many of which will be described below for illustrative purposes. Such techniques are well described in the literature. For example, *Current Protocols in Molecular Biology* or *Current Protocols in Immunology*, John Wiley & Sons, New York, NY (2009); Ausubel et al., *Short Protocols in Molecular Biology*, 3 rdSee also: ed., Wiley & Sons, 1995; Sambrook and Russell, Molecular Cloning: A Laboratory Manual (3rd Edition, 2001); Maniatis et al. Molecular Cloning: A Laboratory Manual (1982); DNA Cloning: A Practical Approach, vol. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed., 1984); Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., 1985); Transcription and Translation (B. Hames & S. Higgins, eds., 1984); Animal Cell Culture (R. Freshney, ed., 1986); Perbal, A Practical Guide to Molecular Cloning (1984) and other similar references.

[0060] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly indicates otherwise.

[0061] Throughout this specification, unless the context requires otherwise, the word “comprise,” or variations such as “comprises” or “comprising,” should be understood to mean that it includes the elements or integers, or groups of elements or integers, that are described, but not exclude any other elements or integers, or groups of elements or integers.

[0062] As used herein and in the appended claims, the terms “3×GS4” or “(GS4)3” are identical unless the context clearly indicates otherwise.

[0063] Each embodiment described herein shall apply mutatis mutandis to all other embodiments unless expressly indicated otherwise.

[0064] Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be carried out according to the manufacturer's specifications, as commonly performed in the art, or as described herein. These and related techniques and procedures may generally be 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. Unless otherwise specified, the nomenclature used in relation to molecular biology, analytical chemistry, synthetic organic chemistry, and medicinal chemistry and pharmaceutical chemistry described herein, as well as their experimental procedures and techniques, are well known and commonly used in the art. Standard techniques may be used for recombinant techniques, molecular biology, microbiology, chemical synthesis, chemical analysis, pharmaceutical preparation, formulation and delivery, and treatment of the subject.

[0065] Embodiments of the present invention relate to polypeptides that bind to one or more FZD receptors and their antigen-binding fragments. Exemplary polypeptide sequences, or their antigen-binding fragments or complementarity-determining regions (CDRs), may be used in or present in the Wnt substitute molecules disclosed herein, as well as those described in any of PCT applications published WO 2019 / 126399, WO 2019 / 126398, and WO 2022 / 192445.

[0066] Embodiments of the present invention relate to polypeptides that bind to LRP5 and / or LRP6 and their antigen-binding fragments. Exemplary antibodies or sequences of their antigen-binding fragments or complementarity-determining regions (CDRs) may be used in or present in the Wnt substitute molecules disclosed herein, as well as in any of the Wnt substitute molecules described in PCT publications WO 2019 / 126401, WO 2019 / 126398, and WO 2022 / 192445.

[0067] As is well known in the art, therapeutic antibodies include fragments such as dAb, Fab, Fab', F(ab')2, Fv, single-chain (scFv), VHH, or single-domain antibodies (Nanobodies®), their synthetic variants, naturally occurring variants, and fusion proteins having a structure that includes an antigen-binding site or fragment (epitope recognition site) with the required specificity. "Diabodies" (WO94 / 13804; P. Holliger et al., Proc. Natl. Acad. Sci. USA 90 6444-6448, 1993), which are multivalent or multispecific fragments constructed by gene fusion, are also a specific form of antibody as envisioned herein. Minibodies containing scFv conjugated to a CH3 domain are also included herein (S. Hu et al., Cancer Res., 56, 3055-3061, 1996). See, for example, Ward, ES et al., Nature 341, 544-546 (1989); Bird et al., Science, 242, 423-426, 1988; Huston et al., PNAS USA, 85, 5879-5883, 1988); PCT / US92 / 09965; WO94 / 13804; P. Holliger et al., Proc. Natl. Acad. Sci. USA 90 6444-6448, 1993; Y. Reiter et al., Nature Biotech, 14, 1239-1245, 1996; S. Hu et al., Cancer Res., 56, 3055-3061, 1996.

[0068] The term “antigen-binding fragment” as used herein refers to a polypeptide fragment containing at least one CDR of immunoglobulin heavy and / or light chains that binds to the antigen of interest, particularly to one or more FZD receptors, or to LRP5 and / or LRP6 receptors. In this regard, the antigen-binding fragments of antibodies described herein may contain one, two, three, four, five, or six CDRs of the VH and VL sequences described herein, or of VH and VL sequences derived from antibodies that bind to one or more FZD receptors or LRP5 and / or LRP6. The antigen-binding fragment of an FZD-specific antibody may bind to one or more FZD receptors. The antigen-binding fragment of an LRP5 and / or LRP6-specific antibody may bind to LRP5 and / or LRP6 receptors. As used herein, this term includes not only isolated fragments but also polypeptides comprising antigen-binding fragments of polypeptides disclosed herein, such as fusion proteins comprising antigen-binding fragments of polypeptides disclosed herein, such as fusion proteins comprising Nanobody® that binds to one or more FZD receptors and Nanobody® that binds to LRP5 and / or LRP6.

[0069] The term "antigen" refers to a molecule or part of a molecule that can be used in animals to produce a polypeptide that can be bound by a selective binding agent, e.g., a polypeptide, and further can bind to the epitope of that antigen. In certain embodiments, a binding agent (e.g., a Wnt substitute molecule or its binding domain) is said to bind specifically to an antigen if it preferentially recognizes its target antigen in a complex mixture of proteins and / or macromolecules. In certain embodiments, a Wnt mimetic or its binding domain (e.g., an antibody or its antigen-binding fragment) has an equilibrium dissociation constant of 10 -7 M or less or 10 -8 It is said that when the M value is less than or equal to M, it specifically binds to the antigen. In some embodiments, the equilibrium dissociation constant is 10 -9 M or less or 10-10 It is also acceptable to have a minimum of M.

[0070] In certain embodiments, the Wnt substitute molecules and polypeptides described herein, as well as their antigen-binding fragments, contain heavy-chain CDR sets and light-chain CDR sets, respectively, sandwiched between heavy-chain framework region (FR) sets and light-chain framework region (FR) sets (which provide support to the CDRs and define the spatial relationships of the CDRs to each other). As used herein, the term “CDR set” refers to three hypervariable regions of the heavy-chain V region or light-chain V region. Starting from the N-terminus of the heavy-chain or light-chain, these regions are denoted “CDR1,” “CDR2,” and “CDR3,” respectively. Thus, the antigen-binding site contains six CDRs, including the CDR sets from the heavy-chain V region and light-chain V region, respectively. A polypeptide containing a single CDR (e.g., CDR1, CDR2, or CDR3) is referred herein to as a “molecular recognition unit.” Crystallographic analysis of several antigen-polypeptide complexes has demonstrated that amino acid residues of the CDRs form extensive contact with the bound antigen, with the most extensive antigen contact being with heavy-chain CDR3. Therefore, molecular recognition units are primarily responsible for the specificity of antigen-binding sites.

[0071] As used herein, the term “FR set” refers to the four adjacent amino acid sequences that constitute the CDR of a CDR set of a heavy chain V region or a light chain V region. Some FR residues may come into contact with the bound antigen; however, FRs, particularly those directly adjacent to the CDR, are primarily responsible for folding the V region into the antigen-binding site. Within the FR, certain amino residues and certain structural features are highly conserved. In this regard, all V region sequences contain an internal disulfide loop of approximately 90 amino acid residues. When the V region folds into the binding site, the CDR is presented as a protruding loop motif that forms the antigen-binding surface. It is generally recognized that there are conserved structural regions of the FR that influence the folded shape of the CDR loop into a certain “standard” structure, regardless of the exact CDR amino acid sequence. Furthermore, it is known that certain FR residues are involved in non-covalent interdomain contacts that stabilize the interaction between the antibody heavy and light chains.

[0072] The structure and location of immunoglobulin CDRs and variable domains can be determined by referring to Kabat, EA et al., Sequences of Proteins of Immunological Interest. 4th Edition. US Department of Health and Human Services. 1987 and its latest edition (currently available online at immuno.bme.nwu.edu).

[0073] However, Fv fragments are more commonly derived using recombinant techniques known in the art. Fv fragments contain a non-covalent VH:VL heterodimer that includes an antigen-binding site that retains much of the antigen recognition and binding ability of the native antibody molecule. Inbar et al. (1972) Proc. Nat. Acad. Sci. USA 69:2659-2662; Hochman et al. (1976) Biochem 15:2706-2710; and Ehrlich et al. (1980) Biochem 19:4091-4096.

[0074] In certain embodiments, single-chain Fv or scFV antibodies are envisioned. For example, kappa bodies (Ill et al., Prot. Eng. 10: 949-57 (1997); mini bodies (Martin et al., EMBO J 13: 5305-9 (1994); dia bodies (Holliger et al., PNAS 90: 6444-8 (1993); or Janusins ​​(Traunecker et al., EMBO J 10: 3655-59 (1991) and Traunecker et al., Int. J. Cancer Suppl. 7: 51-52) (1992) can be prepared using standard molecular biology techniques in accordance with the teachings of this application regarding the selection of antibodies having desired specificity. In yet another embodiment, bispecific or multispecific polypeptides can be made that encompass the ligands of the Disclosure. Bispecific polypeptides can be produced that specifically bind to one or more FZD receptors through one binding domain and specifically bind to a second molecule through a second binding domain. These polypeptides can be produced by recombinant molecular biology techniques or can be physically conjugated together.

[0075] A single-chain Fv(scFv) polypeptide is linked by a linker that encodes a peptide. H and V L Covalently bonded V is expressed from a gene fusion containing the gene encoding the VH ::V L It is a heterodimer. Huston et al. (1988) Proc. Nat. Acad. Sci. USA 85(16):5879-5883. Several methods have been described for identifying the chemical structure to convert naturally aggregated but chemically separated light and heavy polypeptide chains derived from the antibody V region into scFv molecules that fold into a three-dimensional structure substantially similar to the structure of the antigen-binding site. See, for example, U.S. Patent Nos. 5,091,513 and 5,132,405 to Huston et al., and U.S. Patent No. 4,946,778 to Ladner et al.

[0076] In certain embodiments, the Wnt substitute molecule described herein is in the form of a diabody. The diabody may be a polypeptide polymer, each polypeptide comprising a first domain containing an immunoglobulin light chain binding region and a second domain containing an immunoglobulin heavy chain binding region, the two domains linked (e.g., by a peptide linker) but unable to associate with each other to form an antigen-binding site; the antigen-binding site is formed by the association of the first domain of one polypeptide within the polymer with the second domain of another polypeptide within the polymer (WO94 / 13804). Alternatively, the diabody may be formed from intramolecular interactions between two VH domains and two VL domains, which is a tandem diabody. The length and amino acid composition of peptide linkers connecting individual VH and VL domains have an effect on polypeptide folding efficiency, stability, and biological activity (Fabrice Le Gall, et al., Effect of linker sequences between the antibody variable domains on the formation, stability and biological activity of a bispecific tandem diabody. Protein Engineering, Design & Selection vol. 17 no. 4 pp. 357-366, 2004).

[0077] A single-domain antibody fragment (dAb) consists of either a VH domain or a VL domain (Ward, ES et al., Nature 341, 544-546 (1989)).

[0078] Diabodies and scFvs can be constructed using only variable domains, without the Fc region, which may mitigate the effects of anti-idiotype reactions.

[0079] In contrast to bispecific whole antibodies, bispecific diabodies can also be particularly useful because they can be readily constructed and expressed in E. coli or other prokaryotes. Diabodies (and many other polypeptides, such as antibody fragments) with appropriate binding specificity can be readily selected from a library using phage display (see, for example, PCT application publication WO94 / 13804). If one arm of a diabody is kept constant to have specificity for, for example, antigen X, the other arm can be varied to select polypeptides with appropriate specificity and prepare a library.

[0080] In certain embodiments, the polypeptides of this disclosure may take the form of Nanobody®. Nanobody® technology was originally developed after the discovery and identification that camelids (e.g., camels and llamas) possess fully functional antibodies consisting solely of heavy chains and therefore lacking light chains. These heavy-chain-only antibodies contain a single variable domain (VHH) and two constant domains (CH2, CH3). The single variable domains, which have been cloned and isolated, possess full antigen-binding ability and are highly stable. These single variable domains, along with their distinctive structural and functional properties, form the basis of "Nanobodies®". Nanobodies® are encoded by a single gene and are efficiently produced in virtually all prokaryotic and eukaryotic hosts, e.g., E. coli (see, e.g., U.S. Patent No. 6,765,087), fungi (e.g., Aspergillus or Trichoderma), and yeasts (e.g., Saccharomyces, Kluyvermyces, Hansenula, or Pichia (see, e.g., U.S. Patent No. 6,838,254)). The production process is scalable, and several kilograms of Nanobodies® have been produced. Nanobodies® can be formulated as a ready-to-use solution with a long shelf life. Nanoclone® method (e.g., WO (See 06 / 079372) is a proprietary method for generating Nanobodies® against desired targets based on automated high-throughput selection of B cells. Nanobodies® are single-domain antigen-binding fragments of heavy-chain-only antibodies specific to camelids. Nanobodies®, also known as VHH antibodies, are typically small in size, around 15 kDa.

[0081] As mentioned earlier, the variable domains of Fab are responsible for the binding specificity of the entire antibody. Therefore, the smallest unit of Ig with antigen-binding activity is a variable fragment or Fv in which two variable domains (VH and VL) are connected by a disulfide bond. scFv is an engineered form of Fv in which the two variable domains are joined together by a flexible linker instead of a disulfide bond. The length and amino acid composition of this linker play a crucial role in the correct folding of proteins (Ahmad ZA, et al., ScFv antibody: Principles and clinical application. Clin Dev Immunol. 2012;2012 cited 2020 Aug 31), and it is typically 10-25 amino acids long, with a Glu Lys stretch for increased solubility and a Gly Ser stretch for the flexibility of the final protein (Alfthan K, et al., Properties of a single-chain antibody containing different linker peptides. Protein Eng Des Sel. 1995;8(7):725-731, and Whitlow M, et al. An improved linker for single-chain Fv with reduced aggregation and enhance proteolytic stability. Protein Eng Des Sel. 1993;6(8):989-95). Within each of the two variable domains of scFv, there are three hypervariable domains or complementarity-determining regions; there are significant structural differences, and scFv and VHH (nanobody) exhibit distinct properties in vitro and in vivo.

[0082] scFv and VHH(Nb) show significant size differences, with scFv having a weight of approximately 30 kDa, nearly twice the size of Nb (Bannas P, Hambach J, Koch-Nolte F, Johnson M. Nanobodies and nanobody-based human heavy chain antibodies as antitumor therapeutics. Front Immunol. 2017; 8(NOV):1603). This smaller size facilitates the genetic manipulation of VHH (Hassani M, Hajari Taheri F, Sharifzadeh Z, Arashkia A, Hadjati J, van Weerden WM, et al. Construction of a chimeric antigen receptor bearing a nanobody against prostate a specific membrane antigen in prostate cancer. J Cell Biochem. 2019;120(6):10787-95), and the presence of only three antigen-binding loops makes it easy to enhance their inherent tendency to bind to antigens (Hassanzadeh-Ghassabeh G, et al., Nanobodies and their potential applications, Nanomedicine 2013;8:1013-26). Due to the smaller size of VHH, their blood half-life is also shortened by filtration and degradation in the kidneys (Kim TY, Park JH, Shim HE, Choi DS, Lee DE, Song JJ, et al. Prolonged half life of small-sized therapeutic protein using serum albumin-specific protein binder. J Control Release. 2019;315:31-9).

[0083] VHH is more soluble than scFv, and its thermodynamic stability is higher compared to scFv. Therefore, VHH is more resistant to chemical denaturants and proteolytic enzymes (Hussack G, et al., Characterization of single-domain antibodies with an engineered disulfide bond. Methods Mol Biol. 2012;911:417-29) and has higher stability under harsh pH or ionic strength (Van Audenhove I, et al., Nanobodies as Versatile Tools to Understand, Diagnose, Visualize and Treat Cancer. EBioMedicine. 2016;8:40-8). This higher conformational stability is also due to the presence of an extra disulfide bond, which limits the flexibility of VHH by reducing the possibility of heat-induced aggregation. Thus, in scFv, although the hydrophobic interface between VL and VH weakens their stability, this two-domain structure makes them more flexible and more advantageous for some applications.

[0084] Wnt alternative polypeptide This disclosure provides, in certain embodiments, Wnt substitute molecules that bind to one or more FZD receptors and to one or both of LRP5 and / or LRP6. The Wnt substitute molecules may also be referred to as “Wnt mimes” or “Wnt substitute polypeptides.” In certain embodiments, the Wnt substitute molecules bind to one or more human FZD receptors and to one or both of human LRP5 and / or human LRP6.

[0085] In certain embodiments, the Wnt substitute molecule can or modulates Wnt signaling events in cells that come into contact with the Wnt substitute molecule. In certain embodiments, the Wnt substitute molecule increases Wnt signaling. In certain embodiments, the Wnt substitute molecule specifically binds to the human Wnt signaling pathway and modulates its biological activity.

[0086] The Wnt substitute molecules of the present invention are biologically active in binding to one or more FZD receptors, and to one or more of LRP5 and LRP6, and in activating Wnt signaling; in other words, the Wnt substitute molecules are Wnt agonists. The term "Wnt agonist activity" refers to the ability of an agonist to mimic the effect or activity of the WNT protein that binds to Frizzled protein and LRP5 or LRP6. The ability of the Wnt substitute molecules and other Wnt agonists disclosed herein to mimic the activity of WNT can be confirmed by several assays, including but not limited to those disclosed herein. Wnt signaling agonists typically evoke a response or activity similar to or identical to the response or activity evoked by the receptor's native ligand. In particular, the Wnt signaling agonists disclosed herein activate, enhance, or increase the standard WNT / β-catenin signaling pathway. As used herein, the term “enhance” refers to a measurable increase in the level of WNT / β-catenin signaling compared to the level in the absence of a Wnt signaling agonist, e.g., a Wnt substitute molecule disclosed herein.

[0087] In certain embodiments, the Wnt substitute molecules disclosed herein are bispecific, meaning they specifically bind to two or more different epitopes, for example, one or more FZD receptors and LRP5 and / or LRP6.

[0088] In certain embodiments, the Wnt substitute molecules disclosed herein are polyvalent, meaning they each comprise two or more regions that specifically bind to an epitope, the epitopes being the same or different epitopes, e.g., different epitopes on the same target, or even different epitopes on different targets. In certain embodiments, the Wnt substitute molecules disclosed herein comprise two or more regions that each specifically bind to the same epitope, e.g., two or more regions that bind to epitopes in one or more FZD receptors, and / or two or more regions that bind to epitopes in LRP5 and / or LRP6. In certain embodiments, they comprise two or more regions that bind to epitopes in one or more FZD receptors, and two or more regions that bind to epitopes in LRP5 and / or LRP6. In certain embodiments, the ratio of the number of regions that bind to one or more FZD receptors to the number of regions that bind to LRP5 and / or LRP6 in the Wnt substitute molecule may be 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 1:2, 1:3, 1:4, 1:5, or 1:6.

[0089] The Wnt substitute molecules disclosed herein may have any of several different structural forms or configurations. Wnt mimics may include polypeptide and / or non-polypeptide binding moieties, such as small molecules. In certain embodiments, the Wnt substitute molecule includes both a polypeptide region and a non-polypeptide binding moiety. In certain embodiments, the Wnt substitute molecule may contain a single polypeptide, or it may contain two or more, three or more, or four or more polypeptides.

[0090] If the Wnt substitute molecule comprises a single polypeptide, it may be a fusion protein comprising one or more FZD binding domains and one or more LRP5 / 6 binding domains. The binding domains may be directly fused or linked via a linker, for example, a polypeptide linker including, but not limited to, any of those disclosed herein.

