Anti-frizzled antibodies and methods of use

Abstract

To provide anti-frizzled antibodies and methods of use.SOLUTION: The present invention provides anti-Fzd monoclonal antibodies and related compositions, which may be used in any of a variety of therapeutic methods for the treatment of diseases. In one embodiment, the disclosure provides an isolated antibody, or an antigen-binding fragment thereof, that binds to one or more frizzled receptor, comprising a sequence comprising (i) CDRH1, CDRH2 and CDRH3 sequences set forth for any of the antibodies of Table 1A and / or (ii) CDRL1, CDRL2 and CDRL3 sequences set forth for any of the antibodies of Table 1A, or a variant of the antibody, or an antigen-binding fragment thereof, comprising one or more amino acid modifications, wherein the variant comprises less than 8 amino acid substitutions in the CDR sequences.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 607,877, filed December 19, 2017, and U.S. Provisional Application No. 62 / 680,508, filed June 4, 2018, both of which are incorporated herein by reference in their entireties.

[0002] Sequence Listing Description The sequence listing accompanying the present invention is provided in text format in lieu of a paper copy and is incorporated herein by reference. The name of the text file containing the sequence listing is SRZN_004_02WO_ST25.txt. This text file is 527 KB, was created on December 19, 2018, and has been submitted electronically via EFS-Web.

[0003] Technical Field The present invention generally comprises anti-Frizzled antibodies and antigen-binding fragments thereof, compositions, and methods of using same. Such antibodies are useful, for example, in modulating the Wnt signaling pathway. [Background technology]

[0004] Wnt ("Wingless-associated integration site" or "Wingless and Int-1" or "Wingless-Int") ligands and their signals play important roles in regulating 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 (reviewed, e.g., by Clevers, Loh, and Nusse, 2014;346:1248012). Modulation of the Wnt signaling pathway has potential for the treatment of degenerative diseases and tissue injury. One of the challenges of modulating Wnt signaling as a therapeutic agent is the existence of multiple Wnt ligands and Wnt receptors, Frizzled 1-10 (Fzd1-10), and many tissues express multiple overlapping Fzds. Furthermore, in addition to Fzds, canonical Wnt signaling also involves low-density lipoprotein (LDL) receptor-related protein 5 (LRP5) or low-density lipoprotein (LDL) receptor-related protein 6 (LRP6), which are widely expressed in various tissues. Thus, there is a clear need in the art for binding moieties (e.g., antibodies) that specifically bind to one or more Fzds, LRP5, or LRP6. The present invention addresses this need. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Clevers,Loh,and Nusse,2014;346:1248012 Summary of the Invention [Means for solving the problem]

[0006] In various embodiments, the present invention provides anti-Fzd antibodies and antigen-binding fragments thereof and related methods of use.

[0007] In one embodiment, the disclosure provides an isolated antibody or antigen-binding fragment thereof that binds to one or more Frizzled receptors, the antibody or antigen-binding fragment thereof comprising a sequence comprising: (i) the CDRH1, CDRH2, and CDRH3 sequences set forth for any antibody in Table 1A, and / or (ii) the CDRL1, CDRL2, and CDRL3 sequences set forth for any antibody in Table 1A, or a variant or antigen-binding fragment thereof comprising one or more amino acid modifications, wherein the variant or antigen-binding fragment thereof comprises fewer than eight amino acid substitutions in the CDR sequences. In a specific embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence having at least 90% identity to the amino acid sequence set forth in any of SEQ ID NOs: 1-37, 66, or 68, or a heavy chain variable region comprising the amino acid sequence set forth in any of SEQ ID NOs: 1-37, 66, or 68. In certain embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising an amino acid sequence having at least 90% identity to the amino acid sequence set forth in any of SEQ ID NOs: 38-65, 67, or 69, or a light chain variable region comprising the amino acid sequence set forth in any of SEQ ID NOs: 38-65, 67, or 69.

[0008] In certain embodiments, any antibody or antigen-binding fragment thereof is humanized. In certain embodiments, any antibody or antigen-binding fragment thereof is a single-chain antibody, scFv, a monovalent antibody lacking a hinge region, a VHH or a single domain antibody (sdAb), or a minibody. In certain embodiments, any antibody or antigen-binding fragment thereof is a Fab or Fab' fragment.

[0009] In certain embodiments, any antibody or antigen-binding fragment thereof is a fusion protein. In certain embodiments, the antibody or antigen-binding fragment thereof is fused with a polypeptide sequence that binds to LRP5 or LRP6. In certain embodiments, the polypeptide sequence that binds to LRP5 or LRP6 is an antibody or antigen-binding fragment thereof that binds to LRP5 or LRP6.

[0010] In certain embodiments of any antibody or antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof binds to one or more of Frizzled 1 (Fzd1), Frizzled 2 (Fzd2), Frizzled 3 (Fzd3), Frizzled 4 (Fzd4), Frizzled 5 (Fzd5), Frizzled 6 (Fzd6), Frizzled 7 (Fzd7), Frizzled 8 (Fzd8), Frizzled 9 (Fzd9), and Frizzled 10 (Fzd10). In certain embodiments, any antibody or antigen-binding fragment thereof binds to two or more of Frizzled 1 (Fzd1), Frizzled 2 (Fzd2), Frizzled 3 (Fzd3), Frizzled 4 (Fzd4), Frizzled 5 (Fzd5), Frizzled 6 (Fzd6), Frizzled 7 (Fzd7), Frizzled 8 (Fzd8), Frizzled 9 (Fzd9), and Frizzled 10 (Fzd10). In certain embodiments, any antibody or antigen-binding fragment thereof binds to two or more of: (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, Fzd4, Fzd5, and Fzd8; (ix) Fzd5, Fzd8, and Fzd10; (x) Fzd4, Fzd5, and Fzd8; (xi) Fzd1, Fzd5, Fzd7, and Fzd8; or (xii) Fzd1, Fzd2, Fzd4, Fzd5, Fzd7, and Fzd8.

[0011] In a related embodiment, the present disclosure provides an isolated antibody or antigen-binding fragment thereof that competes with any of the antibodies disclosed herein for binding to a human Fzd receptor.

[0012] In certain embodiments, any antibody or antigen-binding fragment thereof binds to Fzd with a KD of 50 μM or less.

[0013] In certain embodiments, any antibody or antigen-binding fragment thereof regulates the Wnt signaling pathway in a cell, optionally in a mammalian cell. In certain embodiments, any antibody or antigen-binding fragment thereof increases signaling via the Wnt signaling pathway in the cell. In certain embodiments, any antibody or antigen-binding fragment thereof decreases signaling via the Wnt signaling pathway in the cell. In certain embodiments, the Wnt signaling pathway is the canonical Wnt signaling pathway or a non-canonical Wnt signaling pathway.