[0091] Wnt-alternative polypeptides can be manipulated to facilitate bonding between two polypeptides. Means of introducing modifications to polypeptides to promote their bonding are known and available in the art. For example, certain amino acids, such as cysteine ​​for forming intermolecular disulfide bonds, can be introduced and used for crosslinking.

[0092] The Wnt substitute molecules described herein may have a variety of different structural forms, including but not limited to those shown in Figures 1-22. It should be understood that the disclosed Wnt substitute molecules are not limited to the structural forms shown in Figures 1-22, and these are merely examples of specific embodiments of various structural forms. For example, the linker present in the exemplary forms may differ from or have a different arrangement than that shown in Figures 1-22.

[0093] In one embodiment, the Wnt substitute molecule includes scFv or its antigen-binding fragment fused with VHH (Nanobody®) or its antigen-binding fragment. In a particular embodiment, scFv specifically binds to one or more FZD receptors, and VHH specifically binds to LRP5 and / or LRP6.

[0094] In certain embodiments, two monomers of a Wnt substitute molecule dimerize to form a polyvalent, multispecific binding composition, including but not limited to those shown in Figures 1A-1D. Dimerization occurs through interactions between VH1 and VL1 of two different monomers. In certain embodiments, a single-chain polypeptide comprises two identical or different scFv or alternatively two diabodies resulting from intramolecular VH / VL interactions, which specifically bind to a VHH having specificity for LRP5 and / or LRP6, and to one or more FZD receptors, including but not limited to those shown in Figures 2-4 and 7B, to form a trivalent, bispecific or triplicate binding molecule.

[0095] In certain embodiments, a single-chain polypeptide comprises two identical or different VHHs that specifically bind to LRP5 and / or LRP6, and two identical or different scFvs that specifically bind to one or more FZDs. In certain embodiments, intramolecular VH-VL interactions result in a tetravalent triple-specificity molecule having identical LRP5 and / or LRP6 binding factors and linked to two different scFv FZD binding factors. In another embodiment, a single-chain polypeptide comprising two different VHHs LRP5 and / or LRP6 and two identical scFv FZD binding factors results in a tetravalent triple-specificity binding molecule, including but not limited to those shown in Figures 5-7A.

[0096] In some embodiments, the single-chain polypeptide comprises one VHH that specifically binds to LRP5 and / or LRP6, and two identical VHHs that bind to one or more FZDs, the presence of identical VHHs leading to a trivalent bispecific binding molecule. In certain embodiments, the VHH FZD binding factors are not identical, and alternative molecules are trivalent triple specific binding molecules, including but not limited to those shown in Figure 8.

[0097] In certain embodiments, the single-chain polypeptide comprises two identical or different VHHs that specifically bind to LRP5 and / or LRP6, and two identical VHHs that specifically bind to one or more FZDs, thereby creating a tetravalent bispecific or triplicate Wnt substitute molecule. In certain embodiments, the single-chain polypeptide comprises two identical or different VHHs that specifically bind to LRP5 and / or LRP6, and two different VHH FZD binding factors, thereby creating tetravalent triplicate and quadruplicate Wnt substitute molecules, including but not limited to those shown in Figure 9.

[0098] In certain embodiments, scFv specifically binds to LRP5 and / or LRP6, and VHH specifically binds to one or more FZD receptors. In certain embodiments, scFv or its antigen-binding fragment is directly fused with VHH or its antigen-binding fragment, while in other embodiments, the two binding regions are fused via a linker moiety. In certain embodiments, scFv comprises either the CDR set described herein or one of the CDR sets described herein. In certain embodiments, VHH comprises either the CDR set described herein or one of the CDR sets disclosed herein.

[0099] In various embodiments, the Wnt surrogate molecule comprises one or more Fabs or antigen-binding fragments thereof that bind to one or more FZD receptors, and one or more Fabs or antigen-binding fragments thereof that bind to LRP5 and / or LRP6. In a particular embodiment, it comprises two types of Fabs or antigen-binding fragments thereof that bind to one or more FZD receptors, and two types of Fabs or antigen-binding fragments thereof that bind to LRP5 and / or LRP6.

[0100] In some embodiments, including but not limited to those shown in Figures 11-13, the Wnt substitute molecule comprises one or more Fab, Fv-Fab or its antigen-binding fragment, and one or more VHH or its antigen-binding fragment. In certain embodiments, the Fab specifically binds to LRP5 and / or LRP6, and the VHH specifically binds to one or more FZD receptors, including but not limited to those shown in Figures 11A-11F. In certain embodiments, the Fab specifically binds to one or more FZD receptors, and the VHH specifically binds to LRP5 and / or LRP6, including but not limited to those shown in Figures 12A-12D and 13A-13D.

[0101] In some embodiments, the Wnt substitute molecule of the present invention comprises a VHH that specifically binds to LRP5 and / or LRP6, and two identical or different VHHs, each bound to one or more FZDs, wherein the VHHs are added from a constant heavy / constant light chain (CH / CL), and non-limiting examples are shown in Figures 14A-14H. In certain embodiments, the Wnt substitute molecule having two identical or different VHHs, each specifically bound to LRP5 and / or LRP6, and two identical or different VHHs, each specifically bound to one or more FZDs, is added from CH / CL, and non-limiting examples are shown in Figures 15A-15F. In certain embodiments, the polypeptide of the present invention comprises one VHH that specifically binds to LRP5 and / or LRP6, added from two FZD binding factors, wherein the FZD binding factors are in the form of scFv-Fab, including but not limited to those shown in Figures 16A-16D. In another embodiment, two scFvs are attached to a Fab having specificity for LRP5 and / or LRP6, and specifically bind to one or more FZDs, as shown in Figures 17A-17D. In some embodiments, the Wnt mimics of the present invention are two identical or different scFvs that bind to one or more FZDs, as well as two identical or different scFvs that bind to LRP5 and / or LRP6, which are attached from CH / CL in cis and trans forms, including but not limited to those shown in Figures 18A-18F.

[0102] In certain embodiments, the Wnt substitute molecules described herein include two identical or different VHHs having specificity for LRP5 and / or LRP6, and two identical or different scFvs, each attached to one or more FZDs, which are added to CH / CL to form cis and trans isomers of the VHH and scFv, including but not limited to those shown in Figures 19-20.

[0103] In some embodiments, the Wnt mimic described herein comprises one VHH attached to a CH or CL having specificity for LRP5 and / or LRP6, and two identical or different scFv attached to a CH and / or CL, including but not limited to those illustrated in Figures 21A-21L, which are bound to one or more FZDs, or the VHH is bound to one of two identical or different scFv attached to a CH or CL, which are bound to one or more FZDs, as shown in Figures 22A-22F.

[0104] In various embodiments, the Wnt substitute molecule comprises one or more of its antigen-binding fragments as disclosed herein. In certain embodiments, the Wnt substitute molecule comprises two or more, three or more, or four or more of its antigen-binding fragments as disclosed herein.

[0105] In certain embodiments, the Wnt substitute molecule includes an FZD-binding region, e.g., its anti-FZD antigen-binding fragment, or an LRP5 and / or LRP6-binding region, e.g., its anti-LRP5 and / or LRP6 antigen-binding fragment, fused to or bound to a polypeptide that specifically binds to one or more FZD receptors. In certain embodiments, the polypeptide that specifically binds to one or more FZD receptors is its polypeptide antigen-binding fragment. In certain embodiments, the antigen-binding fragment is disclosed herein or in PCT application WO 2019 / 126399, “Anti-Frizzled antibodies and Methods of Use,” filed December 19, 2017, which is incorporated herein by reference in its entirety. In certain embodiments, the FZD-binding domain includes three heavy-chain CDRs and / or three light-chain CDRs disclosed for any of the exemplary antibodies or fragments thereof that bind to one or more FZD receptors. In certain embodiments, the FZD-binding domain includes, for example, at least about 1 × 10⁻⁶ -4 M, at least about 1 × 10-5 M, at least about 1 × 10 -6 M, at least about 1 × 10 -7 M, at least about 1 × 10 -8 M, at least about 1 × 10 -9 M, or at least about 1 × 10 -10 M's K D The binding domain can be selected from any binding domain that binds to FZD with such affinity. In a particular embodiment, the FZD binding domain may be, for example, about 1 × 10⁻⁶ -7 Less than M, approximately 1 x 10 -8 Less than M, approximately 1 x 10 -9 Less than M, or approximately 1 × 10 -10 Less than M K D The binding domain can be selected from any binding domains that bind to one or more FZD receptors with high affinity. In a particular embodiment, the FZD binding domain is, for example, about 1 × 10⁻¹⁶ in the context of a Wnt mimetic. -4 Less than or equal to M, approximately 1 × 10 -5 Less than or equal to M, approximately 1 × 10 -6 Less than or equal to M, approximately 1 × 10 -7 Less than or equal to M, approximately 1 × 10 -8 Less than or equal to M, approximately 1 × 10 -9 Less than or equal to M, or at least about 1 × 10⁻⁶ -10 M's K D This high affinity allows for binding to FZD, and any binding domain can be selected.

[0106] Suitable FZD-binding domains include, but are not limited to, de novo-designed FZD-binding proteins, derived binding proteins that specifically bind to one or more FZD proteins, such as scFv, Fab, Diabody, VHH, etc.; binding domains derived from nanobody; Notchin-based engineered scaffolds; norin and engineered binding fragments derived therefrom; and naturally occurring FZD-binding domains. FZD-binding domains may be affinity-selected to enhance binding to one or more desired FZD proteins, for example, to obtain tissue selectivity.

[0107] In some embodiments, the FZD-binding domain binds to one, two, three, four, five or more different frizzled proteins, for example, one or more of the human frizzled proteins FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, and FZD10. In some embodiments, the FZD-binding domain binds to FZD1, FZD2, FZD5, FZD7, and FZD8. In other embodiments, the FZD-binding domain is selective for one or more desired frizzled proteins, for example, having at least 10-fold, 25-fold, 50-fold, 100-fold, 200-fold, or higher specificity for one or more desired frizzled proteins compared to other frizzled proteins.

[0108] In other embodiments, the FZD-binding domain comprises a variable region sequence or its CDR derived from one of several FZD-specific antibodies known in the art, commercially available, or de novo-produced. Any FZD polypeptide can be used as an immunogen or in screening assays for developing Wnt alternative molecules. Non-limiting examples of frizzled-binding domains include antibodies available from Biolegend, e.g., clone CH3A4A7 specific to human frizzled 4 (CD344); clone W3C4E11 specific to human FZD9 (CD349); antibodies available from Abcam, e.g., ab64636 specific to FZD7; ab83042 specific to human FZD4; ab77379 specific to human FZD7; ab75235 specific to human FZD8; and ab102956 specific to human FZD9. Other examples of suitable antibodies are described in particular in U.S. Patent Application No. 20140105917; U.S. Patent Application No. 20130230521; U.S. Patent Application No. 20080267955; U.S. Patent Application No. 20080038272; U.S. Patent Application No. 20030044409, etc., each expressly incorporated herein by reference.

[0109] The FZD binding region of a Wnt mimetic may be an engineered protein selected for its structural homology to the FZD binding region of a WNT protein. Such proteins can be identified by screening a homology-based structural database. In this way, the first protein, for example, the microbial Bh1478 protein, was identified. Subsequently, native proteins can be engineered to produce amino acid substitutions that increase affinity, and further selected by affinity maturation to increase affinity to and binding selectivity to the desired frizzled protein. Non-limiting examples of frizzled binding regions include the Fz27 and Fz27-B12 proteins.

[0110] In certain embodiments, the Wnt substitute molecule includes an LRP5 / 6 binding domain, for example, an anti-LRP5 / 6 antibody or its antigen-binding fragment fused with a polypeptide that specifically binds to one or more FZD receptors. In certain embodiments, the polypeptide that specifically binds to LRP5 / 6 is the antigen-binding fragment. In certain embodiments, it is the antigen-binding fragment disclosed in PCT application WO 2019 / 126401, “Anti-LR5 / 6 Antibodies and Methods of Use,” filed December 19, 2017, which is incorporated herein by reference in its entirety. In certain embodiments, the LRP5 / 6 binding domain includes three heavy-chain CDRs and / or three light-chain CDRs disclosed for any exemplary antibody or fragment thereof that binds to LRP5 and / or LRP6. In certain embodiments, the LRP5 / 6 binding domain includes heavy-chain fragments and / or light-chain fragments of any exemplary antibody or fragment thereof that binds to LRP5 and / or LRP6.

[0111] In a particular embodiment, the LRP5 / 6 binding domain, in the context of the Wnt mimetic, is approximately 1 × 10⁶ -4 Less than or equal to M, approximately 1 × 10 -5 Less than or equal to M, approximately 1 × 10-6 Less than or equal to M, approximately 1 × 10 -7 Less than or equal to M, approximately 1 × 10 -8 Less than or equal to M, approximately 1 × 10 -9 Less than or equal to M, or approximately 1 × 10 -10 K less than or equal to M D The binding domain can be selected from any binding domains that bind to LRP5 or LRP6. In a particular embodiment, the LRP5 / 6 binding domain is approximately 1 × 10⁻¹⁶ in the context of the Wnt mimetic. -4 Greater than or equal to M, approximately 1 × 10 -5 Greater than or equal to M, approximately 1 × 10 -6 Greater than or equal to M, approximately 1 × 10 -7 Greater than or equal to M, approximately 1 × 10 -8 Greater than or equal to M, approximately 1 × 10 -9 Greater than or equal to M, or approximately 1 × 10⁻⁶ -10 K surpasses M D The binding domains that bind to LRP5 or LRP6 can be selected from any available binding domains. In a particular embodiment, the LRP5 / 6 binding domain may be, for example, about 1 × 10⁻⁶ -7 Less than M, approximately 1 x 10 -8 Less than M, approximately 1 x 10 -9 Less than M, or approximately 1 × 10 -10 Less than M K D Any binding domain can be selected that binds to LRP5 or LRP6 with high affinity.

[0112] Other suitable LRP5 / 6 binding domains include, but are not limited to, de novo-designed LRP5 / 6 binding proteins such as scFv, Fab, Diabody, VHH, and other parts of antibodies that specifically bind to one or more LRP proteins; nanobody-derived binding domains; Notchin-based engineered scaffolds; naturally occurring LRP5 / 6 binding factors including, but not limited to, DKK1, DKK2, DKK3, DKK4, and sclerostin; Wise; fusion proteins containing any of the above; derivatives of any of the above; variants of any of the above; and any of the above biologically active fragments. LRP5 / 6 binding domains can be affinity-selected to enhance binding.

[0113] Members of the Dickkopf (DKK) gene family (see Krupnik et al. (1999) Gene 238(2):301-13) include DKK-1, DKK-2, DKK-3, and DKK-4, as well as the DKK-3-related protein Soggy (Sgy). hDKK 1-4 contain two distinct cysteine-rich domains, the positions of which are highly conserved among family members. Exemplary sequences of human Dkk genes and proteins are publicly available, for example, Genbank accession numbers NM_014419(soggy-1); NM_014420(DKK4); AF177394(DKK-1); AF177395(DKK-2); NM_015881(DKK3); and NM_014421(DKK2). In some embodiments of the present invention, the LRP6 binding moiety is a DKK1 peptide, which includes, but is not limited to, the C-terminal domain of human DKK1. The C-terminal domain may include the sequence (SEQ ID NO: 57): KMYHTKGQEGSVCLRSSDCASGLCCARHFWSKICKPVLKEGQVCTKHRRKGSHGLEIFQRCYCGEGLSCRIQKDHHQASNSSRLHTCQRH (see Genbank accession number NP_036374) or a biologically active fragment thereof.

[0114] The binding of the DKK protein to LRP5 / 6 has been discussed, for example, in Brott and Sokol Mol. Cell. Biol. 22 (17), 6100-6110 (2002); and Li et al. J. Biol. Chem. 277 (8), 5977-5981 (2002), each of which is explicitly incorporated herein by reference. The corresponding region of human DKK2 (Genbank reference NP_055236) may contain the sequence:KMSHIKGHEGDPCLRSSDCIEGFCCARHFWTKICKPVLHQGEVCTKQRKKGSHGLEIFQRCDCAKGLSCKVWKDATYSSKARLHVCQK (SEQ ID NO: 58) or its biologically active fragment.

[0115] Antibodies that specifically bind to LRP5 or LRP6 are known in the art, commercially available, or can be produced de novo. LRP5, LRP6, or their fragments can be used as immunogens and / or in screening assays for antibody development. Examples of known antibodies include those described in Gong et al. (2010) PLoS One. 5(9):e12682; Ettenberg et al. (2010) Proc Natl Acad Sci US A. 107(35):15473-8; as well as commercially available antibodies such as Santa Cruz biotechnology antibody clone 1A12; monoclonal antibody 2B11; and Cell Signaling Technology antibody (D80F2), catalog number 5731, which is produced against synthetic human LRP5 / 6 and binds to both full-length and proteolytic fragments of mouse and human LRP6 and LRP5, for example.

[0116] In certain embodiments, the Wnt substitute molecules disclosed herein comprise one or more polypeptides comprising two or more binding domains. For example, the two or more binding domains may be two or more FZD binding domains or one or more LRP5 / 6 binding domains, or they may comprise one or more FZD binding domains and one or more LRP5 / 6 binding domains. The binding domains may be directly joined, contiguous, or separated by linkers, such as polypeptide linkers or non-peptide linkers. The length of the linkers, and therefore the spacing between binding domains, can be used to modulate signal intensity and can be selected depending on the desired use of the Wnt mimetic. The forced distances between binding domains may vary, but in certain embodiments they may be less than about 100 angstroms, less than about 90 angstroms, less than about 80 angstroms, less than about 70 angstroms, less than about 60 angstroms, or less than about 50 angstroms. In some embodiments, the linker is a rigid linker, and in other embodiments, the linker is a flexible linker. In certain embodiments where the linker is a peptide linker, the linker can be about 0, 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 amino acids or longer, and is long and composed of amino acids sufficient to force a distance between binding domains. In some embodiments, the linker contains or consists of one or more glycine residues and / or serine residues. In one embodiment, the peptide linker is a peptide (G4S) (SEQ ID NO: 59) consisting of four consecutive glycine and one serine. In another embodiment, the peptide linker as used herein is a peptide (G4S)3 (SEQ ID NO: 60) consisting of three consecutive G4S peptides. In some embodiments, the peptide linker is ASTKG (Ala-Ser-Thr-Lys-Gly; SEQ ID NO: 61) or DKTHT (Asp-Lys-Thr-His-Thr; SEQ ID NO: 62).In certain embodiments, ASTKG (SEQ ID NO: 61) or DKTHT (SEQ ID NO: 62) is combined with G4S (SEQ ID NO: 59). (Clarissa G. Jakob, et al., Structure reveals function of the dual variable domain immunoglobulin (DVD-Ig). TM ) molecule, mAbs 5:3, 358-363; 2013;Joshua S.Klein, et al., Design and characterization of structured protein linkers with differenting flexibilities; Protein Engineering, Design & Selection vol. 27 no. 10 pp. 325-330, 2014;Weizao Chen, et al.,, Improving the CH1-CK heterodimerization and pharmacokinetics of 4Dm2m, a novel potent CD4 antibody fusion protein against HIV-1, MABS, 2016, VOL. 8, NO. 4, 761-774. )

[0117] In certain embodiments, the Wnt substitute molecule fragment comprises scFv, which contains a linker between its VH and VL domains. In certain embodiments, the linker is of sufficient length and flexibility to allow the VH and VL domains of scFv to bind to each other. In certain embodiments, the linker is a linear peptide linker consisting of approximately 12 to 30 amino acids in length. In one embodiment, the linker is three consecutive peptides, each peptide consisting of four consecutive glycine and one serine (G4S)3 (SEQ ID NO: 60).

[0118] In certain embodiments, a fragment of the Wnt substitute molecule contains VH and VL, and this fragment binds to another fragment containing VH and VL to form a diabody. In this case, in certain embodiments, the linker between VH and VL in the fragment is short enough to prevent VH and VL inside the fragment from binding to each other to form scFv. In certain embodiments, the linker is a linear peptide linker consisting of about 4 to about 12 amino acids in length. In one embodiment, the peptide linker is a peptide (G4S) (SEQ ID NO: 59) consisting of four consecutive glycine and one serine. In some embodiments, the peptide linker is ASTKG (Ala-Ser-Thr-Lys-Gly; SEQ ID NO: 61) or DKTHT (Asp-Lys-Thr-His-Thr; SEQ ID NO: 62). In certain embodiments, ASTKG (SEQ ID NO: 61) or DKTHT (SEQ ID NO: 62) is combined with G4S (SEQ ID NO: 59).

[0119] In certain embodiments, the Wnt substitute molecule comprises a polypeptide sequence having at least 90%, at least 95%, at least 98%, or at least 99% identity to a polypeptide sequence disclosed in any of SEQ ID NOs: 1 to 56, or a polypeptide sequence having at least 90%, at least 95%, at least 98%, or at least 99% identity to an antigen-binding fragment of a polypeptide sequence disclosed in any of SEQ ID NOs: 1 to 56. In certain embodiments, the Wnt substitute molecule comprises or consists of a polypeptide sequence described in any of SEQ ID NOs: 1 to 56 or an antigen-binding fragment thereof.

[0120] In certain embodiments, Wnt substitute molecules directly activate standard Wnt signaling by binding to one or more FZD proteins, as well as to LRP5 and / or 6, particularly by binding to these proteins on the cell surface, e.g., on the surface of human cells. This direct activation of Wnt signaling by Wnt substitute molecules is in contrast to the enhancement of Wnt signaling, which is only enhanced when native WNT proteins are present.

[0121] Wnt surrogate molecules can activate Wnt signaling, for example, by mimicking the effect or activity of the WNT protein that binds to the FZD protein. The ability of the Wnt surrogate molecules of this disclosure to mimic WNT activity can be confirmed by several assays. Wnt surrogate molecules typically elicit a response or activity similar to or identical to that induced by the receptor's native ligand. In particular, the Wnt surrogate molecules of the present invention enhance the standard WNT / β-catenin signaling pathway. As used herein, the term “enhance” refers to a measurable increase in the level of WNT / β-catenin signaling compared to the level in the absence of the Wnt surrogate molecule of the present invention.

[0122] Various methods for measuring the level of standard WNT / β-catenin signaling are known in the art. These include, but are not limited to, the expression of WNT / β-catenin target genes; the expression of TCF reporter genes; β-catenin stabilization; LRP phosphorylation; and assays measuring the translocation of axins from the cytoplasm to the cell membrane and their binding to LRP. The standard WNT / β-catenin signaling pathway ultimately leads to changes in gene expression via the transcription factors TCF7, TCF7L1, TCF7L2, and LEF. The transcriptional response to Wnt activation has been characterized in several cells and tissues. Therefore, comprehensive transcriptional profiling using methods known in the art can be used to assess the activation or inhibition of WNT / β-catenin signaling.

[0123] WNT-responsive gene expression changes are generally mediated by TCF and LEF transcription factors. TCF reporter assays assess transcriptional and translational changes in TCF / LEF regulatory genes to determine the level of WNT / β-catenin signaling. The TCF reporter assay was first described by Korinek, V. et al., 1997. Also known as TOP / FOP, this method involves determining the transactivation activity of endogenous β-catenin / TCF4 by 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 and pFOPFlash, respectively) that activates luciferase expression. A higher ratio of these two reporter activities (TOP / FOP) indicates higher β-catenin / TCF4 activity, while a lower ratio indicates lower β-catenin / TCF4 activity.

[0124] Various other reporter transgenes that respond to Wnt signaling are present in an intact state within the animal body and therefore effectively reflect endogenous WNT signaling. These reporters are based on a multimerized TCF binding site that activates the expression of LacZ or GFP, which can be readily detected by methods known in the art. Examples of these reporter genes include TOP-GAL, BAT-GAL, ins-TOP-EGFP, ins-TOPGAL, LEF-EGFP, Axyn 2-LacZ, Axyn 2-d2EGFP, Lgr5tm1(cre / ERT2), and TOPdGFP.

[0125] The recruitment of dephosphorylated β-catenin to the membrane, the stabilization and phosphorylation state of β-catenin, and the translocation of β-catenin to the nucleus (Klapholz-Brown Z et al., PLoS One. 2(9) e945, 2007) are, in some cases, mediated by complex formation with the TCF transcription factor and TNIK, which is a crucial step in the Wnt signaling pathway. Stabilization is mediated by Disheveled family proteins that inhibit the “disruption” complex, resulting in reduced intracellular β-catenin degradation and subsequent translocation of β-catenin to the nucleus. Therefore, measuring the level and location of β-catenin in cells provides a good reflection of the level of WNT / β-catenin signaling. A non-limiting example of such an assay is the “BioImage β-Catenin Redistribution Assay” (Thermo Scientific), which provides recombinant U2OS cells stably expressing human β-catenin fused to the C-terminus of enhanced green fluorescent protein (EGFP). Imaging and analysis are performed using fluorescence microscopy or a high-content screening (HCS) platform that enables visualization of the level and distribution of EGFP-β-catenin.

[0126] Another way the disruption complex is inhibited is by the removal of axine through its recruitment to the cytoplasmic tail of the WNT coreceptor LRP. Axine has been shown to preferentially bind to the phosphorylated form of the LRP tail. Therefore, visualization of axine translocation using, for example, a GFP-axine fusion protein is another method for assessing the level of WNT / β-catenin signaling.

[0127] In certain embodiments, the Wnt surrogate molecule enhances or increases standard Wnt pathway signaling, such as β-catenin signaling, by at least 30%, 35%, 40%, 45%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 150%, 200%, 250%, 300%, 400%, or 500% compared to β-catenin signaling induced by a neutral substance or negative control, as measured in the above assay, for example, the TOPFlash assay. Negative controls may be included in these assays. In certain embodiments, Wnt mimics can enhance β-catenin signaling by 2-fold, 5-fold, 10-fold, 100-fold, 1000-fold, 10000-fold, or more compared to the activity in the absence of the Wnt substitute molecule, as measured by the above assays, for example, the TOPFlash assay or any of the other assays mentioned herein.

[0128] When used herein, "WNT gene product" or "WNT polypeptide" includes natural sequence WNT polypeptides, WNT polypeptide variants, WNT polypeptide fragments, and chimeric WNT polypeptides. In certain embodiments, the WNT polypeptide is a natural human full-length mature WNT protein.

[0129] For example, the target human natural sequence WNT proteins in this application include the following: WNT-1 (GenBank accession number NM_005430); WNT-2 (GenBank accession number NM_003391); WNT-2B (WNT-13) (GenBank accession number NM_004185 (isoform 1), NM_024494.2 (isoform 2)), WNT-3 (RefSeq.: NM_030753), WNT3a (GenBank accession number NM_033131), WNT-4 (GenBank accession number NM_030761), WNT-5A (GenBank accession number NM_003392), WNT-5B (GenBank accession number NM_032642), WNT-6 (GenBank accession number NM_032642) Accession number NM_006522), WNT-7A (GenBank accession number NM_004625), WNT-7B (GenBank accession number NM_058238), WNT-8A (GenBank accession number NM_058244), WNT-8B (GenBank accession number NM_003393), WNT-9A (WNT-14) (GenBank accession number NM_00 3395), WNT-9B (WNT-15) (GenBank accession number NM_003396), WNT-10A (GenBank accession number NM_025216), WNT-10B ( GenBank accession number NM_003394), WNT-11 (GenBank accession number NM_004626), WNT-16 (GenBank accession number NM_016087)). Each member exhibits varying degrees of sequence identity with the family, but all encode small (i.e., 39–46 kD) acylated and palmitoylated secreted glycoproteins containing 23–24 conserved cysteine ​​residues with highly conserved spacing (McMahon, AP et al., Trends Genet. 1992; 8: 236–242, Miller, J R. Genome Biol. 2002; 3(1): 3001.1–3001.15).Other natural WNT polypeptide sequences of interest include the above-mentioned orthologs derived from any mammal, including domesticated and livestock animals, as well as zoo animals, laboratory animals, or pet animals, such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, rats, mice, frogs, zebrafish, fruit flies, worms, etc.

[0130] In this specification, "Wnt pathway signaling" or "Wnt signaling" refers to the mechanism by which biologically active WNTs exert their effects on cells to modulate cellular activity. WNT proteins modulate cellular activity by binding to WNT receptors, including proteins from the Frizzled (FZD) family, proteins from the ROR family, proteins LRP5, LRP6, FRL1 / crypto, and Derailed / Ryk from the LRP family. Upon activation by WNT binding, WNT receptors activate one or more intracellular signaling cascades, including the standard Wnt signaling pathway; the Wnt / planar cell polarity (Wnt / PCP) pathway; and the Wnt-calcium (Wnt / Ca 2+ Examples include the ) pathway (Giles, RH et al. (2003) Biochim Biophys Acta 1653, 1-24; Peifer, M. et al. (1994) Development 120: 369-380; Papkoff, J. et al (1996) Mol. Cell Biol. 16: 2128-2134; Veeman, MT et al. (2003) Dev. Cell 5: 367-377); and other Wnt signaling pathways well known in the art.

[0131] For example, activation of the standard Wnt signaling pathway leads to inhibition of phosphorylation of the intracellular protein β-catenin, which in turn leads to the accumulation of β-catenin in the cytosol and subsequent translocation to the nucleus, where it interacts with transcription factors, such as TCF / LEF, to activate target genes. Activation of the Wnt / PCP pathway activates the RhoA, c-Jun N-terminal kinase (JNK), and nemo-like kinase (NLK) signaling cascades, regulating biological processes such as tissue polarity and cell migration. For example, Wnt / Ca 2+ Activation of this pathway induces the intracellular release of calcium ions, which in turn activates calcium-sensitive enzymes such as protein kinase C (PKC), calcium-calmodulin-dependent kinase II (CamKII), or calcineurin (CaCN). By assaying the activity of the above signaling pathway, the biological activity of antibodies or their antigen-binding fragments, such as Wnt mimics, can be easily determined.

[0132] In certain embodiments, the functional properties of Wnt substitute molecules can be evaluated using various methods known to those skilled in the art, including, for example, affinity / binding assays (e.g., surface plasmon resonance, competitive inhibition assays), cytotoxicity assays, cell viability assays, cell proliferation or differentiation assays in response to Wnt substitute molecules, and inhibition of cancer cells and / or tumor growth using in vitro or in vivo models, including but not limited to any of those described herein. Furthermore, the Wnt substitute molecules described herein can also be tested for their effects on internal translocation of FZD receptors, in vitro and in vivo efficacy, and the like. Such assays can be performed using well-established protocols known to those skilled in the art (see, for example, Current Protocols in Molecular Biology (Greene Publ. Assoc. Inc. & John Wiley & Sons, Inc., NY, NY); Current Protocols in Immunology (John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M. Shevach, Warren Strober eds. 2001 John Wiley & Sons, NY, NY)) or commercially available kits.

[0133] In certain embodiments, the FZD binding region of the Wnt substitute molecule (e.g., the antigen-binding fragment of an anti-FZD antibody) includes one or more of the CDRs of the anti-FZD antibody described herein. In certain embodiments, the LRP5 / 6 binding region of the Wnt mimetic (e.g., the antigen-binding fragment of an anti-LRP5 / 6 antibody) includes one or more of the CDRs of the anti-LRP5 / 6 antibody described herein. In this regard, it has been shown that in some cases, it is possible to transfer only the VHCDR3 of the antibody while still retaining the desired specific binding (Barbas et al., PNAS (1995) 92: 2529-2533). See also McLane et al., PNAS (1995) 92: 5214-5218 and Barbas et al., J. Am. Chem. Soc. (1994) 116: 2161-2162.

[0134] Also disclosed herein is a method for obtaining a polypeptide or antigen-binding domain specific to an FZD receptor, comprising, optionally, combining the VH domain thus provided with one or more VL domains by adding, deleting, substituting, or inserting one or more amino acids into the amino acid sequence of a VH domain or an amino acid sequence variant of a VH domain as described herein, and testing one or more VH / VL combinations to identify a specific binding member or polypeptide antigen-binding domain that is specific to one or more FZD receptors and optionally has one or more desired properties. The VL domain may substantially have the amino acid sequence described herein. Similar methods may also be used, in which one or more sequence variants of the VL domain disclosed herein are combined with one or more VH domains.

[0135] In certain embodiments, the Wnt substitute molecule is water-soluble. “Water-soluble” means a composition that is soluble in an aqueous buffer in the absence of a surfactant and is typically soluble at concentrations that yield a biologically effective dose of polypeptide. A water-soluble composition forms a substantially homogeneous composition having a specific activity of at least about 5% of the purified starting material, typically at least about 10%, 20%, or 30%, more typically about 40%, 50%, or 60%, and may be about 50%, about 90%, or higher. The Wnt mimics disclosed herein typically form substantially homogeneous aqueous solutions at concentrations of at least 5 μM and higher, e.g., at least 5 μM, 20 μM, or 40 μM, typically at least 60 μM, 70 μM, 80 μM, or 90 μM, sometimes as high as 100 μM, 150 μM, or 250 μM. In other words, the Wnt mimics disclosed herein typically form substantially homogeneous aqueous solutions at concentrations of about 0.4 mg / ml, about 1.0 mg / ml, about 20 mg / ml, or higher.

[0136] An antigen or epitope that “specifically binds” or “preferentially binds” (as used interchangeably herein) to a polypeptide or its antigen-binding fragment is a well-understood term in the art, and methods for determining such specific or preferential binding are also well-known in the art. A molecule, e.g., a Wnt substitute molecule, is said to exhibit “specific binding” or “preferential binding” if it reacts or associates with a particular cell or substance more frequently, more rapidly, for a longer duration, and / or with higher affinity than it reacts or associates with a substitute cell or substance. A molecule or its binding region, e.g., a Wnt substitute molecule or its binding region, “specifically binds” or “preferentially binds” to a target antigen, e.g., an FZD receptor, if it binds with higher affinity, avidity, more readily, and / or for a longer duration than it binds to other substances. For example, a Wnt substitute molecule or its binding domain that specifically or preferentially binds to the FZD1 receptor is a polypeptide that binds to the FZD1 receptor with higher affinity, avidity, more readily, and / or for a longer duration than those that bind to other FZD receptors or non-FZD proteins. By reading this definition, it will be understood that, for example, a Wnt substitute molecule or its binding domain that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. Therefore, "specific binding" or "preferential binding" does not necessarily require (but may include) exclusive binding. In general, though not always, references to binding imply preferential binding.

[0137] In some embodiments, one or more FZD binding domains of the Wnt substitute molecule bind to one, two, three, four, five or more different frizzled proteins, such as one or more of the human frizzled proteins FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, and FZD10. In some embodiments, one or more FZD binding domains bind to FZD1, FZD2, FZD5, FZD7, and FZD8. In various embodiments, any of the FZD binding regions are bound to (i) FZD1, FZD2, FZD7, and FZD9; (ii) FZD1, FZD2, and FZD7; (iii) FZD5 and FZD8; (iv) FZD5, FZD7, and FZD8; (v) FZD1, FZD4, FZD5, and FZD8; (vi) FZD1, FZD2, FZD5, FZD7, and FZD8; (vii) FZD4 and FZD9; (viii) FZD9 and FZD10; (ix) FZD5, FZD8, and FZD10; or (x) FZD4, FZD5, and FZD8; FZD1, FZD5, FZD7, and FZD8. In some embodiments, the FZD binding domain is selective for one or more FZD proteins of interest, for example, having at least 10-fold, 25-fold, 50-fold, 100-fold, 200-fold or higher specificity for one or more desired FZD proteins compared to other FZD proteins. In some embodiments, any one of the one or more FZD binding domains of the Wnt substitute molecule is single-specific, binding to or specifically binding to only one of FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, or FZD10.

[0138] In some embodiments, one or more LRP5 / 6 binding regions of the Wnt substitute molecule bind to one or both of the LRP5 / 6. For convenience, the term "LRP5 / 6" is used to collectively refer to one or both of the LRP5 and / or LRP6.

[0139] Immunological binding generally refers to non-covalent interactions of a type that occur between an immunoglobulin molecule and an antigen to which the immunoglobulin is specific, for example, but not limited to, electrostatic, ionic, hydrophilic and / or hydrophobic attractive or repulsive forces, steric forces, hydrogen bonds, van der Waals forces, and other interactions. The strength or affinity of an immunological binding interaction is determined by the dissociation constant (K) of the interaction. d ) can be expressed in relation to K d A smaller value indicates greater affinity. The immunological binding properties of a selected polypeptide can be quantified using methods well known in the art. One such method involves measuring the rates of antigen-binding site / antigen complex formation and dissociation, whose rates depend on geometric parameters that equally affect the concentration of the complex partner, the affinity of the interaction, and the rates in both directions. Thus, the "on rate constant" (K) is used. on ) and "off-speed constant" (K off Both of these can be determined by calculating the concentrations as well as the actual rates of association and dissociation. off / K on The ratio allows for the cancellation of all parameters unrelated to affinity, and thus the dissociation constant K d It is equivalent to this. For general information, see Davies et al. (1990) Annual Rev. Biochem. 59:439-473.

[0140] In certain embodiments, the Wnt substitute molecules or their binding domains described herein have affinities of less than about 10,000 nM, less than about 1,000 nM, less than about 100 nM, less than about 10 nM, less than about 1 nM, or less than about 0.1 nM, and in some embodiments, the antibody may have even higher affinity for one or more FZD receptors or LRP5 receptors or LRP6 receptors.

[0141] The Wnt substitute molecules disclosed herein may also be modified to include an epitope tag or label for use, for example, in purification or diagnostic applications. There are many linking groups known in the art for making antibody conjugates, including, for example, those disclosed in U.S. Patent No. 5,208,020 or European Patent No. 0 425 235 B1, and Chari et al., Cancer Research 52: 127-131 (1992). Examples of linking groups include disulfide groups, thioether groups, acid-unstable groups, photosensitive groups, peptidase-unstable groups, or esterase-unstable groups, as disclosed in the patents specified above, with disulfide groups and thioether groups being preferred.

[0142] In certain embodiments, the anti-LRP5 / 6 antigen-binding fragment and / or its anti-FZD antigen-binding fragment located within the Wnt substitute molecule are monoclonal. In certain embodiments, they are humanized.

[0143] The present invention further provides isolated nucleic acids encoding polypeptides present in Wnt alternative molecules disclosed herein, in certain specific embodiments. Nucleic acids include DNA and RNA. These and related embodiments may include polynucleotides encoding antibody fragments that bind to one or more FZD receptors and / or LRP5 or LRP6, as described herein. As used herein, the term “isolated polynucleotide” means a polynucleotide of genomic origin, cDNA origin, or synthetic origin, or any combination thereof, and an isolated polynucleotide is, based on its origin, (1) an isolated polynucleotide does not involve all or some of the polynucleotides found in nature; (2) is linked to polynucleotides that are not linked in nature; or (3) does not exist in nature as part of a larger sequence. Isolated polynucleotides may include naturally occurring sequences and / or artificial sequences.

[0144] The term "operably ligated" means that the components to which this term applies are in a relationship that allows them to perform their intrinsic functions under favorable conditions. For example, a protein-coding sequence and an "operably ligated" transcriptional regulatory sequence are ligated to each other so that the expression of the protein-coding sequence is achieved under conditions compatible with the transcriptional activity of the regulatory sequence.

[0145] The term “regulatory sequence,” as used herein, refers to a polynucleotide sequence that can influence the expression, processing, or intracellular localization of a coding sequence to which it is ligated or operably linked. The nature of such a regulatory sequence may be host organism-dependent. In certain embodiments, a prokaryotic transcriptional regulatory sequence may include a promoter, a ribosome binding site, and a transcription termination sequence. In other specific embodiments, a eukaryotic transcriptional regulatory sequence may include a promoter containing one or more recognition sites of a transcription factor, a transcriptional enhancer sequence, a transcription termination sequence, and a polyadenylation sequence. In certain embodiments, the “regulatory sequence” may include a leader sequence and / or a fusion partner sequence.