[0014] In further related embodiments, the present disclosure provides isolated polynucleotides encoding the antibodies or antigen-binding fragments thereof disclosed herein. In certain embodiments, the present disclosure provides expression vectors comprising the isolated polynucleotides, and isolated host cells comprising the expression vectors.

[0015] In another embodiment, the present disclosure provides a pharmaceutical composition comprising a physiologically acceptable excipient, diluent, or carrier and a therapeutically effective amount of an isolated antibody or antigen-binding fragment thereof disclosed herein.

[0016] In a further embodiment, the present disclosure provides a method for stimulating the Wnt signaling pathway in a cell, comprising contacting the cell with an isolated antibody or antigen-binding fragment thereof disclosed herein that increases Wnt signaling. In certain embodiments, the antibody or antigen-binding fragment thereof is a fusion protein comprising a polypeptide sequence that binds to LRP5 or LRP6.

[0017] In another embodiment, the present disclosure provides a method for inhibiting the Wnt signaling pathway in a cell, comprising contacting the cell with an isolated antibody or antigen-binding fragment thereof that inhibits Wnt signaling, as disclosed herein.

[0018] In another embodiment, the present disclosure includes a method for treating a subject having a disease or disorder associated with reduced Wnt signaling, comprising administering to the subject an effective amount of a pharmaceutical composition comprising an isolated antibody or antigen-binding fragment thereof that is an agonist of the Wnt signaling pathway, as disclosed herein.In certain embodiments, the disease or disorder is bone fracture, stress fracture, vertebral compression fracture, osteoporosis, osteoporotic fracture, nonunion 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, 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, stroke, traumatic brain injury, Alzheimer's disease, multiple sclerosis, diseases affecting the blood-brain barrier (BBB), spinal cord injury, spinal cord diseases, oral mucositis, short bowel syndrome, inflammatory bowel disease (IBD), Crohn's disease (CD), ulcerative colitis (UC), especially CD with fistula formation, metabolic syndrome, dyslipidemia, diabetes, pancreatitis, exocrine pancreatic disease dysfunction, wound healing, diabetic foot ulcers, pressure ulcers, venous leg ulcers, epidermolysis bullosa, cutis hypoplasia, myocardial infarction, coronary artery disease, heart failure, hematopoietic cell disorders, 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, liver fibrosis of any cause, portal hypertension, chronic liver failure of any cause, non-alcoholic and / or steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD) (fatty 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, "small for size" syndrome in liver surgery and transplantation, congenital liver disease and liver injury, genetic disease, degeneration, aging, drugs, or any other liver disorder or detection due to injury.

[0019] In another related embodiment, the present disclosure provides a method for treating or preventing a bone disease or bone disorder in a subject in need thereof, comprising providing to the subject an effective amount of a pharmaceutical composition comprising an isolated antibody or antigen-binding fragment thereof disclosed herein that is an agonist of the Wnt signaling pathway. In certain embodiments, the isolated antibody or antigen-binding fragment thereof binds to Fzd1, Fzd2, and FZD7. In certain embodiments, the isolated antibody or antigen-binding fragment thereof binds to Fzd1, Fzd2, FZD7, Fzd5, and Fzd8. Other Fzd molecules that bind to additional Fzd receptors can also be used in conjunction with the LRP5 and / or LRP6 binding agent.

[0020] In another related embodiment, the present disclosure provides a method for increasing bone mineral density, increasing bone volume, increasing bone cortical thickness, increasing bone mineral apposition rate, increasing bone stiffness, increasing bone biomechanical strength, increasing resistance to fracture, or reducing bone loss associated with osteoporosis in a subject in need thereof, comprising providing to the subject an effective amount of a pharmaceutical composition comprising an isolated antibody or antigen-binding fragment thereof that is an agonist of the Wnt signaling pathway, as disclosed herein. In certain embodiments, the isolated antibody or antigen-binding fragment thereof binds to Fzd1, Fzd2, and FZD7. In certain embodiments, the isolated antibody or antigen-binding fragment thereof binds to Fzd1, Fzd2, FZD7, Fzd5, and Fzd8.

[0021] In a related embodiment, the present disclosure provides a method for treating a subject having a disease or disorder associated with increased or enhanced Wnt signaling, comprising administering to the subject an effective amount of a pharmaceutical composition comprising an isolated antibody or antigen-binding fragment thereof disclosed herein that is an inhibitor of the Wnt signaling pathway. In certain embodiments, the disease or disorder is selected from the group consisting of tumors and cancers, degenerative disorders, fibrosis, heart failure, coronary artery disease, heterotopic bone formation, osteoporosis, and congenital high bone mass disorders.

[0022] In further related embodiments, the present disclosure provides isolated antibodies or antigen-binding fragments thereof that bind to one or more Frizzled receptors, wherein the isolated antibodies or antigen-binding fragments thereof bind to an epitope within a region of Frizzled 1 comprising or consisting of amino acid residues 115-230, an epitope within a region of Frizzled 3 comprising or consisting of amino acid residues 29-78, an epitope within a region of Frizzled 4 comprising or consisting of amino acid residues 50-147, an epitope within a region of Frizzled 5 comprising or consisting of amino acid residues 37-149, an epitope within a region of Frizzled 8 comprising or consisting of amino acid residues 55-137, an epitope within a region of Frizzled 9 comprising or consisting of amino acid residues 59-152, or an epitope within a region of Frizzled 10 comprising or consisting of amino acid residues 35-124.