[0146] Where used herein, the term "polynucleotide" refers to a single-stranded or double-stranded nucleic acid polymer. In certain embodiments, the nucleotides constituting the polynucleotide may be ribonucleotides or deoxyribonucleotides, or modified forms of either type of nucleotide. Such modifications include base modifications such as bromouridine, ribose modifications such as arabinosides and 2',3'-dideoxyribose, and internucleotide bond modifications such as phosphorothioates, phosphorodithioates, phosphoroselenoates, phosphorodiselenoates, phosphoranilothioates, phosphoranilideates, and phosphoramidates. Specifically, the term "polynucleotide" includes single-stranded and double-stranded forms of DNA.

[0147] The term "naturally occurring nucleotides" includes deoxyribonucleotides and ribonucleotides. The term "modified nucleotides" includes nucleotides having modified or substituted sugar groups, etc. The term "oligonucleotide linkage" includes oligonucleotide linkages such as phosphorothioates, phosphorodithioates, phosphoroselenoates, phosphorodiselenoates, phosphoranilothioates, phosphoranilideates, and phosphoramidates. For example, see LaPlanche et al., 1986, Nucl. Acids Res., 14:9081; Stec et al., 1984, J. Am. Chem. Soc., 106:6077; Stein et al., 1988, Nucl. Acids Res., 16:3209; Zon et al., 1991, Anti-Cancer Drug Design, 6:539; Zon et al., 1991, OLIGONUCLEOTIDES AND ANALOGUES: A PRACTICAL APPROACH, pp. 87-108 (F. Eckstein, Ed.), Oxford University Press, Oxford England; Stec et al., U.S. Patent No. 5,151,510; Uhlmann and Peyman, 1990, Chemical Reviews, 90:543. These disclosures are incorporated herein by reference for all purposes. Oligonucleotides may contain detectable labels to enable the detection of oligonucleotides or their hybridizations.

[0148] The term "vector" is used to refer to any molecule (e.g., nucleic acid, plasmid, or virus) used to introduce coding information into a host cell. The term "expression vector" refers to a vector containing a nucleic acid sequence that is suitable for transforming host cells and that directs and / or controls the expression of an inserted heterologous nucleic acid sequence. Expression includes, but is not limited to, processes such as transcription, translation, and RNA splicing, if introns are present.

[0149] As will be understood by those skilled in the art, polynucleotides may include genomic sequences, extragenomic sequences, and plasmid-coding sequences, as well as smaller, engineered gene segments, that express or can be adapted to express proteins, polypeptides, peptides, and the like. Such segments may be isolated naturally or may be synthetically modified by those skilled in the art.

[0150] As will also be apparent to those skilled in the art, polynucleotides can be single-stranded (coding or antisense) or double-stranded, and can be DNA molecules (genomic, cDNA, or synthetic) or RNA molecules. RNA molecules may include HnRNA molecules containing introns and corresponding one-to-one with DNA molecules, and mRNA molecules that do not contain introns. Additional coding or non-coding sequences may, but are not required, be present within the polynucleotides of this disclosure, and polynucleotides may, but are not required, be linked to other molecules and / or supporting materials. Polynucleotides may contain native sequences, or sequences that code for variants or derivatives of such sequences.

[0151] Those skilled in the art will understand that, as a result of the degeneracy of the genetic code, there are many nucleotide sequences that encode polypeptides, such as those described herein. Some of these polynucleotides have minimal sequence identity to the nucleotide sequences of the native or original polynucleotide sequences that encode polypeptides within the Wnt substitute molecules. Nevertheless, polynucleotides that vary due to differences in codon usage are expressly assumed in this disclosure. In certain embodiments, sequences that are codon-optimized for mammalian expression are specifically assumed.

[0152] Accordingly, in another embodiment of the present invention, a mutagenesis approach (e.g., site-directed mutagenesis) can be used for the preparation of variants and / or derivatives of the polypeptides described herein. This approach allows for specific modifications in the polypeptide sequence to be made through mutagenesis of the encoding base polynucleotide. These techniques provide a simple approach for preparing and testing sequence variants, incorporating one or more of the aforementioned considerations, for example, by introducing one or more nucleotide sequence changes into the polynucleotide.

[0153] Site-directed mutagenesis enables the production of mutants by providing a specific oligonucleotide sequence encoding the DNA sequence of the desired mutation, as well as a primer sequence of sufficient size and sequence complexity to form a stable double helix on both sides across the deletion junction using a sufficient number of adjacent nucleotides. By using the mutation on a selected polynucleotide sequence, it is possible to improve, alter, reduce, modify, or otherwise change the properties of the polynucleotide itself, and / or alter the properties, activity, composition, stability, or primary sequence of the encoded polypeptide.

[0154] In certain embodiments, the disclosure envisions the mutagenesis of a polynucleotide sequence encoding a polypeptide present in a Wnt substitute molecule to alter one or more properties of the encoded polypeptide, such as binding affinity or function. Site-directed mutagenesis techniques are well known in the art and are widely used to produce variants of both polypeptides and polynucleotides. For example, site-directed mutagenesis is often used to modify a specific portion of a DNA molecule. In such embodiments, primers typically containing about 14 to about 25 nucleotides in length are used, and about 5 to about 10 residues on either side of the junction of the sequence are modified.

[0155] As will be recognized by those skilled in the art, site-directed mutagenesis techniques often utilize phage vectors that exist in both single-stranded and double-stranded forms. Typical vectors useful for site-directed mutagenesis include M13 phages. These phages are readily available commercially, and their use is generally well known to those skilled in the art. Double-stranded plasmids are also conventionally used for site-directed mutagenesis, eliminating the step of transferring the target gene from the plasmid to the phage.

[0156] The preparation of sequence variants of DNA segments encoding selected peptides using site-directed mutagenesis provides a means of producing potentially useful species, but this does not mean it is limited, as other methods exist for obtaining peptide sequence variants and the DNA sequences encoding them. For example, sequence variants can be obtained by treating a recombinant vector encoding a desired peptide sequence with a mutagenic agent such as hydroxylamine. Specific details regarding these methods and protocols can be found in the teachings of Maloy et al., 1994; Segal, 1976; Prokop and Bajpai, 1991; Kuby, 1994; and Maniatis et al., 1982, which are each incorporated herein by reference for their respective purposes.

[0157] In many embodiments, one or more nucleic acids encoding the polypeptide of a Wnt-alternative polypeptide are directly introduced into host cells, and the cells are incubated under conditions sufficient to induce expression of the encoded polypeptide. The Wnt-alternative polypeptides of this disclosure can be prepared using standard techniques well known to those skilled in the art in combination with the polypeptides and nucleic acid sequences provided herein. The polypeptide sequences can be used to determine suitable nucleic acid sequences encoding a particular polypeptide disclosed herein. The nucleic acid sequences can be optimized to reflect a particular codon “priority” for various expression systems according to standard methods well known to those skilled in the art.

[0158] According to certain relevant embodiments, a recombinant host cell comprising one or more constructs described herein, for example, a vector comprising a nucleic acid encoding a Wnt mimetic or its polypeptide; and a method for producing an encoded product, comprising expression from the nucleic acid encoding it. Expression can be successfully achieved by culturing the recombinant host cell containing the nucleic acid under appropriate conditions. After production by expression, the antibody or its antigen-binding fragment can be isolated and / or purified using any preferred technique and subsequently used as desired.

[0159] Polypeptides, as well as the nucleic acid molecules and vectors encoding them, can be isolated and / or purified, for example, from their natural environment in a substantially pure or homogeneous form, or, in the case of nucleic acids, without or substantially without nucleic acids or genes of origin other than the sequence encoding the polypeptide having the desired function. Nucleic acids may include DNA or RNA, and may be entirely or partially synthesized. References to nucleotide sequences described herein include DNA molecules having the given sequence and RNA molecules having the given sequence in which T is substituted with U, unless the context requires otherwise.

[0160] Systems for the cloning and expression of polypeptides in various different host cells are well known. Suitable host cells include bacteria, mammalian cells, yeast, and baculovirus systems. Mammalian cell lines available in the art for heterologous polypeptide expression include HEK293 cells, Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells, NSO mouse melanoma cells, and many others. Prokaryotic expression includes, but is not limited to, the bacterial host E. coli.

[0161] For example, the expression of polypeptides and their antigen-binding fragments in prokaryotic cells such as E. coli is well established in the art. For a review, see, for example, Pluckthun, A. Bio / Technology 9: 545-551 (1991). Expression in eukaryotic cells under culture is also available to those skilled in the art as an option for the production of antibodies or their antigen-binding fragments. For recent reviews, see, for example, Ref, ME (1993) Curr. Opinion Biotech. 4: 573-576; Trill JJ et al. (1995) Curr. Opinion Biotech 6: 553-560.

[0162] Suitable vectors containing appropriate regulatory sequences, including, as appropriate, promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes, and other sequences, can be selected or constructed. The vector may, as appropriate, be a plasmid, virus, e.g., phage, or phagemid. For further details, see, for example, Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrook et al., 1989, Cold Spring Harbor Laboratory Press. Many known techniques and protocols for handling nucleic acids in the preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and protein analysis are described in detail in Current Protocols in Molecular Biology, Second Edition, Ausubel et al. eds., John Wiley & Sons, 1992, or subsequent revisions.

[0163] The term “host cell” is used to refer to a cell into which a nucleic acid sequence encoding one or more polypeptides described herein has been introduced, or can be introduced, and which further expresses, or is capable of expressing, a selected gene of interest, e.g., a gene encoding any polypeptide described herein. The term includes offspring of parental cells, regardless of whether the offspring are identical to the original parent in morphology or genetic makeup, as long as the selected gene is present. Methods involving the introduction of such nucleic acids into host cells are also conceivable. Any available technique can be used for introduction. For eukaryotic cells, preferred techniques include calcium phosphate transfection, DEAE-dextran, electroporation, liposome-mediated transfection, and transduction using retroviruses or other viruses, e.g., vaccinia, or, in the case of insect cells, baculoviruses. For bacterial cells, preferred techniques include calcium chloride transformation, electroporation, and transfection using bacteriophages. Following introduction, expression from the nucleic acid can be induced or enabled, for example, by culturing the host cell under conditions for gene expression. In one embodiment, the nucleic acid is incorporated into the genome (e.g., chromosomes) of the host cell. Integration can be facilitated by including sequences that promote recombination with the genome, according to standard techniques.

[0164] The present invention also provides, in certain embodiments, a method comprising using the constructs described above in an expression system to express a specific polypeptide, such as a Wnt substitute molecule as described herein. The term “transduction” is typically used to refer to the transfer of genes from one bacterium to another by a phage. “Transduction” also refers to the acquisition and transfer of eukaryotic cell sequences by retroviruses. The term “transfection” is used to refer to the uptake of exogenous or extrinsic DNA by a cell, and a cell is “transfected” when the extrinsic DNA is introduced inside the cell membrane. Several transfection techniques are well known in the art and are disclosed herein. See, for example, Graham et al., 1973, Virology 52:456; Sambrook et al., 2001, MOLECULAR CLONING, A LABORATORY MANUAL, Cold Spring Harbor Laboratories; Davis et al., 1986, BASIC METHODS IN MOLECULAR BIOLOGY, Elsevier; and Chu et al., 1981, Gene 13:197. Such techniques can be used to introduce one or more exogenous DNA segments into suitable host cells.

[0165] As used herein, the term “transformation” refers to a change in the genetic characteristics of a cell, and a cell is transformed if it has been modified to contain new DNA. For example, a cell is transformed if it has been genetically modified from its native state. After transfection or transduction, the transformed DNA may be recombined with the cell’s DNA by being physically incorporated into the cell’s chromosomes, or it may be transiently maintained as an episomal element without replication, or it may be independently replicated as a plasmid. A cell is considered stably transformed if the DNA is replicated with cell division. The terms “naturally occurring” or “synthetic” as used herein refer to biomaterials, such as nucleic acid molecules, polypeptides, and host cells, that are found in nature and have not been manipulated by humans. Similarly, “not naturally occurring” or “unnatural” as used herein refer to materials that are not found in nature or have been structurally modified or synthesized by humans.

[0166] The terms “polypeptide,” “protein,” “peptide,” and “glycoprotein” are used interchangeably and refer to polymers of amino acids, not limited to any particular length. These terms do not exclude modifications such as myristylation, sulfation, glycosylation, phosphorylation, and addition or deletion of signal sequences. The term “polypeptide” or “protein” refers to one or more chains of amino acids, each chain comprising amino acids covalently linked by peptide bonds, and such polypeptide or protein may include multiple chains non-covalently and / or covalently linked by peptide bonds, having the sequence of a natural protein, i.e., a protein naturally occurring in cells, particularly non-recombinant cells, or produced by genetically engineered or recombinant cells; it may include molecules having the amino acid sequence of a natural protein, or molecules having deletions, additions, and / or substitutions of one or more amino acids of the natural sequence. The terms “polypeptide” and “protein” specifically include Wnt substitute molecules, their FZD binding domains, their LRP5 / 6 binding domains, antibodies that bind to the FZD receptor or LRP5 or LRP6 receptor as disclosed herein, and their antigen-binding fragments, or sequences having one or more amino acid deletions, additions, and / or substitutions of any of these polypeptides. Thus, a “polypeptide” or “protein” may include one (referred to as a “monomer”) or multiple (referred to as “multimer”) amino acid chains.

[0167] The terms “isolated protein,” “isolated Wnt substitute polypeptide,” or “isolated polypeptide” as used herein mean that the subject protein, a Wnt mimetic, (1) does not contain at least some other proteins typically found in nature; (2) essentially does not contain other proteins from the same source, e.g., the same species; (3) is expressed by cells of a different species; (4) is isolated from at least about 50 percent of the polynucleotides, lipids, carbohydrates or other material found in nature; (5) does not involve (by covalent or non-covalent interactions) any portion of the protein that is found in nature with the “isolated protein”; (6) is operably accompanied (by covalent or non-covalent interactions) a polypeptide not found in nature; or (7) is not naturally occurring. Such isolated proteins may be encoded by genomic DNA, cDNA, mRNA, or other RNA, or may be of synthetic origin, or any combination thereof. In certain embodiments, isolated proteins may contain naturally occurring and / or artificial polypeptide sequences. In certain embodiments, the isolated protein is substantially free from proteins or polypeptides or other contaminants found in its natural environment that would be considered to interfere with its use (for therapeutic, diagnostic, preventive, research, or otherwise).

[0168] Modification of the amino acid sequence of any of the polypeptides described herein (e.g., Wnt substitute polypeptides or their FZD binding region or LRP5 / 6 binding region) is envisioned. For example, it may be desirable to improve the binding affinity and / or other biological properties of the Wnt substitute molecule. For example, amino acid sequence variants of Wnt mimetic molecules can be prepared by introducing appropriate nucleotide changes into the antibody or the polynucleotide encoding its chain, or by peptide synthesis. Such modifications include, for example, deletions from and / or insertions of residues within the amino acid sequence of the antibody, and / or substitutions thereof. Any combination of deletions, insertions, and substitutions can be performed to arrive at the final Wnt substitute molecule, provided that the final construct possesses the desired characteristics (e.g., high affinity binding to one or more FZD and LRP5 / 6 receptors). Amino acid changes may also alter the post-translational process of the antibody, for example, by changing the number or position of glycosylation sites. Any of the above variations and modifications of the polypeptides of the present invention can be incorporated into the antibodies of the present invention.

[0169] This disclosure provides variants of any of the polypeptides disclosed herein (e.g., Wnt substitute molecules or their FZD-binding domain or LRP5 / 6-binding domain, or antibodies or their antigen-binding fragments). In certain embodiments, the variants have at least 90%, at least 95%, at least 98%, or at least 99% identity with the polypeptides disclosed herein. In certain embodiments, such variant polypeptides bind to one or more FZD receptors and / or one or more LRP5 / 6 receptors to a degree of at least about 50%, at least about 70%, and in certain embodiments at least about 90%, similar to the Wnt substitute molecules specifically described herein. In further embodiments, such variant Wnt surrogate molecules bind to one or more FZD receptors and / or one or more LRP5 / 6 receptors with higher affinity than the Wnt surrogate molecules described herein, quantitatively binding to, for example, at least about 105%, 106%, 107%, 108%, 109%, or 110%, comparable to the antibody sequences specifically described herein.

[0170] In certain embodiments, the Wnt substitute molecule or its binding region, e.g., Fab, scFv, or VHH, may include a) a heavy chain variable region comprising: ai. a CDR1 region having the same amino acid sequence as the heavy chain CDR1 region of the selected antibody described herein; ii. a CDR2 region having the same amino acid sequence as the heavy chain CDR2 region of the selected antibody; and iii. a CDR3 region having the same amino acid sequence as the heavy chain CDR3 region of the selected antibody; and / or b) a light chain variable domain comprising: ai. a CDR1 region having the same amino acid sequence as the light chain CDR1 region of the selected antibody; ii. a CDR2 region having the same amino acid sequence as the light chain CDR2 region of the selected antibody; and iii. a CDR3 region having the same amino acid sequence as the light chain CDR3 region of the selected antibody, wherein the antibody specifically binds to a selected target (e.g., one or more FZD receptors or LRP5 receptors or LRP6 receptors). In a further embodiment, the polypeptide or antigen-binding fragment is a variant antigen-binding fragment, the variant comprising a heavy chain and a light chain identical to the selected antibody except for up to one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen or more amino acid substitutions in the CDR regions of the VH and VL regions. In a further embodiment, the polypeptide or antigen-binding fragment is a variant having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the polypeptide or its antigen-binding fragment, and comprising up to one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen or more amino acid substitutions in the CDR regions of the VH and / or VL regions. In this regard, the CDR region of the selected antibody may contain one, two, three, four, five, six, seven, eight, or, in certain embodiments, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, or more amino acid substitutions. The substitutions may be within the CDR in either the VH and / or VL region. (See, for example, Muller, 1998, Structure 6:1153-1167).

[0171] In certain embodiments, the Wnt substitute molecule or its binding region, e.g., Fab, scFv, or VHH, may have: a) a heavy chain variable region having an amino acid sequence that is at least 80%, at least 95%, at least 90%, at least 95%, or at least 98% or 99% identical to the heavy chain variable region of the antibody or antigen-binding fragment described herein; and / or b) a light chain variable region having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% or 99% identical to the light chain variable region of the antibody or antigen-binding fragment described herein. In certain embodiments, the light chain variable region and / or the heavy chain variable region each independently comprises at least one, at least two, or all three CDR sequences of the light chain variable region or heavy chain variable region disclosed herein. In certain embodiments, the light chain variable region and / or heavy chain variable region each independently contain 0, less than 2, less than 3, less than 4, less than 5, less than 6, less than 7, or less than 8 amino acid substitutions in the CDR. In certain embodiments, the Wnt agonist contains 0, less than 2, less than 3, less than 4, less than 5, less than 6, less than 7, less than 8, less than 9, less than 10, less than 11, or less than 12 amino acid substitutions in the CDR.

[0172] A polypeptide has a certain percentage of "sequence identity" with another polypeptide, which means the percentage of amino acids that are the same when the two sequences are aligned and compared. Sequence similarity can be determined in several different ways. To determine sequence identity, sequences can be aligned using methods and computer programs, including BLAST, available on the World Wide Web at ncbi.nlm.nih.gov / BLAST / . Another alignment algorithm is FASTA, available in the Genetics Computing Group (GCG) package from Madison, Wisconsin, USA, a wholly owned subsidiary of Oxford Molecular Group, Inc. Other techniques for alignment are described in Methods in Enzymology, vol. 266: Computer Methods for Macromolecular Sequence Analysis (1996), ed. Doolittle, Academic Press, Inc., a division of Harcourt Brace & Co., San Diego, Calif., USA. Of particular interest are alignment programs that allow gaps in sequences. Smith-Waterman is one type of algorithm that allows gaps in sequence alignment. See Meth. Mol. Biol. 70: 173-187 (1997). Sequence alignment can also be performed using a GAP program that employs the Needleman and Wunsch alignment method. See J. Mol. Biol. 48: 443-453 (1970).

[0173] Of interest is the BestFit program, which uses the local homology algorithm of Smith and Waterman (Advances in Applied Mathematics 2: 482-489 (1981)) to determine sequence identity. The gap generation penalty is generally in the range of 1 to 5, usually 2 to 4, and 3 in many embodiments. The gap expansion penalty is generally in the range of about 0.01 to 0.20, and often 0.10. The program has default parameters determined by the input sequences to be compared. Preferably, sequence identity is determined using the default parameters determined by the program. This program is also available from the Genetics Computing Group (GCG) package in Madison, Wisconsin, USA.

[0174] Another program of interest is the FastDB algorithm. FastDB is described in *Current Methods in Sequence Comparison and Analysis, Macromolecule Sequencing and Synthesis, Selected Methods and Applications*, pp. 127-149, 1988, Alan R. Liss, Inc. The sequence identity percentage is calculated by FastDB based on the following parameters: Mismatch penalty: 1.00; Gap penalty: 1.00; Gap size penalty: 0.33; and Joint penalty: 30.0.

[0175] The determination of the three-dimensional structure of a representative polypeptide (e.g., a variant FZD binding domain or LRP5 / 6 binding domain of a Wnt substitute molecule provided herein) can be carried out by conventional methodologies, which allow for the virtual modeling of substitutions, additions, deletions, or insertions of one or more amino acids by selected native or non-native amino acids, for the purpose of determining whether the thus derived structural variant retains the space-filling properties of the species disclosed herein. For example, Donate et al., 1994 Prot. Sci. 3:2378;Bradley et al., Science 309: 1868-1871 (2005);Schueler-Furman et al., Science 310:638 (2005);Dietz et al., Proc. Nat. Acad. Sci. USA 103:1244 (2006); Dodson et al., Nature 450:176 (2007); Qian et al., Nature 450:259 (2007); Raman et al. Science 327:1014-1018 (2010). For these embodiments and related embodiments, some further non-limiting examples of computer algorithms that can be used, for example, for the rational design of binding regions include VMD, a molecular visualization program that uses 3-D graphics and built-in scripting to display, animate, and analyze large biomolecular systems (see the website of Theoretical and Computational Biophysics Group, University of Illinois at Urbana-Champagne, ks.uiuc.edu / Research / vmd / ).Many other computer programs are known in the art and available to those skilled in the art that enable the determination of atomic dimensions (van der Waals radii) from space-filling models of energy-minimizing conformations; GRID for determining regions of high affinity for different chemical groups and thereby attempting to strengthen bonds; Monte Carlo search for calculating mathematical alignments; and CHARMM (Brooks et al. (1983) J. Comput. Chem. 4:187-217) and AMBER (Weiner et al. (1981) J. Comput. Chem. 106:765) (Eisenfield et al. (1991) Am. J. Physiol. 261:C376-386; Lybrand (1991) J. Pharm. Belg. 46:49-54; Froimowitz (1990) Biotechniques 8:640-644; Burbam et al. (See also (1990) Proteins 7:99-111; Pedersen (1985) Environ. Health Perspect. 61:185-190; and Kini et al. (1991) J. Biomol. Struct. Dyn. 9:475-488). Various suitable computer programs for calculation are also commercially available, for example, from Schroedinger (Munich, Germany).

[0176] composition Pharmaceutical compositions comprising a Wnt substitute molecule described herein and one or more pharmaceutically acceptable diluents, carriers, or additives are also disclosed. In certain embodiments, the pharmaceutical composition further comprises one or more WNT polypeptides or Norrin polypeptides.

[0177] In further embodiments, pharmaceutical compositions are also disclosed comprising a polynucleotide containing a nucleic acid sequence encoding a Wnt substitute molecule as described herein, and one or more pharmaceutically acceptable diluents, carriers, or additives. In certain embodiments, the pharmaceutical composition further comprises one or more polynucleotides containing a nucleic acid sequence encoding a WNT polypeptide or a Norrin polypeptide. In certain embodiments, the polynucleotide is DNA or mRNA, e.g., modified mRNA. In certain embodiments, the polynucleotide is modified mRNA further comprising a 5' cap sequence and / or a 3' tailing sequence, e.g., a poly-A tail. In other embodiments, the polynucleotide is an expression cassette comprising a promoter operably ligated to the coding sequence. In certain embodiments, the nucleic acid sequence encoding a Wnt mimetic and the nucleic acid sequence encoding a WNT polypeptide or a Norrin polypeptide are present in the same polynucleotide.

[0178] In further embodiments, a pharmaceutical composition is also disclosed comprising an expression vector, e.g., a viral vector, comprising a polynucleotide containing a nucleic acid sequence encoding a Wnt substitute molecule described herein, and one or more pharmaceutically acceptable diluents, carriers, or additives. In certain embodiments, the pharmaceutical composition further comprises an expression vector, e.g., a viral vector, comprising a polynucleotide containing a nucleic acid sequence encoding a WNT polypeptide or a Norrin polypeptide. In certain embodiments, the nucleic acid sequence encoding the Wnt mimetic and the nucleic acid sequence encoding the WNT polypeptide or Norrin polypeptide are present in the same polynucleotide, e.g., an expression cassette.

[0179] The present invention further envisions a pharmaceutical composition comprising a cell comprising an expression vector comprising a polynucleotide containing a promoter operably linked to a nucleic acid encoding a Wnt substitute molecule, and one or more pharmaceutically acceptable diluents, carriers, or additives. In certain embodiments, the pharmaceutical composition further comprises a cell comprising an expression vector comprising a polynucleotide containing a promoter operably linked to a nucleic acid sequence encoding a WNT polypeptide or a Norrin polypeptide. In certain embodiments, the nucleic acid sequence encoding the Wnt mimetic and the nucleic acid sequence encoding the WNT polypeptide or Norrin polypeptide are present in the same polynucleotide, e.g., in the expression cassette and / or within the same cell. In certain embodiments, the cell is a heterogeneous cell or an autologous cell obtained from a subject to be treated. In certain embodiments, the cell is a stem cell, e.g., adipose-derived stem cell or hematopoietic stem cell.

[0180] This disclosure envisions a pharmaceutical composition comprising a first molecule for the delivery of a Wnt substitute molecule as a first activator, and a second molecule for the delivery of a WNT polypeptide or a Norrin polypeptide. The first and second molecules may be of the same type or different types. For example, in certain embodiments, the first and second molecules may be independently selected from the following types of molecules: polypeptides, small organic molecules, nucleic acids encoding the first or second activator (DNA or mRNA as needed, modified RNA as needed), vectors containing nucleic acid sequences encoding the first or second activator (expression vectors or viral vectors as needed), and cells, eukaryotic cells or prokaryotic cells containing nucleic acid sequences encoding the first or second activator (expression cassettes as needed).

[0181] The subject molecules can be used alone or in combination with pharmaceutically acceptable carriers, diluents, additives, and reagents that are generally safe, non-toxic, and useful for preparing desired formulations, including additives that are acceptable for use in mammals, e.g., humans or primates. Such additives may be solid, liquid, semi-solid, or, in the case of aerosol compositions, gas. Examples of such carriers, diluents, and additives include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Auxiliary active compounds may also be incorporated into the formulations. Solutions or suspensions used in the formulation include sterile diluents, such as water for injection, saline solution, fixing oil, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antimicrobial compounds, such as benzyl alcohol or methylparaben; antioxidants, such as ascorbic acid or sodium bisulfite; chelate compounds, such as ethylenediaminetetraacetic acid (EDTA); buffers, such as acetate buffer, citrate buffer, or phosphate buffer; surfactants to prevent aggregation, such as Tween® 20; and compounds to adjust tonicity, such as sodium chloride or dextrose. pH can be adjusted using an acid or base such as hydrochloric acid or sodium hydroxide. In certain embodiments, the pharmaceutical composition is sterile.

[0182] The pharmaceutical composition may further comprise a sterile aqueous solution or dispersion, and a sterile powder for the immediate preparation of a sterile injectable solution or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, or phosphate-buffered saline (PBS). In some cases, the composition should be sterile and fluid so that it can be drawn into a syringe or delivered from a syringe to a target. In certain embodiments, the composition is stable under manufacturing and storage conditions and preserved against microbial contamination such as bacteria and fungi. The carrier may be, for example, a solvent or dispersion medium containing water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of a dispersion, and by the use of surfactants. Prevention of microbial activity can be achieved by various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it is preferable to include isotonic agents, such as sugars, polyalcohols like mannitol and sorbitol, and sodium chloride in the composition. By including absorption-delaying agents, such as aluminum monostearate and gelatin, in the composition, sustained absorption of the internal composition can be achieved.

[0183] Sterile solutions can be prepared by incorporating the required amount of Wnt substitute molecules or their antigen-binding fragments (or encoding polynucleotides or cells containing them) into a suitable solvent, along with one or a combination of the components listed above, as needed, followed by sterilization by filtration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other required components listed above. In the case of sterile powders for the preparation of sterile injection solutions, the preparation methods are vacuum drying and lyophilization, thereby obtaining powders of the active ingredient and any additional desired components from the pre-sterilized filtered solution.

[0184] In one embodiment, the pharmaceutical composition is prepared using a controlled-release formulation that includes a carrier, such as an implant and a microencapsulation delivery system, which protects the antibody or its antigen-binding fragment from rapid removal from the body. Biodegradable, biocompatible polymers, such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid, can be used. Methods for preparing such formulations will be apparent to those skilled in the art. The materials can also be commercially available. Liposome suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art.

[0185] For ease of administration and uniformity of dosage, it may be advantageous to formulate pharmaceutical compositions in dosage unit forms. When used herein, a dosage unit form refers to a physically distinct unit suitable as a unit dose for the subject to be treated, each unit containing a predetermined amount of an active antibody or its antigen-binding fragment, calculated to produce the desired therapeutic effect, along with the required pharmaceutical carrier. The specifications of a dosage unit form are determined and directly depend upon the characteristics specific to the antibody or its antigen-binding fragment, the particular therapeutic effect to be achieved, and the constraints inherent in the art for which such an active antibody or its antigen-binding fragment is formulated for the treatment of an individual.

[0186] The pharmaceutical composition may be included in a container, pack, or dispenser, such as a syringe, such as a pre-filled syringe, along with instructions for administration.

[0187] The pharmaceutical compositions of the present invention include any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compounds that can (directly or indirectly) provide a biologically active antibody or an antigen-binding fragment thereof upon administration to an animal, including a human.

[0188] The present invention comprises pharmaceutically acceptable salts of the Wnt substitute molecules described herein. The term “pharmaceutically acceptable salt” means a physiologically and pharmaceutically acceptable salt of the compound of the present invention, i.e., a salt that retains the desired biological activity of the parent compound and does not impart any undesirable toxicological effects to it. Various pharmaceutically acceptable salts are known in the art and are described, for example, in “Remington's Pharmaceutical Sciences”, 17th edition, Alfonso R. Gennaro (Ed.), Mark Publishing Company, Easton, PA, USA, 1985 (and more recent editions), “Encyclopaedia of Pharmaceutical Technology”, 3rd edition, James Swarbrick (Ed.), Informa Healthcare USA (Inc.), NY, USA, 2007, and J. Pharm. Sci. 66: 2 (1977). For a review of suitable salts, see also "Handbook of Pharmaceutical Salts: Properties, Selection, and Use," Stahl and Wermuth (Wiley-VCH, 2002).

[0189] Pharmaceutically acceptable base addition salts are formed using metals or amines, such as alkali and alkaline earth metals, or organic amines. Metals used as cations include sodium, potassium, magnesium, and calcium. Amines include N-N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine (see, e.g., Berge et al., "Pharmaceutical Salts," J. Pharma Sci., 1977, 66, 119). The base addition salts of the acidic compounds are prepared in the conventional manner by contacting the free acid form with a sufficient amount of the desired base to produce the salt. The free acid form can be regenerated in the conventional manner by contacting the salt form with the acid to isolate the free acid. The free acid forms differ somewhat from their respective salt forms in certain physical properties, such as solubility in polar solvents, but in other respects, for the purposes of this invention, the salts are equivalent to their respective free acids.

[0190] In some embodiments, the pharmaceutical compositions provided herein comprise a therapeutically effective amount of a Wnt substitute molecule or a pharmaceutically acceptable salt thereof, mixed with a pharmaceutically acceptable carrier, diluent and / or additive, such as saline, phosphate-buffered saline, phosphates and amino acids, polymers, polyols, sugars, buffers, preservatives and other proteins. Exemplary amino acids, polymers and sugars include octylphenoxypolyethoxyethanol compounds, polyethylene glycol monostearate compounds, polyoxyethylene sorbitan fatty acid esters, sucrose, fructose, dextrose, maltose, glucose, mannitol, dextran, sorbitol, inositol, galactitol, xylitol, lactose, trehalose, bovine or human serum albumin, citrates, acetates, Ringer's solution and Hanks' solution, cysteine, arginine, carnitine, alanine, glycine, lysine, valine, leucine, polyvinylpyrrolidone, polyethylene and glycol. Preferably, the formulation is stable at 4°C for at least 6 months.

[0191] In some embodiments, the pharmaceutical compositions provided herein include buffers, such as phosphate-buffered saline (PBS) or sodium phosphate / sodium sulfate, Tris buffer, glycine buffer, sterile water, and other buffers known to those skilled in the art, such as those described by Good et al. (1966) Biochemistry 5:467. The pH of the buffer may be in the range of 6.5 to 7.75, preferably 7 to 7.5, and most preferably 7.2 to 7.4.

[0192] How to use This disclosure also provides methods for using the Wnt substitute molecules disclosed herein, for example, to modulate the Wnt signaling pathway, for example, to increase Wnt signaling, and administration of the Wnt mimetic disclosed herein in various therapeutic settings. This specification provides methods for treatment using the Wnt mimetic. In one embodiment, the Wnt mimetic is provided to subjects having a disease involving inappropriate or deregulated Wnt signaling, for example, reduced Wnt signaling.

[0193] Increasing Wnt pathway signaling and related therapeutic methods In certain embodiments, Wnt substitute molecules can be used to increase Wnt signaling in tissues or cells. Accordingly, in some embodiments, the present invention provides a method for increasing or enhancing Wnt signaling in tissues or cells, comprising contacting the tissue or cells with an effective amount of a Wnt mimetic disclosed herein or a pharmaceutically acceptable salt thereof, wherein the Wnt substitute molecule is a Wnt signaling pathway agonist. In some embodiments, the contact is performed in vitro, ex vivo, or in vivo. In certain embodiments, the cells are cultured cells, and the contact is performed in vitro. In certain embodiments, the method further comprises contacting the tissue or cells with one or more WNT polypeptides or Norrin polypeptides.

[0194] In related embodiments, the present invention provides a method for increasing Wnt signaling in a tissue or cell, comprising contacting the tissue or cell with a polynucleotide comprising an effective amount of a Wnt substitute molecule disclosed herein. In certain embodiments, the target tissue or cell is also contacted with a polynucleotide comprising a nucleic acid sequence encoding a WNT polypeptide or a Norrin polypeptide. In certain embodiments, the polynucleotide is DNA or mRNA, e.g., modified mRNA. In certain embodiments, the polynucleotide is modified mRNA further comprising a 5' cap sequence and / or a 3' tailing sequence, e.g., a poly-A tail. In other embodiments, the polynucleotide is an expression cassette comprising a promoter operably ligated to the coding sequence. In certain embodiments, the nucleic acid sequence encoding a Wnt mimetic and the nucleic acid sequence encoding a WNT polypeptide or a Norrin polypeptide are present in the same polynucleotide.

[0195] In related embodiments, the present invention provides a method for increasing Wnt signaling in a tissue or cell, comprising contacting the tissue or cell with a vector containing an effective amount of a nucleic acid sequence encoding a Wnt substitute molecule. In certain embodiments, the tissue or cell is also contacted with a vector containing a nucleic acid sequence encoding a WNT polypeptide or a Norrin polypeptide. In certain embodiments, the vector is an expression vector and may include a promoter operably linked to the nucleic acid sequence. In certain embodiments, the vector is a viral vector. In certain embodiments, the nucleic acid sequence encoding the Wnt substitute molecule and the nucleic acid sequence encoding the WNT polypeptide or Norrin polypeptide are present in the same vector, for example, in the same expression cassette.

[0196] In related embodiments, the present invention provides a method for increasing Wnt signaling in a tissue, comprising contacting the tissue with cells containing an effective amount of a nucleic acid sequence encoding a Wnt substitute molecule of the present invention. In certain embodiments, the tissue is also contacted with cells containing a nucleic acid sequence encoding a WNT polypeptide or a Norrin polypeptide. In certain embodiments, the nucleic acid sequence encoding the Wnt mimetic and the nucleic acid sequence encoding the WNT polypeptide or Norrin polypeptide are present in the same cell. In certain embodiments, the cells are heterologous cells or autologous cells obtained from the subject being treated. In certain embodiments, the cells have been transduced with a vector containing an expression cassette encoding the Wnt mimetic or a WNT polypeptide or a Norrin polypeptide. In certain embodiments, the cells are stem cells, e.g., adipose-derived stem cells or hematopoietic stem cells.

[0197] The Wnt substitute molecules disclosed herein can be used to treat diseases, disorders, or conditions, for example, by increasing Wnt signaling in targeted cells, tissues, or organs. Accordingly, in some embodiments, the present invention provides a method for treating a disease or condition in a subject requiring it, for example, a disease or disorder associated with decreased Wnt signaling or in which increased Wnt signaling provides therapeutic benefits, comprising contacting the subject with an effective amount of the composition of this disclosure. In certain embodiments, the composition is a pharmaceutical composition comprising any of the following: a Wnt substitute molecule; a nucleic acid sequence encoding the Wnt substitute molecule, for example, a polynucleotide including DNA or mRNA, optionally modified mRNA; a vector containing a nucleic acid sequence encoding the Wnt substitute molecule, for example, an expression vector or viral vector; or a cell containing a nucleic acid sequence encoding the Wnt substitute molecule, for example, a cell transduced with an expression vector or viral vector encoding the Wnt substitute molecule. In certain embodiments, the disease or condition is a pathological disease or disorder, or an injury, for example, an injury resulting from a wound. In certain embodiments, the wound may be the result of another therapeutic treatment. In certain embodiments, the disease or condition involves impaired function of tissue repair, healing, or regeneration, or would benefit from increased tissue repair, healing, or regeneration. In some embodiments, contact is performed in vivo, i.e., the subject composition is administered to the subject.

[0198] In certain embodiments, the method comprises further contacting the subject with a pharmaceutical composition comprising one or more WNT polypeptides or Norrin polypeptides. The disclosure assumes that the subject is contacted with a first molecule for the delivery of a Wnt substitute molecule as a first activator, and a second molecule for the delivery of a WNT polypeptide or Norrin polypeptide. The first and second molecules may be of the same type or different types. For example, in certain embodiments, the first and second molecules may be independently selected from the following types of molecules: polypeptides, small organic molecules, nucleic acids encoding the first or second activator (DNA or mRNA as needed, modified RNA as needed), vectors containing nucleic acid sequences encoding the first or second activator (expression vectors or viral vectors as needed), and cells containing nucleic acid sequences encoding the first or second activator (expression cassettes as needed).

[0199] In related embodiments, the present invention provides a method for treating a disease or condition, for example, a disease or disorder associated with decreased Wnt signaling or in which increased Wnt signaling provides therapeutic benefits, comprising contacting a subject in need with a pharmaceutical composition comprising a polynucleotide comprising a nucleic acid sequence encoding a Wnt substitute molecule disclosed herein. In certain embodiments, the subject is also contacted with a pharmaceutical composition comprising a polynucleotide comprising a nucleic acid sequence encoding a WNT polypeptide or Norrin polypeptide in an effective amount. In certain embodiments, the polynucleotide is DNA or mRNA, for example, modified mRNA. In certain embodiments, the polynucleotide is modified mRNA further comprising a 5' cap sequence and / or a 3' tailing sequence, for example, a poly-A tail. In other embodiments, the polynucleotide is an expression cassette comprising a promoter operably ligated to the coding sequence. In certain embodiments, the nucleic acid sequence encoding the Wnt substitute molecule and the nucleic acid sequence encoding the WNT polypeptide or Norrin polypeptide are present in the same polynucleotide.