[0023] In certain embodiments, the present disclosure provides an isolated antibody or antigen-binding fragment thereof that binds to one or more frizzled receptors, wherein the antibody or antigen-binding fragment thereof contacts the frizzled receptor at any pair of amino acid residues shown in Table 3 at a distance of less than 5 angstroms. In certain embodiments, for example, the following are provided: (Item 1) 1. An isolated antibody or antigen-binding fragment thereof that binds to one or more Frizzled receptors, comprising the sequence: (i) the CDRH1, CDRH2, and CDRH3 sequences set forth for any antibody in Table 1A; (ii) the CDRL1, CDRL2, and CDRL3 sequences set forth for any antibody in Table 1A, and / or (iii) an isolated antibody or antigen-binding fragment thereof comprising a CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 sequence set forth in Table 2; Alternatively, a variant of said antibody or antigen-binding fragment thereof comprising one or more amino acid modifications, wherein said variant of said antibody or antigen-binding fragment thereof comprises fewer than eight amino acid substitutions in said CDR sequences. (Item 2) 2. The isolated antibody or antigen-binding fragment thereof according to Item 1, comprising a heavy chain variable region comprising an amino acid sequence having at least 90% identity to any of the amino acid sequences set forth in SEQ ID NOs: 1 to 37, 66, 68, or 1477. (Item 3) Item 4. The isolated antibody or antigen-binding fragment thereof according to Item 2, comprising a heavy chain variable region comprising an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 37, 66, 68, or 1477. 4. The isolated antibody or antigen-binding fragment thereof according to any one of Items 1 to 3, comprising a light chain variable region comprising an amino acid sequence having at least 90% identity to the amino acid sequence set forth in any one of SEQ ID NOs: 38 to 65, 67, 69, or 1476. (Item 5) 5. The isolated antibody or antigen-binding fragment thereof according to Item 4, comprising a light chain variable region comprising an amino acid sequence set forth in any one of SEQ ID NOs: 38 to 65, 67, 69, or 1476. (Item 6) 6. The isolated antibody or antigen-binding fragment thereof according to any of items 1 to 5, wherein the antibody or antigen-binding fragment thereof is humanized. (Item 7) 7. The isolated antibody or antigen-binding fragment thereof according to any one of items 1 to 6, wherein the antibody or antigen-binding fragment thereof is a single-chain antibody, an scFv, a monovalent antibody lacking a hinge region, a VHH or sdAb, or a minibody. (Item 8) 8. The isolated antibody or antigen-binding fragment thereof of item 7, wherein the antibody or antigen-binding fragment thereof is a VHH or an sdAb. (Item 9) 2. The isolated antibody or antigen-binding fragment thereof of item 1, wherein the antibody or antigen-binding fragment thereof is a Fab or Fab' fragment. (Item 10) 10. The isolated antibody or antigen-binding fragment thereof according to any one of items 1 to 9, wherein the antibody or antigen-binding fragment thereof is a fusion protein. (Item 11) 11. The isolated antibody or antigen-binding fragment thereof of claim 10, wherein the antibody or antigen-binding fragment thereof is fused to a polypeptide sequence that binds to LRP5 or LRP6. (Item 12) 12. The isolated antibody or antigen-binding fragment thereof of item 11, wherein the polypeptide sequence that binds to LRP5 or LRP6 is an antibody or antigen-binding fragment thereof that binds to LRP5 or LRP6. (Item 13) 13. The isolated antibody or antigen-binding fragment thereof according to any of items 1 to 12, wherein the antibody or antigen-binding fragment thereof binds to one or more of Frizzled 1 (Fzd1), Frizzled 2 (Fzd2), Frizzled 3 (Fzd3), Frizzled 4 (Fzd4), Frizzled 5 (Fzd5), Frizzled 6 (Fzd6), Frizzled 7 (Fzd7), Frizzled 8 (Fzd8), Frizzled 9 (Fzd9), and Frizzled 10 (Fzd10). (Item 14) 14. The isolated antibody or antigen-binding fragment thereof of item 13, wherein the antibody or antigen-binding fragment thereof binds to two or more of Frizzled 1 (Fzd1), Frizzled 2 (Fzd2), Frizzled 3 (Fzd3), Frizzled 4 (Fzd4), Frizzled 5 (Fzd5), Frizzled 6 (Fzd6), Frizzled 7 (Fzd7), Frizzled 8 (Fzd8), Frizzled 9 (Fzd9), and Frizzled 10 (Fzd10). (Item 15) The antibody or antigen-binding fragment thereof is selected from the group consisting of: (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; and (vii) Fzd. (viii) Fzd9 and Fzd10; (ix) Fzd5, Fzd8, and Fzd10; (x) Fzd4, Fzd5, and Fzd8; (xi) Fzd1, Fzd5, Fzd7, and Fzd8; or (xii) Fzd1, Fzd2, Fzd4, Fzd5, Fzd7, and Fzd8. (Item 16) 16. An isolated antibody or antigen-binding fragment thereof that competes with the antibody of any of items 1 to 15 for binding to human Frizzled. (Item 17) 17. The isolated antibody or antigen-binding fragment thereof according to any one of items 1 to 16, which binds to the Fzd with a KD of 50 μM or less. (Item 18) 18. The isolated antibody or antigen-binding fragment thereof of any of items 1 to 17, which modulates the Wnt signaling pathway in a cell, optionally in a mammalian cell. (Item 19) 20. The isolated antibody or antigen-binding fragment thereof of item 18, which increases signaling through the Wnt signaling pathway in the cell. (Item 20) 20. The isolated antibody or antigen-binding fragment thereof of claim 18, which reduces signaling through the Wnt signaling pathway in the cell. (Item 21) 21. The isolated antibody or antigen-binding fragment thereof according to any of items 18 to 20, wherein the Wnt signaling pathway is the canonical Wnt signaling pathway. (Item 22) 21. The isolated antibody or antigen-binding fragment thereof according to any of items 18 to 20, wherein the Wnt signaling pathway is a non-canonical Wnt signaling pathway. (Item 23) 23. An isolated polynucleotide encoding the isolated antibody or antigen-binding fragment thereof according to any one of items 1 to 22. (Item 24) 24. An expression vector comprising the isolated polynucleotide of Item 23. (Item 25) 25. An isolated host cell comprising the expression vector of item 24. (Item 26) 37. A pharmaceutical composition comprising a physiologically acceptable excipient, diluent, or carrier and a therapeutically effective amount of the isolated antibody or antigen-binding fragment thereof according to any of items 1 to 22 or 36. (Item 27) 20. A method for stimulating the Wnt signaling pathway in a cell, comprising contacting the cell with the isolated antibody or antigen-binding fragment thereof of item 19. (Item 28) 27. The method of claim 26, wherein the antibody or antigen-binding fragment thereof is a fusion protein comprising a polypeptide sequence that binds to LRP5 or LRP6. (Item 29) 21. A method for inhibiting the Wnt signaling pathway in a cell, comprising contacting the cell with the isolated antibody or antigen-binding fragment thereof of paragraph 20. (Item 30) 27. A method for treating a subject having a disease or disorder associated with decreased Wnt signaling, comprising administering to the subject an effective amount of the pharmaceutical composition of item 26, wherein the isolated antibody or antigen-binding fragment thereof is an agonist of the Wnt signaling pathway. (Item 31) The disease or disorder is selected from the group consisting of fractures, stress fractures, vertebral compression fractures, osteoporosis, osteoporotic fractures, nonunion fractures, delayed union fractures, spinal fusion, preoperative optimization for spinal surgery, osteonecrosis, osseointegration of implants or orthopedic devices, osteogenesis imperfecta, bone grafting, tendon repair, tendon-osseous integration, tooth growth and regeneration, 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, and age-related macular degeneration. AMD, vitreoretinopathy, retinopathy, diabetic retinopathy, retinal degenerative diseases, Fuchs' dystrophy, corneal diseases, stroke, traumatic brain injury, Alzheimer's disease, multiple sclerosis, diseases affecting the blood-brain barrier (BBB), spinal cord injury, spinal cord diseases, oral mucositis, short bowel syndrome, inflammatory bowel disease (IBD), Crohn's disease (CD), ulcerative colitis (UC), especially CD with fistula formation, metabolic syndrome, dyslipidemia, diabetes, pancreatitis, exocrine pancreatic insufficiency, wound Wound healing, diabetic foot ulcers, pressure ulcers, venous leg ulcers, epidermolysis bullosa, cutis hypoplasia, myocardial infarction, coronary artery disease, heart failure, hematopoietic cell disorders, 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, liver fibrosis of any cause, portal hypertension, chronic liver failure of any cause, non-alcoholic fatty liver disease 31. The method of claim 30, wherein the liver damage is selected from the group consisting of nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD) (fatty 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 transplant, "small for size" syndrome in liver surgery and transplant, congenital liver disease and liver injury, genetic disease, degeneration, aging, drugs, or any other liver damage or detection due to injury. (Item 32) 32. The method of claim 30 or 31, wherein the disease or disorder is a bone disease or disorder and the Wnt surrogate molecule binds to Fzd1, Fzd2, and FZD7. (Item 33) 33. The method of item 32, wherein the Wnt surrogate molecule also binds to Fzd5 and Fzd8. (Item 34) 27. A method for treating a subject having a disease or disorder associated with increased or enhanced Wnt signaling, comprising administering to the subject an effective amount of the pharmaceutical composition of claim 26, wherein the isolated antibody or antigen-binding fragment thereof is an inhibitor of the Wnt signaling pathway. (Item 35) 32. The method according to item 31, wherein the disease or disorder is selected from the group consisting of tumors and cancers, degenerative disorders, fibrosis of any organ or tissue, idiopathic pulmonary fibrosis, renal fibrosis, heart failure, coronary artery disease, osteoarthritis, heterotopic ossification, osteoporosis, congenital high bone mass disorders. (Item 36) 1. An isolated antibody or antigen-binding fragment thereof that binds to one or more Frizzled receptors, (a) an epitope within a region of Frizzled 1 comprising or consisting of amino acid residues 115 to 230, or within the corresponding region of Fzd2 or Fzd7; (b) an epitope within a region of Frizzled 3 comprising or consisting of amino acid residues 29-78; (c) an epitope within a region of Frizzled 4 comprising or consisting of amino acid residues 50 to 147; (d) an epitope within a region of Frizzled 5 comprising or consisting of amino acid residues 37 to 149, or within the corresponding region of Fzd1, Fzd2, Fzd7, or Fzd8; (e) an epitope within a region of Frizzled 8 comprising or consisting of amino acid residues 55 to 137, or within the corresponding region of Fzd5; (f) an epitope within a region of Frizzled 9 comprising or consisting of amino acid residues 59 to 152, or within the corresponding region of Fzd10; or (g) An isolated antibody or antigen-binding fragment thereof that binds to an epitope within a region of Frizzled 10 comprising or consisting of amino acid residues 35 to 124. (Item 37) An isolated antibody or antigen-binding fragment thereof that binds to one or more frizzled receptors, wherein the antibody or antigen-binding fragment thereof contacts the frizzled receptor at any set of amino acid residues shown in Table 3 at a distance of less than 5 angstroms. [Brief explanation of the drawings]