[0200] In related embodiments, the present invention provides a method for treating a disease or condition, for example, a disease or disorder associated with reduced Wnt signaling or in which increased Wnt signaling provides therapeutic benefits, comprising contacting a subject in need with a pharmaceutical composition comprising a vector comprising a nucleic acid sequence encoding an effective amount of a Wnt substitute molecule. In certain embodiments, the subject is also contacted with a pharmaceutical composition comprising a vector comprising a nucleic acid sequence encoding an effective amount of a WNT polypeptide or a Norrin polypeptide. In certain embodiments, the vector is an expression vector and may include a promoter operably linked to the nucleic acid sequence. In certain embodiments, the vector is a viral vector. In certain embodiments, the nucleic acid sequence encoding the Wnt mimetic and the nucleic acid sequence encoding the WNT polypeptide or Norrin polypeptide are present in the same vector, for example, in the same expression cassette.

[0201] In a relevant embodiment, the present invention provides a method for treating a disease or condition, for example, a disease or disorder associated with decreased Wnt signaling or in which increased Wnt signaling provides therapeutic benefits, comprising contacting a subject in need of such treatment with a pharmaceutical composition comprising cells comprising a nucleic acid sequence encoding an effective amount of a Wnt substitute molecule. In a particular embodiment, the subject is also contacted with cells comprising a nucleic acid sequence encoding a WNT polypeptide or a Norrin polypeptide. In a particular embodiment, the nucleic acid sequence encoding the Wnt mimetic and the nucleic acid sequence encoding the WNT polypeptide or Norrin polypeptide are present in the same cell. In a particular embodiment, the cells are heterologous cells or autologous cells obtained from the subject to be treated. In a particular embodiment, the cells have been transduced with a vector comprising an expression cassette encoding a Wnt mimetic or a WNT polypeptide or a Norrin polypeptide. In a particular embodiment, the cells are stem cells, for example, adipose-derived stem cells or hematopoietic stem cells.

[0202] Wnt signaling plays a crucial role in the developmental processes and maintenance of stem cells. Reactivation of Wnt signaling is associated with the regeneration and repair of most tissues after injury and disease. Wnt surrogate molecules are expected to provide healing and tissue repair benefits in response to injury and disease. Causes of tissue damage and loss include, but are not limited to, aging, degeneration, genetic conditions, infections and inflammation, traumatic injury, toxin / metabolism-induced toxicity, or other pathological conditions. Wnt signaling and Wnt signaling enhancers have been shown to activate adult tissue-resident stem cells. In some embodiments, the compounds of the present invention are administered for use in the treatment of affected or damaged tissue, for use in tissue regeneration, and for use in cell growth and proliferation, and / or for use in tissue manipulation.

[0203] Human diseases associated with mutations in the Wnt pathway provide strong evidence for enhancing Wnt signaling in the treatment and prevention of these diseases. Preclinical in vivo and in vitro studies have provided further evidence of the involvement of Wnt signaling in many disease states, further supporting the use of Wnt surrogate molecules in various human diseases. For example, the compositions of the present invention can be used to promote or increase bone growth or regeneration, bone grafting, fracture healing, treatment of osteoporosis and osteoporotic fractures, spinal fusion, osseointegration of orthopedic devices, tendon-osseointegration, tooth growth and regeneration, dental implants, periodontal disease, maxillofacial reconstruction, and osteonecrosis of the jaw. Furthermore, the compositions of the present invention can also be used for the treatment of alopecia; for the enhancement of regeneration of sensory organs, such as the treatment of hearing loss, vestibular dysfunction, macular degeneration, vitreoretinopathy, other retinal degenerative diseases, Fuchs dystrophy, and other corneal diseases; for the treatment of stroke, traumatic brain injury, Alzheimer's disease, multiple sclerosis, and other conditions affecting the blood-brain barrier; and for the treatment of spinal cord injury and other spinal cord diseases. Furthermore, the compositions of the present invention can also be used for the treatment of oral mucositis, short bowel syndrome, inflammatory bowel disease (IBD), and other gastrointestinal disorders; treatment of metabolic syndrome; treatment of diabetes, pancreatitis, and conditions in which the exocrine or endocrine pancreatic tissue is damaged; treatment of conditions in which enhanced epidermal regeneration is desired, such as the healing of epidermal wounds, diabetic foot ulcers, syndromes involving tooth, nail or skin malformations, and conditions in which angiogenesis is beneficial; treatment of myocardial infarction, coronary artery disease, and heart failure; treatment of hematopoietic cell growth, such as enhanced hematopoietic stem cell transplantation from bone marrow or mobilized peripheral blood, and treatment of immunodeficiency and graft-versus-host disease; treatment of acute kidney injury and chronic kidney disease; and treatment of lung disease, chronic obstructive pulmonary disease (COPD), and enhancement of lung tissue regeneration. Furthermore, the compositions of the present invention can also be used to enhance liver cell regeneration (e.g., liver regeneration), treat cirrhosis, enhance liver transplantation, treat acute liver failure, treat chronic liver disease accompanied by hepatitis C or hepatitis B virus infection or after antiviral therapy, alcoholic liver disease, and non-alcoholic liver disease accompanied by steatohepatitis or fatty liver disease. The compositions of the present invention can treat diseases and disorders including, but not limited to, conditions in which regenerative cell growth is desired.

[0204] Human genetics involving loss-of-function or gain-of-function mutations in components of Wnt signaling provides strong evidence supporting the enhancement of Wnt signaling for bone growth. Conditions in which enhanced bone growth is desired include, but are not limited to, fractures, grafts, periprosthetic device implantation, osteoporosis, osteoporotic fractures, spinal fusion, osteonecrosis of the jaw, dental implants, periodontal disease, and maxillofacial reconstruction. Wnt surrogate molecules enhance and promote Wnt signaling, which is crucial in promoting bone regeneration. Methods for bone tissue regeneration benefit from the administration of the compounds of the present invention, which may be systemic or topical. In some embodiments, bone marrow cells are exposed to the molecules of the present invention, resulting in the activation of stem cells in the bone marrow.

[0205] In some embodiments, bone regeneration is enhanced by contacting responsive cell populations, such as bone marrow, bone progenitor cells, or bone stem cells, with an effective dose of a Wnt substitute molecule disclosed herein. Methods for bone tissue regeneration benefit from the administration of a Wnt mimetic, which may be systemic or topical. In some such embodiments, the contact is performed in vivo. In other such embodiments, the contact is performed ex vivo. The molecule can be localized to the site of action by loading it into a matrix, for example, which is biodegradable as needed and provides sustained release of the active agent as needed. Examples of matrix carriers include, but are not limited to, absorbable collagen sponges, ceramics, hydrogels, polymer microspheres, nanoparticles, and bone cement.

[0206] Compositions comprising one or more Wnt substitute molecules disclosed herein can be used for in vivo treatment of skeletal tissue defects. "Skeletal tissue defect" means a defect of bone or other skeletal connective tissue at any site where restoration of bone or connective tissue is desired, regardless of how the defect arose, for example, as a result of surgical intervention, tumor removal, ulceration, implantation, fracture, or other traumatic or degenerative condition. The compositions of the present invention can be used as part of a regimen for repairing defects or lesions in cartilage tissue, such as degenerative wear and arthritis, trauma to the tissue, replacement of a torn meniscus, meniscectomy, joint dislocation due to ligament rupture, joint misalignment, fracture, or due to genetic disease, in order to restore cartilage function to the connective tissue.

[0207] Furthermore, Wnt substitute molecules can also be used for the treatment of periodontal disease, a leading cause of tooth loss and associated with several systemic conditions. In some embodiments, tooth or underlying bone regeneration is enhanced by contact with a responsive cell population. In some such embodiments, the contact is performed in vivo. In other such embodiments, the contact is performed ex vivo, followed by the transplantation of activated stem cells or progenitor cells. The molecule can be localized to the site of action by loading it into a matrix that is, for example, biodegradable as needed and provides sustained release of activating factors as needed. Examples of matrix carriers include, but are not limited to, absorbable collagen sponges, ceramics, hydrogels, bone cements, polymer microspheres, and nanoparticles.

[0208] Studies have shown that Wnt signaling and the biology of R-spongin can promote the regeneration of sensory hair cells in the inner ear after injury, aging, or degeneration. Loss of sensory hair cells in the inner ear that is associated with hearing loss or vestibular dysfunction can also benefit from the compositions of the present invention. In the inner ear, the auditory organs house mechanosensitive hair cells necessary for converting sound vibrations into electrical impulses. The vestibular organs, consisting of the semicircular canals (SSCs), utricle, and saccule, also contain sensory hair cells for detecting head position and movement. The compositions of the present invention can be used, for example, by injection; in a matrix or other depot system; or by other topical application to the ear, for enhancing auditory regeneration.

[0209] Furthermore, Wnt substitute molecules can also be used for the regeneration of retinal tissue. In the adult mammalian retina, Müller glial cells can regenerate retinal cells containing photoreceptors, for example, after neurotoxic injury in vivo. Wnt signaling and Wnt signaling enhancers can promote the proliferation of Müller glial-derived retinal precursors after injury or during degeneration. The compositions of the present invention can also be used for the regeneration of intraocular tissues and other cell types. For example, age-related macular degeneration (AMD), other retinal degenerative diseases, corneal diseases, Fuchs dystrophy, vitreoretinopathy, and genetic diseases can benefit from the compositions of the present invention. AMD is characterized by a gradual decline in central visual field and visual acuity. Fuchs dystrophy is characterized by the progressive loss of corneal endothelial cells. Wnt signaling and Wnt signaling enhancement can promote the regeneration of corneal endothelium, retinal epithelium, and other ocular tissues. In other embodiments, the compositions of the present invention can be used, for example, for retinal regeneration and the treatment of macular degeneration, by injection; in a matrix or other depot system; or by other topical application to the eye.

[0210] Specific populations of proliferative cells for homeostatic regeneration of hepatocytes have been identified through lineage tracking studies, including, for example, Axyn 2-positive cells in the pericentral region. Lineage tracking studies have also identified further potential hepatic progenitor cells, including, but not limited to, Lgr-positive cells. Autoregenerating hepatocytes, as well as other populations of potential progenitor cells, including Lgr5-positive and Axyn 2-positive cells, have been identified as being able to regenerate after injury in response to Wnt signaling and / or R-spondin. Numerous preclinical models of acute liver injury and hepatic failure, as well as chronic liver disease, have shown that hepatocyte recovery and regeneration benefit from enhanced Wnt signaling. The compositions of the present invention can be used for the treatment of acute liver failure, acute alcoholic liver injury, chronic liver disease with or after hepatitis C or B virus infection or antiviral therapy, chronic alcoholic liver disease, non-alcoholic fatty liver disease and non-alcoholic steatohepatitis (NASH), cirrhosis, and severe chronic liver disease of any cause, as well as for enhancing hepatocyte regeneration. Methods for regenerating liver tissue can be benefited by administering the compound of the present invention, which may be systemic or local. These methods include, but are not limited to, systemic and local administration, such as injection into liver tissue, injection into a vein or blood vessel connected to the liver, or implantation of a sustained-release formulation.

[0211] Wnt signaling plays a crucial role in the regeneration of various epithelial tissues. Various epidermal conditions benefit from treatment with the compounds of the present invention. Mucositis occurs when the rapidly dividing epithelial cells lining the inside of the gastrointestinal tract break down, making the mucosal tissue susceptible to ulceration and infection. The inner epithelial layer lining the mouth, known as the oral mucosa, is one of the most sensitive parts of the body and is particularly vulnerable to chemotherapy and radiation. Oral mucositis is perhaps the most common debilitating complication of cancer treatment, especially chemotherapy and radiation. In addition, the compositions of the present invention may also be beneficial in the treatment of short bowel syndrome, inflammatory bowel disease (IBD), or other gastrointestinal disorders. Other epidermal conditions include epidermal wound healing, diabetic foot ulcers, and syndromes involving malformations of teeth, nails, or skin. The molecules of the present invention can be used in all these conditions where regenerating cells come into contact with the compounds of the present invention. Methods for the regeneration of epithelial tissue benefit from the administration of the compounds of the present invention, which may be systemic or topical. The contact is localized, including intradermal and subcutaneous, and can be applied to targeted areas as a gel, lotion, cream, etc.

[0212] Wnt signaling, as well as its enhancement and promotion, plays a crucial role in the repair and regeneration of tissues including the pancreas, kidneys, and lungs, in preclinical models, in addition to the skin and gastrointestinal tract. Wnt substitute molecules may be beneficial for various disease conditions involving the exocrine and endocrine parts of the pancreas, kidneys, or lungs. Wnt mimes can be used in the treatment of metabolic syndrome; diabetes mellitus; acute or chronic pancreatitis; exocrine pancreatic insufficiency; acute kidney injury; chronic kidney disease; lung diseases including but not limited to chronic obstructive pulmonary disease (COPD); and other conditions causing loss of lung epithelial tissue. Methods for the regeneration of these tissues can be benefited by the administration of the compounds of the present invention, which may be systemic or topical.

[0213] Epidermal Wnt signaling, in coordination with signaling mediated by other developmental factors, is crucial for hair follicle regeneration in adults. Hair loss is a common problem, androgenic alopecia, often referred to as male pattern baldness, is the most common form of hair loss in men. In some embodiments, hair follicle regeneration is enhanced by contacting a responsive cell population with the molecule of the present invention. In some such embodiments, the contact is performed in vivo. In other such embodiments, the contact is performed ex vivo. This molecule can be localized to the site of action, for example, as a topical lotion, gel, cream, etc.

[0214] Stroke, traumatic brain injury, Alzheimer's disease, multiple sclerosis, and other conditions affecting the blood-brain barrier (BBB) ​​can be treated using Wnt substitute molecules. Angiogenesis is crucial for ensuring the supply of oxygen and nutrients to many tissues throughout the body, and is particularly important for the central nervous system (CNS) because nerve tissue is highly susceptible to hypoxia and ischemia. CNS endothelial cells that form the BBB are highly polarized cells held together by tight junctions and differ from endothelial cells in non-nerve tissues in that they express specific transporters. Wnt signaling regulates CNS angiogenesis and / or function. Conditions with impaired BBB can benefit from the administration of the compounds of the present invention, which may be systemic or topical, for example, by direct injection, intrathecal administration, or implantation of a sustained-release formulation. In addition, Wnt signaling is actively involved in neurogenesis and plays a role in post-injury neuroprotection. The compositions of the present invention can also be used to treat spinal cord injury, other spinal cord diseases, stroke, traumatic brain injury, and other conditions.

[0215] Wnt signaling also plays a role in angiogenesis. Wnt substitute molecules may be beneficial in conditions where angiogenesis is advantageous, such as in the treatment of myocardial infarction, coronary artery disease, heart failure, and conditions caused by genetic disorders. Methods for the regeneration of these tissues can be benefited by the administration of the compounds of the present invention, which may be systemic or topical.

[0216] In certain embodiments, the methods of the present invention promote tissue regeneration, for example, in tissue that has been damaged or has suffered a reduction or loss of tissue or cells. The loss or damage may be any reduction in cell number, including disease or injury. For example, accidents, autoimmune disorders, side effects of treatment, or disease conditions may constitute trauma. Tissue regeneration increases the number of cells in the tissue, preferably allowing connections between cells in the tissue to be re-established, and more preferably allowing the tissue to regain its functionality.

[0217] The terms “administer,” “introduce,” or “deliver,” as used herein, refer to the delivery of a composition to cells, to cells, tissues and / or organs of interest, or to the subject. Such administration or introduction may be carried out in vivo, in vitro, or ex vivo.

[0218] In certain embodiments, the pharmaceutical composition is administered parenterally, for example, intravenously, orally, rectally, or by injection. In some embodiments, the pharmaceutical composition is administered topically, for example, locally or intramuscularly. In some embodiments, the composition is administered to target tissue, for example, bone, joint, ear tissue, eye tissue, gastrointestinal tract, skin, wound site, or spinal cord. The methods of the present invention can be carried out in vivo or ex vivo. In some embodiments, contact of target cells or tissue with the Wnt substitute molecule is performed ex vivo, followed by transplantation of cells or tissue, for example, activated stem cells or progenitor cells, to the target. Those skilled in the art can determine the appropriate site and route of administration based on the disease or disorder being treated.

[0219] Dosage and drug regimens may depend on various factors readily determined by the physician, such as the nature of the disease or disorder, the characteristics of the subject, and the subject's medical history. In certain embodiments, the amount of Wnt substitute molecule administered or provided to the subject is in the range of about 0.0001 mg to about 50 mg, about 0.001 mg to 50 mg, 0.1 mg to about 500 mg, or about 0.1 mg to about 50 mg per kg of the subject's body weight.

[0220] Terms such as “treatment” and “to treat” are used herein in general to mean obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic in that it completely or partially prevents the disease or its symptoms, e.g., by reducing the likelihood of the disease or its symptoms occurring in a subject, and / or therapeutic in that it partially or completely cures the disease and / or adverse effects resulting from the disease. As used herein, “treatment” encompasses any treatment of a disease in a mammal, including: (a) preventing the onset of the disease in a subject that may be predisposed to the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease, i.e., suppressing its onset; or (c) alleviating the disease, i.e., causing its regression. Therapeutic agents (e.g., Wnt mimes) may be administered before, during, or after the onset of a disease or injury. Treatment of a disease in progress is of particular interest if the treatment stabilizes or reduces undesirable clinical symptoms in the patient. Such treatment is preferably performed before the function of the affected tissue is completely lost. The treatment of the subject is preferably administered during the symptomatic stage of the disease, and in some cases after the symptomatic stage of the disease. In some embodiments, the method of the subject results in therapeutic benefits, such as prevention of the onset of disability, cessation of the progression of disability, or reversal of the progression of disability. In some embodiments, the method of the subject includes a step of detecting that therapeutic benefits have been achieved. Those skilled in the art will understand that such measures of therapeutic effectiveness are applicable to the specific disease being modified and will recognize appropriate detection methods used to measure therapeutic effectiveness.

[0221] Other embodiments relate, in part, to the use of the Wnt substitute molecules disclosed herein to promote or enhance the growth or proliferation of cells, tissues, and organoids, for example, by contacting cells or tissues with one or more Wnt substitute molecules, optionally in combination with Norrin or R-sponge polypeptides. In certain embodiments, cells or tissues are contacted ex vivo, in vitro, or in vivo. Such methods can be used, for example, to generate cells, tissues, or organoids for therapeutic use, which are to be transplanted or grafted into a subject. Such methods can also be used to generate cells, tissues, or organoids for research use. Wnt mimics have broad applications in non-therapeutic methods, such as in vitro research methods.

[0222] The present invention provides a method for tissue regeneration of damaged tissue, such as the tissues discussed above, comprising administering a Wnt substitute molecule to cells. The Wnt mimetic may be administered directly to cells in vivo, orally, intravenously, or by other methods known in the art to a subject, or ex vivo to cells. In some embodiments in which the Wnt mimetic is administered to cells ex vivo, these cells can be transplanted to a subject before, after, or during the administration of the Wnt mimetic.

[0223] Wnt signaling is a crucial component of stem cell culture, as described in, for example, WO2010 / 090513, WO2012 / 014076, Sato et al., 2011 (GASTROENTEROLOGY 201 1; 141: 1762-1772) and Sato et al., 2009 (Nature 459, 262-5). The Wnt substitute molecules disclosed herein are suitable substitutes for or can be combined with R-sponge for use in these stem cell culture media.

[0224] Accordingly, in one embodiment, the Disclosure provides a method for enhancing the proliferation of stem cells, comprising contacting stem cells with one or more Wnt mimics disclosed herein. In one embodiment, the Disclosure provides a cell culture medium comprising one or more Wnt substitute molecules disclosed herein. In some embodiments, the cell culture medium may be any cell culture medium already known in the Art, which typically comprises WNT or R-spongin, but in which WNT or R-spongin is replaced (completely or partially) or supplemented with Wnt mimics disclosed herein. For example, the culture medium may be such as those described in WO2010 / 090513, WO2012 / 014076, Sato et al., 2011 (GASTROENTEROLOGY 2011; 141: 1762-1772) and Sato et al., 2009 (Nature 459, 262-5), which are incorporated herein by reference in their entirety.

[0225] Stem cell culture media often contain additional growth factors. Therefore, this method may further involve supplying growth factors to stem cells. Commonly used growth factors in cell culture media include epidermal growth factor (EGF, Peprotech), transforming growth factor-alpha (TGF-alpha, Peprotech), basic fibroblast growth factor (bFGF, Peprotech), brain-derived neurotrophic factor (BDNF, R&D Systems), hepatocyte growth factor (HGF), and keratinocyte growth factor (KGF, Peprotech, also known as FGF7). EGF is a potent mitogenic factor for various cultured ectoderm and mesoderm cells and has a significant effect on the differentiation of certain cells in vivo and in vitro, as well as the differentiation of certain fibroblasts under cell culture conditions. EGF precursors exist as membrane-bound molecules that are cleaved by proteolysis to produce a 53-amino acid peptide hormone that stimulates cells. EGF or other mitogenic growth factors can thus be supplied to stem cells. During stem cell culture, mitogenic growth factors can be added to the culture medium every two days, and it is preferable to replace the culture medium every four days. Generally, mitogenic growth factors are selected from the group consisting of i) EGF, TGF-alpha, and KGF; ii) EGF, TGF-alpha, and FGF7; iii) EGF, TGF-alpha, and FGF; iv) EGF and KGF; v) EGF and FGF7; vi) EGF and FGF; vii) TGF-alpha and KGF; viii) TGF-alpha and FGF7, or ix) TGF-alpha and FGF. In certain embodiments, the disclosure includes a stem cell culture medium comprising, for example, a Wnt substitute molecule disclosed herein, in combination with, if necessary, one or more of the growth factors or combinations thereof described herein.

[0226] These methods for enhancing stem cell proliferation can be used to grow new organoids and tissues from stem cells, as described, for example, in WO2010 / 090513, WO2012 / 014076, Sato et al., 2011 (GASTROENTEROLOGY 2011; 141: 1762-1772) and Sato et al., 2009 (Nature 459, 262-5).

[0227] In some embodiments, Wnt substitute molecules are used to enhance stem cell regeneration. Exemplary stem cells of interest include, but are not limited to, muscle satellite cells; hematopoietic stem cells and their progenitor cells (U.S. Patent No. 5,061,620); neural stem cells (see Morrison et al. (1999) Cell 96: 737-749); embryonic stem cells; mesenchymal stem cells; mesodermal stem cells; liver stem cells; adipose tissue-derived stem cells, etc.

[0228] Other embodiments of the present invention relate, in part, to diagnostic applications for detecting the presence of cells or tissues expressing one or more FZD receptors and / or LRP5 and / or LRP6 receptors. Accordingly, the present disclosure provides methods for detecting one or more FZD receptors or LRP5 receptors or LRP6 receptors in a sample, for example, methods for detecting cells or tissues expressing FZD1. Such methods can be applied, for example, by detecting the binding of Wnt mimetic molecules in a variety of known detection methods, including but not limited to immunohistochemistry (IHC), immunocytochemistry (ICC), in situ hybridization (ISH), whole-mount in situ hybridization (WISH), fluorescent DNA in situ hybridization (FISH), flow cytometry, enzyme immunoassay (EIA), and enzyme-conjugated immunoassay (ELISA).

[0229] ISH is a type of hybridization that uses labeled complementary DNA or RNA strands (i.e., primary binding agents) to identify the location of specific DNA or RNA sequences in a cell or a part or section of tissue (in situ), or the whole tissue (whole-mount ISH) if the tissue is small enough. Those skilled in the art will understand that this is distinct from immunohistochemistry, which uses antibodies as primary binding agents to identify the location of proteins in tissue sections. DNA ISH can be used on genomic DNA to determine the structure of chromosomes. Fluorescent DNA ISH (FISH) can be used, for example, in medical diagnostics to assess the integrity of chromosomes. RNA ISH (hybridization histochemistry) is used to measure and localize mRNA and other transcripts within tissue sections or whole mounts.

[0230] In various embodiments, the Wnt surrogate molecules described herein are conjugated to a detectable label that can be detected directly or indirectly. In this regard, an antibody "conjugate" refers to a Wnt mimic that is covalently linked to a detectable label. In the present invention, DNA probes, RNA probes, monoclonal antibodies, their antigen-binding fragments, and antibody derivatives thereof, such as single-chain variable fragment antibodies or epitope-tagged antibodies, can all be covalently linked to a detectable label. In "direct detection", only one detectable antibody, i.e., a detectable primary antibody, is used. Thus, direct detection means that an antibody conjugated to a detectable label can be detected by itself without the addition of a second antibody (secondary antibody).

[0231] A "detectable label" is a molecule or material that can generate a detectable (e.g., visually, electronically, or otherwise) signal indicating the presence and / or concentration of a label in a sample. When conjugated to an antibody, the detectable label can be used for localizing and / or quantifying the target to which the specific antibody is directed. Thereby, the presence and / or concentration of the target in the sample can be detected by detecting the signal generated by the detectable label. Detectable labels can be detected directly or indirectly, and several different detectable labels conjugated to different specific antibodies can be used in combination to detect one or more targets.

[0232] Examples of detectable labels that can be detected directly include fluorescent dyes, radioactive substances, and metal particles. In contrast, indirect detection requires the application of one or more additional antibodies (i.e., secondary antibodies) after the application of the primary antibody. Thus, detection is performed by detecting the binding of the secondary antibody or binding agent to the detectable primary antibody. Examples of detectable primary binding agents or antibodies that require the addition of a secondary binding agent or antibody include detectable enzyme binding agents and detectable hapten binding agents or antibodies.

[0233] In some embodiments, the detectable label is conjugated to a nucleic acid polymer comprising a first binding agent (e.g., in the processes of ISH, WISH, or FISH). In other embodiments, the detectable label is conjugated to an antibody comprising a first binding agent (e.g., in the IHC process).

[0234] Examples of detectable labels that can be conjugated to the Wnt surrogate molecules used in the methods of the present disclosure include fluorescent labels, enzyme labels, radioisotopes, chemiluminescent labels, electrochemiluminescent labels, bioluminescent labels, polymers, polymer particles, metal particles, haptens, and dyes.

[0235] Examples of fluorescent labels include 5-(and 6)-carboxyfluorescein, 5- or 6-carboxyfluorescein, 6-(fluorescein)-5-(and 6)-carboxamidehexanoic acid, fluorescein isothiocyanate, rhodamine, tetramethylrhodamine, as well as dyes such as Cy2, Cy3 and Cy5, optionally substituted coumarins including AMCA, phycobiliproteins including PerCP, R-phycoerythrin (RPE) and allophycoerythrin (APC), Texas Red, Princeton Red, green fluorescent protein (GFP) and its analogues, as well as conjugates of R-phycoerythrin or allophycoerythrin, inorganic fluorescent labels, and particles based on semiconductor materials such as coated CdSe nanocrystals.

[0236] Examples of polymer particle labeling include polystyrene, PMMA, or silica microparticles or latex particles that can be embedded with fluorescent dyes, or polymer micelles or capsules containing dyes, enzymes, or substrates.

[0237] Examples of metal particle labeling include gold particles and coated gold particles, which can be converted by silver staining. Examples of haptens include DNP, fluorescein isothiocyanate (FITC), biotin, and digoxigenin. Examples of enzyme labeling include horseradish peroxidase (HRP), alkaline phosphatase (ALP or AP), β-galactosidase (GAL), glucose-6-phosphate dehydrogenase, β-N-acetylglucosaminidase (acetylglucosaminidase), β-glucuronidase, invertase, xanthine oxidase, firefly luciferase, and glucose oxidase (GO). Examples of substrates commonly used for horseradish peroxidase include 3,3'-diaminobenzidine (DAB), nickel-fortified diaminobenzidine, 3-amino-9-ethylcarbazole (AEC), benzidine dihydrochloride (BDHC), Hanker-Yates reagent (HYR), Indofan blue (IB), tetramethylbenzidine (TMB), 4-chloro-1-naphthol (CN), alpha-naphtholpyronin (alpha-NP), o-dianisidine (OD), 5-bromo-4-chloro-3-indolyl phosphate (BCIP), nitrobluetetrazolium (NBT), 2-(p-iodophenyl)-3-p-nitrophenyl-5-phenyltetrazolium chloride (INT), tetranitrobluetetrazolium (TNBT), and 5-bromo-4-chloro-3-indoxyl-beta-D-galactoside / ferro-felicyanide (BCIG / FF).

[0238] Examples of substrates commonly used for alkaline phosphatases include naphthol-AS-B1-phosphate / Fastred TR (NABP / FR), naphthol-AS-MX-phosphate / Fastred TR (NAMP / FR), naphthol-AS-B1-phosphate / -Fastred TR (NABP / FR), naphthol-AS-MX-phosphate / Fastred TR (NAMP / FR), naphthol-AS-B1-phosphate / new fuschin (NABP / NF), bromochloroindolyl phosphate / nitrobluetetrazolium (BCIP / NBT), and 5-bromo-4-chloro-3-indolyl-b--d-galactopyranoside (BCIG).

[0239] Examples of luminescent labels include luminol, isoluminol, acridinium esters, 1,2-dioxetane, and pyridopyridazine. Examples of electrochemiluminescent labels include ruthenium derivatives. Examples of radioactive labels include radioisotopes of iodide, cobalt, selenium, tritium, carbon, sulfur, and phosphorus.

[0240] A detectable label can be conjugated to an antibody described herein, or any other molecule that specifically binds to the biological marker of interest, such as an antibody, nucleic acid probe, or polymer. Furthermore, those skilled in the art will understand that a detectable label can also be conjugated to a second and / or third and / or fourth and / or fifth binding agent or antibody, etc. Furthermore, those skilled in the art will understand that each additional binding agent or antibody used to characterize the biological marker of interest may act as a signal amplification step. The biological marker can be detected visually, for example, using an optical microscope, fluorescence microscope, or electron microscope, in which case the detectable substance may be, for example, a dye, gold colloid particles, or a luminescent reagent. Alternatively, a visually detectable substance bound to a biological marker can be detected using a spectrophotometer. If the detectable substance is a radioactive isotope, detection can be performed visually by autoradiography or non-visually using a scintillation counter. For example, see Larsson, 1988, Immunocytochemistry: Theory and Practice, (CRC Press, Boca Raton, Fla.); Methods in Molecular Biology, vol. 80 1998, John D. Pound (ed.) (Humana Press, Totowa, NJ).

[0241] The present invention further provides a kit for detecting one or more FZD or LRP5 / 6 receptors in a sample, or cells or tissues expressing one or more FZD or LRP5 / 6 receptors, the kit comprising at least one antibody, polypeptide, polynucleotide, vector, or host cell as described herein. In certain embodiments, the kit may include a buffer, enzyme, label, substrate, beads or other surfaces to which the antibody of the present invention is attached, and instructions for use.

[0242] All of the above-mentioned U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications, PCT application WO 2019 / 126399 titled "Anti-Frizzled Antibodies", PCT application WO 2019 / 126401 titled "Anti-LRP5 / 6 Antibodies and Methods of Use", PCT application WO 2019 / 126398 titled "WNT Surrogate Molecules and Uses Thereof", and PCT application WO 2020 / 010308 titled "Multi-Specific WNT Surrogate Molecule and Uses Thereof" are incorporated herein by reference in their entirety.

[0243] From the above, although specific embodiments of the present invention have been described herein for illustrative purposes, it will be understood that various modifications can be made without departing from the spirit and scope of the invention. Therefore, the present invention is not limited except as provided for by the appended claims.

[0244] All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications mentioned herein and / or listed in the application data sheet are incorporated herein by reference in their entirety. Furthermore, to provide further embodiments, aspects of the embodiments may be modified to adopt various patent, application, and publication concepts as necessary.

[0245] Further embodiments can be provided by combining the various embodiments described above. These and other modifications can be made to the embodiments in light of the detailed description above. In general, the terms used in the following claims should not be construed as limiting the claims to the specific embodiments disclosed herein and in the claims, but rather as encompassing all possible embodiments, along with the entire scope of equivalents of such claims. Thus, the claims are not limited by this disclosure. [Examples]

[0246] Reagents and materials Wnt substitute polypeptides or Wnt mimics, collectively referred to as mini-SWAP molecules ("mini-SWAPs") based on several parental IgG-based SWAP molecules, have been synthesized in various different forms. Examples of complete IgG SWAP molecules from which they are derived include, but are not limited to, R2M13-26, which contains an anti-FZD Fv domain with an attached VHH that binds to one or more FZDs and LRP5 and / or LRP6.

[0247] DNA fragments encoding mini-SWAP polypeptides, including those shown in Figures 10A-10D, were synthesized based on peptide sequences (IDT) and cloned into pcDNA3.1(+)(ThermoFisher). All DNA constructs contain a human kappa light chain leader peptide sequence (MDMRVPAQLLGLLLLWLRGARC) at the 5' end of the coding sequence (CDS) for efficient secretion of recombinant proteins expressed in mammalian cells. A C-terminal 6× histidine tag (His6) was added to the polypeptides to facilitate purification.

[0248] All recombinant proteins were expressed in Expi293F cells (Thermo Fisher Scientific) after transient transfection and then secreted. The proteins were first purified using nickel-immobilized metal affinity chromatography, followed by further purification by size exclusion chromatography (SEC) on a Superdex 200 Increase 10 / 300 GL column (Cytiva) using 1× HBS buffer (20 mM HEPES pH 7.5, 150 mM NaCl). SEC allowed for assessment of polydispersity by several proteins eluting with adequate retention, which were pooled for further characterization (Figure 23); some proteins did not form diabodies and / or were not well expressed and / or formed aggregates. The proteins were examined by SDS-polyacrylamide gel electrophoresis and / or used in other activity and characterization assays. Shaded fractions were analyzed by non-reducing (NR) and reducing (R) SDS-PAGE, as shown in Figure 24A. SDS-PAGE gels of other purified proteins are shown in Figures 24B and 24C.

[0249] Analysis of the hydrophobicity of polypeptides The hydrophobicity of purified mini-swap samples was analyzed by hydrophobic interaction chromatography (HIC)-HPLC on an Agilent HPLC system. Briefly, samples were diluted with 3 parts Buffer B (15.4 mM K2HPO4, 9.6 mM KH2PO4, 2 M (NH4)2SO4, pH 7.2), and 10 μL or more was loaded onto a Tosoh Bioscience TSKgel Butyl-NPR column (0.58 mL) equilibrated with 80% Buffer B. Elution was performed by a 12 mL linear gradient to 40% Buffer B and to 0% Buffer B or 100% Buffer A (15.4 mM K2HPO4, 9.6 mM KH2PO4, pH 7.2), and monitored by OD280. Peak retention was recorded compared to standard mAbs with known retention, where later elutions were more hydrophobic.

[0250] Table 1 shows the hydrophobicity of the polypeptide.

Table 1

[0251] Super Top Flash (STF) assay WNT signaling activity was measured using Huh7 cells containing a luciferase reporter gene controlled by a WNT-responsive promoter (Super Top Flash reporter assay, STF) as previously reported (Chen et al., (2020) STAR Protocols, 1: 100043). Briefly, cells were seeded in 96-well plates at a density of 1,000 cells / well and treated with 3 μM IWP2 24 hours later to inhibit the production of endogenous WNT. Subsequently, recombinant Wnt replacement molecules were added to the cells overnight. Cells were lysed with Luciferase Cell Culture Lysis Reagent (Promega), and luciferase activity was measured using the Luciferase Assay System (Promega) according to the procedure suggested by the supplier.

[0252] Figures 25 and 26 and Table 3 show various miniSWAPs with STF activity equivalent to or improved compared to the IgG-based parental molecule R2M13-26 (also known as SZN-1326).

[0253] In Figure 25, SZN-1326 is the parental molecule of the following samples: VHH26-R2M13-diabody (SZP31557), VHH26-tandem-R2M13-diabody (SZP31558), VHH26-tandem-R2M13-scFv with a 5mer linker (SZP31559), VHH26-tandem-R2M13-scFv with a 15mer linker (SZP31560).

[0254] In Figure 26, SZN-1326 is the parent molecule of the diabody, VHH26_R2M13-VL_R2M13-VH(SZP31770). VHH26-R2M13-diabody(SZP31557) was included in this assay as a control.

[0255] GFP Reporter Assay WNT signaling activity was measured using Hek293T cells containing a GFP reporter gene regulated by a WNT-responsive promoter (GFP reporter assay). Briefly, cells were seeded at a density of 20,000 cells / well in 96-well plates and treated in the presence of 3 μM IWP2 after 24 hours to inhibit endogenous WNT production. Recombinant Wnt mimetic cells were then added to the cells overnight. After removing the culture medium, GFP fluorescence was measured using a microplate reader.

[0256] Tm / Tagg and Dynamic Light Scattering (DLS) of WNT Alternative Molecules The melting temperature was measured using an UNcle instrument (UNchained Labs) by monitoring intrinsic fluorescence changes. Protein concentrations ranged from 0.08 to 0.5 mg / mL in HBS, pH 7.4, and the temperature was increased from 15°C to 95°C at a rate of 1°C / min. In the same experiment, the aggregation onset temperature (Tagg) was determined by static light scattering while increasing particle size. The protein diameter in solution was measured using dynamic light scattering (DLS) at 15°C with the same instrument. All measurements were performed in pairs. Table 2 shows the representative Tm1 (first melting temperature) and Tag for each sample, as well as the range of the main peak observed by DLS, along with the diameter and correlated estimated MW. For all molecules, Tm1 was higher than 54.8°C and Tag was lower than 65°C. DLS results showed that SZP31557 formed an intermolecular diabody, and the observed MW was approximately twice that of the monomeric form, confirming that there was no dimerization of the monomeric mini-SWAP. Examples of Tm and Tag data are shown in Figure 27.

[0257] Table 2 shows the Tm / tag and DLS measurements. [Table 2] Table 2

[0258] In vitro efficacy of Wnt polypeptide in organoid cultures Mouse small intestinal organoids (Stemcell Technologies, 70931) were grown and subculturised as needed until sufficient organoids were available for each efficacy assay. For the efficacy assays, the organoids were divided in a 1:6 ratio, seeded in 48-well tissue culture plates, and grown overnight at 37°C in complete mouse IntestiCult® Organoid Growth Medium (Stemcell Technologies, 06005). The following day, the wells were treated at 37°C for 3 days with fresh Wnt substitute molecules in a 10-0.01 nM 10-fold dilution series in a basal medium containing 1 μM Wnt-producing inhibitor (IWP2) (Tocris, 686770-61-6), or with a control, with 6 wells per well, followed by replacement with a fresh protein dilution series or control, and grown for a further 2 days. On day 7, all viable cells in each well were evaluated using the CellTiter-Glo 3D cell viability assay (Promega, G9682). An example of assay readings is shown in Figure 28. Nonlinear regression analysis was performed using Graphpad Prism to determine the EC (Ethical Cell Viability). 50 The calculations are shown in Table 3.