[0024] [Figure 1] (A) Diagrammatic representation of the Fzd1:1RC07 complex. Fzd1 is shown in light gray, and the heavy and light chains of 1RC07 are shown in medium and dark gray, respectively. (B) A more detailed view of the Fzd1:1RC07 interface, with the CRD loop positions marked. (C) A more detailed view of Zn+2 binding with 2mFo-DFc (2.0σ) and the anomalous difference map (15.0σ), shown in blue and yellow meshes, respectively. [Figure 2] (A) Overall structure of the Fzd1:R2M9 complex. The molecular surface of Fzd1 is shown as a light gray transparent surface. The heavy and light chains of R2M9 are colored in dark and light black tones, respectively. Lipids (palmitoleic acid; PAM) observed in the structure of Wnt8:Fzd8 (PDB code: 4F0A) are shown as light gray spheres. (C) Detailed view of the Fzd1:R2M9 interface, with the positions of the CDR loops of H1, H2, and H3 of the heavy chain and L1, L2, and L3 of the light chain marked. [Figure 3] (A) Overall structure of the Fzd4:3SD10 complex. The molecular surface of Fzd4 is shown as a light gray transparent surface. The heavy and light chains of 3SD10 are colored in dark and light black shades. The lipid (palmitoleic acid; PAM) observed in the structure of Wnt8:Fzd8 (PDB code: 4F0A) is shown as a light gray sphere. (B) Detailed view of the Fzd4:3SD10 interface, with the position of the CRD loop marked. [Figure 4](A) Overall structure of the Fzd5:R2M3 complex. The molecular surface of Fzd5 is shown as a light gray transparent surface. The heavy and light chains of R2M3 are colored in dark and light black tones, respectively. Lipids (palmitoleic acid; PAM) observed in the structure of Wnt8:Fzd8 (PDB code: 4F0A) are shown as light gray spheres. (C) Detailed view of the Fzd5:R2M3 interface, with the positions of the CDR loops of H1, H2, and H3 of the heavy chain and L1, L2, and L3 of the light chain marked. [Figure 5] (A) Overall structure of the Fzd8:005S-H05 complex. The molecular surface of Fzd8 is shown as a light gray transparent surface. The heavy and light chains of 005S-H05 are colored in dark and light black tones, respectively. Lipids (palmitoleic acid; PAM) observed in the structure of Wnt8:Fzd8 (PDB code: 4F0A) are shown as light gray spheres. (C) Detailed view of the Fzd8:005S-H05 interface, with the positions of the CDR loops of H1, H2, and H3 of the heavy chain and L1, L2, and L3 of the light chain marked. [Figure 6] (A) Overall structure of the Fzd5:004S-E05 complex. The molecular surface of Fzd5 is shown as a light gray transparent surface. The heavy and light chains of 004S-E05 are colored in dark and light black tones, respectively. Lipids (palmitoleic acid; PAM) observed in the structure of Wnt8:Fzd8 (PDB code: 4F0A) are shown as light gray spheres. (C) Detailed view of the Fzd5:004S-E05 interface, with the positions of the CDR loops of H1, H2, and H3 of the heavy chain and L1, L2, and L3 of the light chain marked. [Figure 7](A) Overall structure of the Fzd5:4A12 complex. The molecular surface of Fzd5 is shown as a light gray transparent surface. The heavy and light chains of 4A12 are colored in dark and light black tones, respectively. Lipids (palmitoleic acid; PAM) observed in the structure of Wnt8:Fzd8 (PDB code: 4F0A) are shown as light gray spheres. (C) Detailed view of the Fzd5:4A12 interface, with the positions of the CDR loops of H1, H2, and H3 of the heavy chain and L1, L2, and L3 of the light chain marked. [Figure 8] (A) Overall structure of the Fzd9:014S-B06 complex. The molecular surface of Fzd5 is shown as a light gray transparent surface. The heavy and light chains of 014S-B06 are colored in dark and light black tones, respectively. Lipids (palmitoleic acid; PAM) observed in the structure of Wnt8:Fzd8 (PDB code: 4F0A) are shown as light gray spheres. (C) Detailed view of the Fzd5:014S-B06 interface, with the positions of the CDR loops of H1, H2, and H3 of the heavy chain and L1, L2, and L3 of the light chain marked. [Figure 9] (A) Overall structure of the Fzd10:005S-A07 complex. The molecular surface of Fzd5 is shown as a light gray transparent surface. The heavy and light chains of 005S-A07 are colored in dark and light black tones, respectively. Lipids (palmitoleic acid; PAM) observed in the Wnt8:Fzd8 (PDB code: 4F0A) structure are shown as light gray spheres. (C) Detailed view of the Fzd10:005S-A07 interface, with the positions of the CDR loops of H1, H2, and H3 of the heavy chain and L1, L2, and L3 of the light chain marked. [Figure 10](A) Overall structure of the Fzd10:005S-E12 complex. The molecular surface of Fzd5 is shown as a light gray transparent surface. The heavy and light chains of 005S-E12 are colored in dark and light black tones, respectively. Lipids (palmitoleic acid; PAM) observed in the structure of Wnt8:Fzd8 (PDB code: 4F0A) are shown as light gray spheres. (C) Detailed view of the Fzd10:005S-E12 interface, with the positions of the CDR loops of H1, H2, and H3 of the heavy chain and L1, L2, and L3 of the light chain marked. [Figure 11] (A) Overall structure of the Fzd3:029S-E03 complex. The molecular surface of Fzd5 is shown as a light gray transparent surface. The heavy and light chains of 029S-E03 are colored in dark and light black tones, respectively. Lipids (palmitoleic acid; PAM) observed in the structure of Wnt8:Fzd8 (PDB code: 4F0A) are shown as light gray spheres. (C) Detailed view of the Fzd3:029S-E03 interface, with the positions of the CDR loops of H1, H2, and H3 of the heavy chain and L1, L2, and L3 of the light chain marked. DETAILED DESCRIPTION OF THE INVENTION