[0259] Table 3. EC of STF Wnt signaling assay 50 and EC of organoid culture assays of Wnt substitute molecules 50 [Table 3-1] [Table 3-2]

[0260] Table 4 lists various mini-SWAP molecules and presents the polypeptide sequences present within them. The various domains present within the Wnt-mimicking polypeptide are shown below: VHH03 or VHH26 are highlighted in bold, R2M13-VH in light gray, R2M13-VL in black, the G4S linker (sequence number 59) and 3×G4S linker (sequence number 60) are in light gray, and CDRs based on Kabat's definition are double-underlined. Table 4 also shows the parent molecules from which the mini-SWAPs are derived, and provides the figure numbers illustrating the structures of the mini-SWAPs. Table 4 also includes the domain sequences of the parent SWAP molecules.

[0261] Table 4. Mini-SWAP and parent array [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5] [Table 4-6] [Table 4-7] [Table 4-8] [Table 4-9] [Table 4-10] [Table 4-11] [Table 4-12] [Table 4-13] [Table 4-14] [Table 4-15]

[0262] Those skilled in the art will readily understand that the above constructs can be modified and expressed as various homologs and isoforms, edited in the unbound domain sequence, and expressed using various suitable expression vector systems and various synonymous nucleotide sequences. References [ka] [ka]

Claims

1. A WNT substitute molecule, wherein the WNT substitute molecule is a)N’ AB1 ｎ -X-AB2 ｍ -Y-AB3 ｐ -Z-AB4 ｑ C’ Polypeptides having the structure; or b) Each N’ AB1 ｎ -X-AB2 ｍ -Y-AB3 ｐ -Z-AB4 ｑ C’ Dimers of first and second polypeptide monomers having the structure Including, in the formula, AB1 is an antigen-binding antibody fragment selected from the group consisting of Fab, a single-chain Fv fragment (scFv), a variable heavy chain (VH) and a variable light chain (VL) linked via a linker in any order, and a VH or VL or a single-domain antibody (VHH), which binds to one or more FZD receptors or to LRP5 and / or LRP6; AB2 is an antigen-binding antibody fragment selected from the group consisting of a variable heavy chain (VH) and a variable light chain (VL) linked via a linker in either the order Fab, scFv, and a VH or VL or a single-domain antibody (VHH), which binds to one or more FZD receptors or to LRP5 and / or LRP6; AB3 is an antigen-binding antibody fragment selected from the group consisting of a variable heavy chain (VH) and a variable light chain (VL) linked via a linker in either the order Fab, scFv, and a VH or VL or a single-domain antibody (VHH), which binds to one or more FZD receptors or to LRP5 and / or LRP6; AB4 is an antigen-binding antibody fragment selected from the group consisting of a variable heavy chain (VH) and a variable light chain (VL) linked via a linker in either the order Fab, scFv, and a VH or VL or a single-domain antibody (VHH), which binds to one or more FZD receptors or to LRP5 and / or LRP6; X, Y, and Z are either nonexistent or peptide linkers, and if there are no adjacent fragments to both the N-terminus and C-terminus of each of X, Y, or Z, then each of X, Y, and Z is nonexistent; n is 0, 1, or 2; m is 0, 1, or 2; p is 0, 1, or 2; q is 0, 1, or 2. Each polypeptide comprises at least two fragments selected from the group consisting of AB1, AB2, AB3, and AB4. A WNT substitute molecule comprising one or more antigen-binding antibody fragments that bind to one or more FZD receptors, and one or more antigen-binding fragments that bind to LRP5 and / or LRP6.

2. The molecule according to claim 1, wherein the peptide linker is approximately 0 to approximately 100 amino acids or approximately 1 to approximately 100 amino acids.

3. The molecule according to claim 2, wherein the peptide linker comprises one or more glycine and / or serine residues.

4. The peptide linker between the VH chain and VL chain of X, Y, Z, or the scFv or diabody, a) Glycine x 4-serine: (G4S) (Sequence ID 59); b) (Glycine x 4 - Serine) x 3: (G4S) 3 (Sequence ID 60); c) ASTKG (Sequence No. 61); d) DKTHT (Sequence ID 62); e) G4S (SEQ ID NO: 59) and ASTKG (SEQ ID NO: 61); and f) G4S (Sequence ID 59) and DKTHT (Sequence ID 62) The molecule according to claim 3, which is a peptide selected from the group consisting of the following.

5. The molecule according to claim 1, wherein the molecule is the dimer, and the dimer is formed by one or more interactions between VH and VL present in the first monomer and VH and VL present in the second monomer.

6. The molecule according to claim 1, comprising a diabody, wherein the diabody comprises an intramolecular pairing of VH and VL present in the polypeptide, or an intermolecular or intramolecular pairing of VH and VL present in the first monomer and VH and VL present in the second monomer, wherein, optionally, the VH of the first monomer binds to the VL of the second monomer to form a first antigen-binding domain, and the VL of the first monomer binds to the VH of the second monomer to form a second antigen-binding domain.

7. The molecule is the dimer, and for each polypeptide monomer, AB1 is VHH; AB2 are VH and VL connected via a linker in any order; n = 1; m = 1; p = 0; q = 0; X exists; Y and Z do not exist, The molecule according to any one of claims 1 to 6, wherein the VH and VL of the first monomer bond to the VL and VH of the second monomer to form a diamond body.

8. The molecule is the dimer, and for each polypeptide monomer, AB1 is VH and VL connected via a linker in any order; AB2 is VHH; n = 1; m = 1; p = 0; q = 0; X exists; Y and Z do not exist. The molecule according to any one of claims 1 to 6, wherein the VH and VL of each monomer bond to the VL and VH of the other monomer to form a diabody.

9. The molecule according to any one of claims 7 to 8, wherein the VH of the first monomer binds to the VL of the second monomer to form a first binding domain, and the VH of the second monomer binds to the VL of the first monomer to form a second binding domain.

10. The molecule according to any one of claims 7 to 8, wherein the dimer is a homodimer and the molecule is a polyvalent, multispecific binding molecule.

11. The molecule according to any one of claims 7 to 8, wherein the molecule is a heterodimer, the VH of the first monomer is bound to the VL of the second monomer to form a first antigen-binding domain, the VL of the first monomer is bound to the VH of the second monomer to form a second antigen-binding domain, the first antigen-binding domain is bound to a first set of one or more antigens, and the second antigen-binding domain is bound to a second set of one or more antigens, and the first and second sets are the same or different antigens, or different sets of antigens, which may overlap as needed.

12. The molecule according to any one of claims 7 to 11, wherein the VHH binds to LRP5 or LRP6, and the VH and VL form a diabody that binds to one or more FZD receptors.

13. The molecule according to claim 1, wherein the molecule is a single polypeptide.

14. AB1 is VHH; AB2 are VH and VL connected via a linker in any order; AB3 are VH and VL connected via a linker in any order; n = 1; m = 1; p = 1; q = 0; X and Y exist, and Z does not exist. The VH and VL of AB2 bind to the VL and VH of AB3 to form a diamond body, the VH of AB1 binds to the VL of AB2 to form a first antigen-binding domain, and the VL of AB1 binds to the VH of AB2 to form a second antigen-binding domain. The molecule according to claim 13.

15. The molecule according to claim 13 or claim 14, wherein the VH and VL of AB2 are the same as the VH and VL of AB3.

16. The molecule according to claim 13 or claim 14, wherein the VH and VL of AB2 are different from the VH and VL of AB3.

17. The molecule according to claim 16, wherein the VH of AB2 binds to the VL of AB3 to form a first antigen-binding domain, the VL of AB2 binds to the VH of AB3 to form a second antigen-binding domain, the first antigen-binding domain binds to a first set of one or more antigens, and the second antigen-binding domain binds to a second set of one or more antigens, wherein the first and second sets are the same or different antigens, or different sets of antigens, which may overlap as needed.

18. The molecule according to any one of claims 14 to 17, wherein, for each of AB2 and AB3, VH is located on the amino-terminal side relative to VL.

19. The molecule according to any one of claims 14 to 17, wherein, for each of AB2 and AB3, VL is located on the amino-terminal side relative to VH.

20. The molecule according to any one of claims 14 to 19, wherein the VHH binds to LRP5 and / or LRP6, and the diabody binds to one or more FZD receptors.

21. AB1 is VHH; AB2 is scFv; AB3 is scFv; n = 1; m = 1; p = 1; q = 0, The molecule according to claim 13, wherein X and Y are present, and Z is not present.

22. The molecule according to claim 21, wherein the first and second scFv are the same.

23. The molecule according to claim 21, wherein the first and second scFv are not the same.

24. The molecule according to claim 23, wherein the molecule is a trivalent triple-specific polypeptide.

25. The molecule according to any one of claims 21 to 24, wherein AB1 binds to LRP5 and / or LRP6, and AB2 and AB3 each independently bind to one or more FZD receptors.

26. AB1 is VHH; AB2 are VH and VL connected via a linker in any order; AB3 are VH and VL connected via a linker in any order; AB4 is VHH; n = 1; m = 1; p = 1; q = 1, X, Y, and Z exist, The molecule according to claim 13, wherein the VH and VL of AB2 are bonded to the VL and VH of AB3 to form a diamond body.

27. The molecule according to claim 26, wherein the VH and VL of AB2 are the same as the VH and VL of AB3.

28. The molecule according to claim 26, wherein the VH and VL of AB2 are different from the VH and VL of AB3.

29. The molecule according to claim 28, wherein the VH of AB2 binds to the VL of AB3 to form a first antigen-binding domain, the VL of AB2 binds to the VH of AB3 to form a second antigen-binding domain, the first antigen-binding domain binds to a first set of one or more antigens, the second antigen-binding domain binds to a second set of one or more antigens, and the first and second sets are the same or different antigens, or different sets of antigens, which may overlap as needed.

30. The molecule according to any one of claims 26 to 29, wherein, for each of AB2 and AB3, VH is located on the amino-terminal side relative to VL.

31. The molecule according to any one of claims 26 to 29, wherein, for each of AB2 and AB3, VL is located on the amino-terminal side relative to VH.

32. The molecule according to any one of claims 26 to 31, wherein the VHH of AB1 is the same as the VHH of AB4.

33. The molecule according to claim 32, wherein the molecule is a triple-specific tetravalent bond molecule.

34. The molecule according to any one of claims 26 to 32, wherein the VHH of AB1 is different from the VHH of AB4.

35. The molecule according to claim 34, wherein the molecule is a quadruple-specific tetravalent bond molecule.

36. The molecule according to any one of claims 26 to 35, wherein AB1 and AB4 are bound to LRP5 and / or LRP6, and the diabody is bound to one or more FZD receptors.

37. AB1 is VHH; AB2 is scFv; AB3 is scFv; AB4 is VHH; n = 1; m = 1; p = 1; q = 1; The molecule according to claim 13, wherein X, Y, and Z are present.

38. The molecule according to claim 37, wherein the scFv of AB2 is the same as the scFv of AB3.

39. The molecule according to claim 38, wherein the molecule is a tetravalent bispecific binding molecule.

40. The molecule according to claim 37, wherein the scFv of AB2 is not the same as the scFv of AB3.

41. The molecule according to any one of claims 37 to 40, wherein the VHH of AB1 is the same as the VHH of AB4.

42. The molecule according to any one of claims 37 to 40, wherein the VHH of AB1 is not the same as the VHH of AB4.

43. The molecule according to claim 42, wherein the molecule is a tetravalent quadruspecific polypeptide or a tetravalent tripuspecific polypeptide.

44. The molecule according to any one of claims 38 to 43, wherein AB1 and AB4 each independently bind to LRP5 and / or LRP6, and AB2 and AB3 each independently bind to one or more FZD receptors.

45. AB1 is VHH; AB2 is scFv; AB3 is VHH; AB4 is scFv; n = 1; m = 1; p = 1; q = 1; The molecule according to claim 13, wherein X, Y, and Z are present.

46. The molecule according to claim 45, wherein AB2 and AB4 are the same.

47. The molecule according to claim 46, wherein AB1 and AB3 are the same, and the molecule is a tetravalent bispecific binding molecule.

48. The molecule according to claim 46, wherein AB1 and AB3 are not the same, and the molecule is a tetravalent triple-specific binding molecule.

49. The molecule according to any one of claims 45 to 48, wherein AB2 and AB4 are not the same.

50. The molecule according to claim 49, wherein AB1 and AB3 are the same, and the molecule is a tetravalent triple-specific binding molecule.

51. The molecule according to claim 49, wherein AB1 and AB3 are not the same, and the molecule is a tetravalent quadruspecific binding molecule.

52. The molecule according to any one of claims 44 to 51, wherein AB1 and AB3 each independently bind to LRP5 and / or LRP6, and AB2 and AB4 each independently bind to one or more FZD receptors.

53. AB1 is scFv; AB2 is VHH; AB3 is scFv; n = 1; m = 1; p = 1; q = 0; The molecule according to claim 13, wherein X and Y are present, and Z is not present.

54. The molecule according to claim 53, wherein AB1 and AB3 are the same.

55. The molecule according to claim 54, wherein the molecule is a trivalent bispecific binding molecule.

56. The molecule according to claim 53, wherein AB1 and AB3 are not the same.

57. The molecule according to claim 53, wherein the molecule is a trivalent triple-specific binding molecule.

58. The molecule according to any one of claims 53 to 57, wherein AB1 and AB3 each independently bind to one or more FZD receptors, and AB2 binds to LRP5 and / or LRP6.

59. AB1 is VHH; AB2 is VHH; AB3 is VHH; n = 1; m = 1; p = 1; q = 0; The molecule according to claim 13, wherein X and Y are present, and Z is not present.

60. The molecule according to claim 59, wherein AB1, AB2, and AB3 each independently bind to LRP5 and / or LRP6, or one or more FZD receptors.

61. The molecule according to any one of claims 59 to 60, wherein AB2 and AB3, or AB1 and AB2, or AB1 and AB3 are the same.

62. The molecule according to claim 61, wherein the molecule is a trivalent bispecific binding molecule.

63. The molecule according to any one of claims 59 to 62, wherein AB2 and AB3, AB1 and AB2, or AB1 and AB3 are not the same.

64. The molecule according to claim 63, wherein the molecule is a trivalent triple-specific binding molecule.

65. The molecule according to claim 63 or claim 64, wherein AB1, AB2, and AB3 each independently bind to the same or different FZD receptors, or to different, optionally overlapping sets of FZD receptors.

66. AB1 is VHH; AB2 is VHH; AB3 is VHH; AB4 is VHH; n = 1; m = 1; p = 1; q = 1; The molecule according to claim 13, wherein X, Y, and Z are present.

67. The molecule according to claim 66, wherein the molecule is a tetravalent bispecific binding molecule having two VHHs that bind to the same FZD receptor and two VHHs that each bind to LRP5 and / or LRP6.

68. The molecule according to claim 66, wherein the molecule is a tetravalent, triple-specific binding molecule having two VHHs that bind to different FZD receptors or different epitopes on the same FZD receptor, and two VHHs that bind to the same LRP5 and / or LRP6.

69. The molecule according to claim 66, wherein the molecule is a tetravalent, triple-specific binding molecule having two identical VHHs that bind to the FZD receptor and two different VHHs that bind to different LRP5 and / or LRP6, or to different epitopes of the same LRP5 or LRP6.

70. The molecule according to claim 66, wherein the molecule is a tetravalent, quadruple-specific binding molecule having two VHHs that each bind to a different FZD receptor or a different combination of FZD receptors, and two VHHs that each bind to a different LRP5 and / or LRP6.

71. The molecule according to any one of claims 1 to 70, wherein the molecule modulates the Wnt signaling pathway in cells, and optionally in mammalian cells.

72. The molecule according to claim 71, wherein the molecule increases signal transduction via the Wnt signaling pathway in the cell.

73. The molecule according to claims 71 to 72, wherein the Wnt signaling pathway is a standard Wnt signaling pathway.

74. The molecule according to claims 71 to 72, wherein the Wnt signaling pathway is a non-standard Wnt signaling pathway.

75. A molecule according to any one of claims 74, comprising a polypeptide shown in any one of Sequence IDs 1 to 56 or a polypeptide having at least 90% or 95% identity with respect to an internal domain of any one of Sequence IDs 1 to 56.

76. A polypeptide comprising a CDR present in any one of SEQ ID NOs: 1 to 56, or a variant thereof having less than one, less than two, less than three, less than four, less than five, less than six, less than seven, or less than eight amino acid substitutions within the CDR of the polypeptide, according to any one of claims 1 to 75.

77. Isolated polynucleotides encoding a polypeptide of a molecule according to any one of claims 1 to 76, wherein the isolated polynucleotide is optionally mRNA and optionally modified mRNA.

78. An expression vector comprising an isolated polynucleotide as described in claim 77.

79. An isolated host cell comprising the expression vector according to claim 78, wherein the isolated host cell is a prokaryotic cell or a eukaryotic cell.

80. The host cell according to claim 79, which is a bacterial cell, a fungal cell, or a mammalian cell.

81. A pharmaceutical composition comprising a physiologically acceptable additive, diluent or carrier, and a therapeutically effective amount of a molecule according to any one of claims 1 to 76, a polynucleotide according to claim 77, an expression vector according to claim 88, or a host cell according to claim 79 or 80.

82. A method for stimulating the Wnt signaling pathway in a cell, comprising contacting the cell with a molecule according to any one of claims 1 to 76 or a pharmaceutical composition according to claim 81, wherein the molecule is an agonist of the Wnt signaling pathway.

83. A method for treating a disease or disorder related to a decrease or dysfunction of Wnt signaling in a subject requiring treatment of such disease or disorder, comprising providing the subject with a molecule according to any one of claims 1 to 76 or a pharmaceutical composition according to claim 81, wherein the molecule is an agonist of the Wnt signaling pathway.

84. The aforementioned disease or disorder is a fracture, stress fracture, vertebral compression fracture, osteoporosis, osteoporotic fracture, non-union fracture, delayed union fracture.Fracture, spinal fusion, preoperative optimization for spinal surgery, osteonecrosis, osseointegration of implants or orthopedic devices, osteogenesis imperfecta, bone grafting, tendon repair, tendon-osseointegration, tooth growth and regeneration, salivary gland disorders, maxillofacial surgery, dental implants, periodontal disease, maxillofacial reconstruction, osteonecrosis of the jaw, hip or femoral head, avascular necrosis, alopecia, hearing loss, vestibular dysfunction, macular degeneration, age-related macular degeneration (AMD), vitreoretinopathy, retinopathy, diabetic retinopathy, retinal degenerative diseases, Fuchs dystrophy, corneal diseases, dry eye and lacrimal gland disorders including Sjögren's syndrome, stroke, traumatic brain injury, Alzheimer's disease, multiple sclerosis, muscular dystrophy, muscle atrophy due to sarcopenia or cachexia, diseases affecting the blood-brain barrier (BBB), spinal cord injury, spinal cord diseases, oral mucositis, short bowel syndrome, Crohn's disease and ulcerative colitis, inflammatory bowel disease (IBD), metabolic syndrome, diabetes mellitus, dyslipidemia, pancreatitis, exocrine pancreatic insufficiency, impaired wound healing, diabetic wound healing, diabetic foot ulcers, pressure ulcers, venous lower extremity ulcers Cerebral ulcer, epidermolysis bullosa, cutaneous dysplasia, myocardial infarction, coronary artery disease, heart failure, hematopoietic cell damage, immunodeficiency, graft-versus-host disease, acute kidney injury, chronic kidney disease, chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis, acute liver failure of any cause, drug-induced acute liver failure, alcoholic liver disease, chronic liver failure of any cause, cirrhosis, hepatic fibrosis of any cause, portal hypertension, chronic liver dysfunction of any cause, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD) (fatty liver), alcoholic The method according to claim 83, selected from the group consisting of hepatitis, hepatitis C virus-induced liver disease (HCV), hepatitis B virus-induced liver disease (HBV), other viral hepatitis (e.g., hepatitis A virus-induced liver disease (HAV) and hepatitis D virus-induced liver disease (HDV)), primary biliary cirrhosis, autoimmune hepatitis, liver surgery, liver injury, liver transplantation, "slightly under-grafted" syndrome in liver surgery and transplantation, congenital liver disease and disorder, genetic disorder, degeneration, aging, drugs and injury, or any other liver disorder or defect.

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