[0025] The present disclosure relates to antibodies and antigen-binding fragments thereof that specifically bind to one or more Fzd receptors, including antibodies with particular Fzd receptor specificity and / or functional properties. One embodiment of the present invention encompasses certain humanized antibodies and fragments thereof that can bind to one or more Fzd receptors and modulate downstream Wnt pathway signaling and associated biological effects.

[0026] Embodiments of the present invention relate to the use of anti-Fzd antibodies, or antigen-binding fragments thereof, for the diagnosis, evaluation, and treatment of diseases and disorders associated with the Wnt signaling pathway. In certain embodiments, the subject antibodies and antigen-binding fragments thereof are used to modulate the Wnt signaling pathway in cells or tissues. In certain embodiments, the subject antibodies and antigen-binding fragments thereof are used in the treatment or prevention of diseases and disorders associated with abnormal or dysregulated (e.g., increased or decreased) Wnt signaling, or in which decreasing or increasing Wnt signaling is believed to provide a therapeutic benefit.

[0027] The practice of the present invention will employ, unless expressly indicated to the contrary, conventional methods of virology, immunology, microbiology, molecular biology, and recombinant DNA techniques within the skill of those in the art, many of which are described below for illustrative purposes. Such techniques are fully explained in the literature, e.g., 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 rd 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 See 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.

[0028] As used in this specification and the appended claims, the singular forms "a / an" and "the" include plural referents unless the content clearly dictates otherwise.

[0029] Throughout this specification, unless the context requires otherwise, "comprise" or variations such as "comprises" or "comprising" should be understood to mean the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

[0030] Each embodiment herein is intended to apply mutatis mutandis to every other embodiment unless otherwise stated.

[0031] Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques can be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. These and related techniques and procedures can generally be performed according to conventional methods known in the art and as described in various general and more specific references cited and discussed throughout the specification. Unless specific definitions are provided, the nomenclature utilized in connection with molecular biology, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein, as well as the laboratory procedures and techniques within these fields, are those well known and commonly used in the art. Standard techniques can be used for recombinant technology, molecular biology, microbiology, chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, and delivery, and treatment of subjects.

[0032] Embodiments of the present invention relate to antibodies and antigen-binding fragments thereof that bind to one or more Fzd receptors. Exemplary antibodies, or antigen-binding fragments or complementarity-determining regions (CDRs) thereof, are set forth in Table 1A and SEQ ID NOs: 1-65.

[0033] As is well known in the art, an antibody is an immunoglobulin molecule capable of specifically binding to a target (e.g., carbohydrate, polynucleotide, lipid, polypeptide, etc.) via at least one epitope recognition site within the variable region of the immunoglobulin molecule. As used herein, the term encompasses not only intact polyclonal or monoclonal antibodies, but also fragments thereof (e.g., dAb, Fab, Fab', F(ab')2, Fv), single chain (scFv), VHH or sdAb (also known as nanobodies), synthetic variants thereof, naturally occurring variants, fusion proteins comprising an antibody or its antigen-binding fragment, humanized antibodies, chimeric antibodies, and any other modified configuration of an immunoglobulin molecule comprising an antigen-binding site or fragment (epitope recognition site) with the required specificity. "Diabodies," which are multivalent or multispecific fragments constructed by gene fusion (WO 94 / 13804; P. Holliger et al., Proc. Natl. Acad. Sci. USA 90 6444-6448, 1993), are also a specific form of antibody contemplated herein. Minibodies, including scFvs linked to CH3 domains, are also included herein (S. Hu et al., Cancer Res., 56, 3055-3061, 1996). For example, Ward,ESet 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. 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 See Res.,56,3055-3061,1996.

[0034] The term "antigen-binding fragment," as used herein, refers to a polypeptide fragment comprising at least one CDR of an immunoglobulin heavy and / or light chain that binds to an antigen of interest, particularly one or more Fzd receptors. In this regard, antigen-binding fragments of the antibodies described herein can comprise one, two, three, four, five, or all six CDRs of the VH and VL sequences described herein from an antibody that binds to one or more Fzd receptors. Antigen-binding fragments of the Fzd-specific antibodies described herein are capable of binding to an Fzd receptor. As used herein, the term encompasses not only isolated fragments but also polypeptides comprising antigen-binding fragments of the antibodies disclosed herein, for example, fusion proteins comprising antigen-binding fragments of the antibodies disclosed herein.

[0035] In certain embodiments, the antibody or antigen-binding fragment thereof modulates Wnt signaling events in cells contacted with the antibody or antigen-binding fragment. In certain embodiments, the antibody or antigen-binding fragment thereof increases Wnt signaling, while in other embodiments, it decreases Wnt signaling. In certain embodiments, the antibody or antigen-binding fragment thereof specifically binds to or modulates the biological activity of the human Wnt signaling pathway.

[0036] The term "antigen" refers to a molecule or portion of a molecule that can be bound by a selective binding agent (e.g., an antibody) and that can be used in an animal to generate antibodies capable of binding to an epitope of that antigen. In certain embodiments, an antibody is said to specifically bind an antigen if it preferentially recognizes the target antigen within a complex mixture of proteins and / or macromolecules. In certain embodiments, an antibody has an equilibrium dissociation constant of ≦10 -7 or 10 -8 It is said to specifically bind to an antigen when M. In some embodiments, the equilibrium dissociation constant is ≦10 -9 M or 10 -10It can be M.

[0037] In certain embodiments, the antibodies and antigen-binding fragments thereof described herein comprise a set of heavy and light chain CDRs, each interposed between a set of heavy and light chain framework regions (FRs). The set of FRs supports the CDRs and defines their spatial relationship to one another. As used herein, the term "CDR set" refers to the three hypervariable regions of a heavy or light chain V region. These regions are designated "CDR1," "CDR2," and "CDR3," respectively, proceeding from the N-terminus of the heavy or light chain. Thus, an antigen-binding site comprises six CDRs, including the CDR sets from each of the heavy and light chain V regions. A polypeptide comprising a single CDR (e.g., CDR1, CDR2, or CDR3) is referred to herein as a "molecular recognition unit." Crystallographic analysis of multiple antigen-antibody complexes has demonstrated that amino acid residues in the CDRs form extensive contacts with the bound antigen, with the most extensive antigen contact occurring through heavy chain CDR3. Thus, the molecular recognition unit is primarily responsible for the specificity of the antigen-binding site.

[0038] As used herein, the term "FR set" refers to four adjacent amino acid sequences that frame the CDRs of a CDR set of a heavy or light chain V region. While some FR residues may contact the bound antigen, FRs are primarily responsible for folding the V region into the antigen-binding site, specifically the FR residues directly adjacent to the CDRs. Within FRs, certain amino acid 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 CDRs are presented as protruding loop motifs that form the antigen-binding surface. Generally, regardless of the detailed CDR amino acid sequence, it is recognized that there are conserved structural regions of FRs that influence the folded shape of the CDR loops to adopt a specific "canonical" structure. Furthermore, certain FR residues are known to participate in noncovalent interdomain contacts that stabilize the interaction between the heavy and light chains of an antibody.

[0039] The structure and location of the CDRs and variable domains of immunoglobulins can be determined by reference to Kabat, EA et al., Sequences of Proteins of Immunological Interest. 4th Edition. US Department of Health and Human Services. 1987 and updates thereto (available on the Internet at immuno.bme.nwu.edu). Alternatively, CDRs can be determined by using IMGT® (International ImMunoGeneTics Information System®), available at http: / / www.imgt.org (see, e.g., Lefranc, M.-P. et al. (1999) Nucleic Acids Res., 27:209-212; Ruiz, M. et al. (2000) Nucleic Acids Res., 28:219-221; Lefranc, M.-P. (2001) Nucleic Acids Res., 29:207-209; Lefranc, M.-P. (2003) Nucleic Acids Res., 31:307-310; Lefranc, M.-P. et al. (2004) In Silico Biol.,5,0006[Epub],5:45-60(2005)];Lefranc,M.-P.et al.(2005)Nucleic Acids Res.,33:D593-597;Lefranc,M.-P.et al.(2009)Nucleic Acids Res.,37:D1006-1012;Lefranc,M.-P.et al. (2015) Nucleic Acids Res., 43:D413-422).

[0040] A "monoclonal antibody" refers to a homogeneous antibody population composed of amino acids (natural or non-natural) involved in selective binding of an epitope. Monoclonal antibodies are highly specific and directed against a single epitope. The term "monoclonal antibody" encompasses not only intact and full-length monoclonal antibodies, but also fragments thereof (e.g., Fab, Fab', F(ab')2, Fv), single-chain (scFv), VHH or sdAb, variants thereof, fusion proteins containing an antigen-binding fragment of a monoclonal antibody, humanized monoclonal antibodies, chimeric monoclonal antibodies, and any other modified configuration of an immunoglobulin molecule containing an antigen-binding fragment (epitope recognition site) with the required specificity and ability to bind to the epitope. It is not intended to be limiting with respect to the source of the antibody or the manner in which it is made (e.g., hybridoma, phage selection, recombinant expression, transgenic animals, etc.). The term encompasses whole immunoglobulins as well as fragments, etc., as described above in the definition of "antibody."

[0041] The proteolytic enzyme papain preferentially cleaves IgG molecules to produce several fragments, two of which (F(ab) fragments) each contain a covalently linked heterodimer with an intact antigen-binding site. The enzyme pepsin can cleave IgG molecules to produce several fragments, including F(ab')2, which contains both antigen-binding sites. Fv fragments for use in accordance with certain embodiments of the invention can be produced by preferential proteolytic cleavage of IgM immunoglobulin molecules, and more rarely, IgG or IgA immunoglobulin molecules. However, Fv fragments are more commonly derived using recombinant techniques known in the art. Fv fragments are non-covalently linked V fragments containing an antigen-binding site that retain much of the antigen recognition and binding capacity of the native antibody molecule. H ::V L Heterodimers are included. 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.

[0042] In certain embodiments, single-chain Fv antibodies, i.e., scFv antibodies, are contemplated, such as kappa bodies (Ill et al., Prot. Eng. 10:949-57 (1997)); minibodies (Martin et al., EMBO J 13:5305-9 (1994)); diabodies (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 1999). Suppl. 7:51-52 (1992)) can be prepared using standard molecular biology techniques, following the teachings of the present application for selection of antibodies with the desired specificity. In yet other embodiments, bispecific or chimeric antibodies can be produced that encompass the ligands of the present disclosure. For example, chimeric antibodies can comprise CDRs and framework regions from different antibodies, while generating bispecific antibodies that specifically bind to one or more Fzd receptors through one binding domain and to a second molecule through a second binding domain. These antibodies can be produced through recombinant molecular biology techniques or can be physically conjugated together.

[0043] Single-chain antibody Fv (scFv) polypeptides consist of V fragments linked by a peptide-encoding linker. H- and V L- Covalently linked V expressed from a gene fusion containing the coding gene H ::V LIt is a heterodimer. Huston et al. (1988) Proc. Nat. Acad. Sci. USA 85(16):5879-5883. Several methods have been described for identifying chemical structures for converting naturally aggregated (but chemically separated) polypeptide light and heavy chains from antibody V regions into scFv molecules that fold into a three-dimensional structure substantially similar to the structure of an 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.

[0044] In certain embodiments, the Fzd-binding antibodies described herein are in the form of diabodies. Diabodies are multimers of polypeptides, each of which comprises a first domain comprising an immunoglobulin light chain binding region and a second domain comprising an immunoglobulin heavy chain binding region, the two domains being 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 a first domain of one polypeptide within the multimer with a second domain of another polypeptide within the multimer (WO94 / 13804).

[0045] A dAb fragment of an antibody consists of the VH domain (Ward, ES et al., Nature 341, 544-546 (1989)).

[0046] When bispecific antibodies are used, they can be identified by various methods (Holliger, P. and The bispecific antibody may be a conventional bispecific antibody that can be produced (e.g., prepared chemically or from a hybrid hybridoma) by conventional techniques (e.g., by conventional techniques, e ...

[0047] Bispecific diabodies may also be particularly useful because, in contrast to bispecific whole antibodies, they can be easily constructed and expressed in E. coli. Diabodies (and many other polypeptides, e.g., antibody fragments) of appropriate binding specificities can be readily selected from libraries using phage display (WO 94 / 13804). Libraries can be generated in which one arm of the diabody remains constant, e.g., specificity directed against antigen X, while the other arm is varied, allowing selection of antibodies of appropriate specificity. Bispecific whole antibodies can be generated by knobs-into-holes engineering (J.B.B. Ridgeway et al., Protein Eng., 9, 616-621, 1996).

[0048] In certain embodiments, the antibodies described herein can be provided in the form of a UniBody®. A UniBody® is an IgG4 antibody with the hinge region removed (see GenMab Utrecht, The Netherlands; see also, e.g., US20090226421). This proprietary antibody technology creates a smaller, more stable antibody format with a longer predictive therapeutic window than current small antibody formats. IgG4 antibodies are considered inert and therefore do not interact with the immune system. Fully human IgG4 antibodies can be modified by removing the hinge region of the antibody, resulting in half-molecule fragments with different stability characteristics relative to the corresponding intact IgG4 (GenMab, Utrecht). Having an IgG4 molecule leaves only one area on the UniBody® that can bind to its cognate antigen (e.g., disease target), so the UniBody® binds monovalently to only one site on the target cell.

[0049] In certain embodiments, the antibodies of the present disclosure can take the form of a VHH or sdAb. VHH or sdAb technology was originally developed after the discovery and identification of camelids (e.g., camels and llamas) as possessing fully functional antibodies consisting only of heavy chains and lacking light chains. Such heavy chain-only antibodies comprise a single variable domain (V HH ) and two constant domains (C H 2. C H 3). Cloned and isolated single variable domains have full antigen-binding capacity and are highly stable. These single variable domains form the basis of "VHHs or sdAbs" due to their unique structural and functional properties. VHHs or sdAbs are encoded by a single gene and are efficiently produced in almost all prokaryotic and eukaryotic hosts, such as E. coli (see, e.g., U.S. Pat. No. 6,765,087), molds (e.g., Aspergillus or Trichoderma), and yeasts (e.g., Saccharomyces, Kluyveromyces, Hansenula, or Pichia) (see, e.g., U.S. Pat. No. 6,838,254). The production process is scalable, and multi-kilogram quantities of VHHs or sdAbs have been produced. VHHs or sdAbs can be formulated as ready-to-use solutions with long shelf lives. The Nanoclone® method (see, e.g., WO06 / 079372) is a unique method for generating VHHs or sdAbs against desired targets, based on automated high-throughput selection of B cells. VHHs or sdAbs are single-domain antigen-binding fragments of heavy-chain-only antibodies, which are unique to camelids. VHHs or sdAbs are typically small, approximately 15 kDa in size.

[0050] In certain embodiments, the anti-Fzd antibodies or antigen-binding fragments thereof disclosed herein are humanized. This refers to chimeric molecules, which are generally prepared using recombinant techniques, having an antigen-binding site derived from an immunoglobulin from a non-human species, with the remainder of the immunoglobulin structure of the molecule based on the structure and / or sequence of a human immunoglobulin. The antigen-binding site may comprise either complete variable domains fused to constant domains or only CDRs grafted onto appropriate framework regions within the variable domains. The epitope-binding site may be wild-type or modified by one or more amino acid substitutions. This eliminates the constant region as an immunogen in human individuals, but the possibility of an immune response to the external variable region remains (LoBuglio, AF et al., (1989) Proc Natl Acad Sci USA 86:4220-4224; Queen et al., PNAS (1988) 86:10029-10033; Riechmann et al., Nature (1988) 332:323-327). Exemplary methods for humanizing the anti-Fzd antibodies disclosed herein include those described in U.S. Pat. No. 7,462,697.

[0051] Another approach focuses not only on providing human-derived constant regions but also on modifying the variable regions to reshape them as closely as possible to human form. Both heavy and light chain variable regions are known to contain three complementarity-determining regions (CDRs). The CDRs vary in response to the epitope of interest, determining binding ability, and are flanked by four framework regions (FRs). The FRs are relatively conserved in a given species and are presumed to provide scaffolding for the CDRs. When a non-human antibody is prepared against a specific epitope, the variable region can be "reshaped" or "humanized" by grafting CDRs from the non-human antibody onto the FRs present in the human antibody to be modified. The application of this approach to various antibodies has been reported in the following publications: Sato, K., et al., (1993) Cancer Res 53:851-856; Riechmann, L., et al., (1988) Nature 332:323-327; Verhoeyen, M., et al., (1988) Science 239:1534-1536; Kettleborough, CA, et al., (1991) Protein Engineering 4:773-3783; Maeda, H., et al., (1991) Human Antibodies Hybridoma 2:124-134; Gorman, SD, et al., (1991) Proc Natl Acad Sci USA 88:4181-4185; Tempest, PR, et al., (1991) Bio / Technology 9:266-271; Co, MS, et al., (1991) Proc Natl Acad Sci USA 88:2869-2873; Carter, P., et al., (1992) Proc Natl Acad Sci USA 89:4285-4289; and Co, MS et al., (1992) J Immunol 148:1149-1154. In some embodiments, humanized antibodies preserve all CDR sequences (e.g., a humanized mouse antibody that contains all six CDRs from the mouse antibodies).In other embodiments, a humanized antibody has one or more (1, 2, 3, 4, 5, 6) CDRs that are altered relative to the original antibody, also referred to as one or more CDRs "derived from" one or more CDRs from the original antibody.

[0052] In certain embodiments, the antibodies of the present disclosure may be chimeric antibodies. In this regard, chimeric antibodies are composed of an antigen-binding fragment of an anti-Fzd antibody operably linked to or fused with a heterologous Fc portion of a different antibody. In certain embodiments, the heterologous Fc domain is of human origin. In other embodiments, the heterologous Fc domain may be from a different Ig class from the patient's antibody, including IgA (including subclasses IgA1 and IgA2), IgD, IgE, IgG (including subclasses IgG1, IgG2, IgG3, and IgG4), and IgM. In further embodiments, the heterologous Fc domain may be composed of CH2 and CH3 domains from one or more different Ig classes. As described above for humanized antibodies, an anti-Fzd antigen-binding fragment of a chimeric antibody may include only one or more of the CDRs of an antibody described herein (e.g., one, two, three, four, five, or six CDRs of an antibody described herein) or may include the entire variable domain (VL, VH, or both).

[0053] In certain embodiments, the antibodies or antigen-binding fragments thereof disclosed herein comprise fusion proteins, such as Wnt signaling pathway agonist fusion proteins, also referred to herein as "Wnt surrogates." The Wnt surrogates of the present invention are typically biologically active in binding to their cognate Frizzled receptors and activating Wnt signaling; i.e., the surrogates are Wnt agonists. The term "Wnt agonist activity" refers to the ability of an agonist to mimic the effect or activity of a Wnt protein binding to a Frizzled protein. The ability of the agonists of the present invention to mimic Wnt activity can be confirmed by multiple assays. Agonists of the present invention typically initiate a similar or identical reaction or activity to that initiated by the receptor's natural ligand. Specifically, agonists of the present invention enhance the canonical 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 agonist of the present invention.

[0054] In certain embodiments, the Wnt signaling pathway agonist fusion protein (or Wnt surrogate) comprises an anti-Fzd antibody or antigen-binding fragment thereof fused to a polypeptide that specifically binds LRP5 and / or LRP6, as disclosed herein. In certain embodiments, the polypeptide that specifically binds LRP5 and / or LRP6 is an antibody or antigen-binding fragment thereof. In certain embodiments, this is an antibody or antigen-binding fragment thereof disclosed in U.S. Provisional Patent Application No. 62 / 607,879, filed December 19, 2017, attorney docket number SRZN-005 / 00US, entitled "Anti-LRP5 / 6 antibodies and Methods of Use," which is incorporated herein by reference in its entirety.

[0055] Suitable LRP5 / 6 binding domains include, but are not limited to, newly designed LRP5 / 6 binding proteins, antibody-derived binding proteins (e.g., scFv, Fab, etc.), and other portions of antibodies that specifically bind to one or more Fzd receptors; VHH- or sdAb-derived binding domains; knottin-based engineered scaffolds; native LRP5 / 6 (including, but not limited to, DKK1, DKK2, DKK3, DKK4, sclerostin); Wise; fusion proteins comprising any of the above; derivatives of any of the above; variants of any of the above; and biologically active fragments of any of the above. The LRP5 / 6 binding domain can be affinity-selective to enhance binding.

[0056] 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). hDKK1-4 contain two distinct cysteine-rich domains in which the positions of 10 cysteine residues are highly conserved among family members. Exemplary sequences of human DKK genes and proteins are publicly available, e.g., 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 invention, the Lrp6-binding moiety is a DKK1 peptide, including but not limited to the C-terminal domain of human DKK1. The C-terminal domain may comprise the sequence: KMYHTKGQEGSVCLRSSDCASGLCCARHFWSKICKPVLKEGQVCTKHRRKGSHGLEIFQRCYCGEGLSCRIQKDHHQASNSSRLHTCQRH (SEQ ID NO: 70; see Genbank Accession No. NP_036374), or a biologically active fragment thereof.

[0057] The binding of DKK proteins to LRP5 / 6 is 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 expressly incorporated herein by reference. The corresponding region of human DKK2 (Genbank reference NP_055236) can comprise the sequence: KMSHIKGHEGDPCLRSSDCIEGFCCARHFWTKICKPVLHQGEVCTKQRKKGSHGLEIFQRCDCAKGLSCKVWKDATYSSKARLHVCQK (SEQ ID NO: 71), or a biologically active fragment thereof.

[0058] Antibodies that specifically bind to LRP5 or LRP6 are known in the art, are commercially available, or can be newly generated. LRP5, LRP6, or fragments thereof can be used as immunogens or in screening assays to develop antibodies. Examples of known antibodies include, but are not limited to, 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; and commercially available antibodies from, for example, Santa Cruz biotechnology antibody clone 1A12 (raised against synthetic LRP5 / 6 of human origin and binds to both full-length and proteolytic fragments of LRP6 and LRP5 of mouse and human origin); monoclonal antibody 2B11; Cell Signaling Technology antibody specific for LRP5 (D80F2), catalog number 5731;

[0059] In some embodiments, the LRP5 / 6 binding domain or element binds with high affinity, e.g., at least about 1×10 -7 M, at least 1 × 10 -8M, at least 1 × 10 -9 M, at least 1 × 10 -10 K of M D The LRP5 / 6-binding domain can be selected from any domain that binds to LRP5 / 6 at the target site. Suitable LRP5 / 6-binding domains include, but are not limited to, newly designed LRP5 / 6-binding proteins, antibody-derived binding proteins (e.g., scFv, Fab, etc.), and other portions of antibodies that specifically bind to one or more Fzd receptors; VHH- or sdAb-derived binding domains; knottin-based engineered scaffolds; natural LRP5 / 6-binding proteins or polypeptides (including, but not limited to, Norrin, DKK1, DKK2, DKK3, DKK4, and sclerostin); and the like. In certain embodiments, the LRP5 / 6-binding domain is the C-terminal portion of DKK1. The LRP5 / 6-binding domain can be affinity-selective to enhance binding.

[0060] The anti-Fzd antibody or antigen-binding fragment thereof and the LRP5 / 6-binding domain may be directly linked or separated by a linker (e.g., a polypeptide linker or a non-peptide linker). The region of the Wnt surrogate that binds to one or more Fzd receptors and the polypeptide that binds to LRP5 and / or LRP6 may be contiguous or separated by a linker (e.g., a polypeptide linker or a non-peptide linker). The length of the linker, i.e., the spacing between the binding domains, can be used to adjust signal strength and can be selected depending on the desired use of the Wnt surrogate. The required distance between the binding domains can vary, but in certain embodiments, it can 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. When the linker is a peptide linker, the linker can be about 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 or more amino acids in length, and of a length and amino acid composition sufficient to enhance the distance between the binding domains. In some embodiments, the linker comprises or consists of one or more glycine and / or serine residues.

[0061] Wnt surrogates can be multimerized, for example, via the Fc domain, by linkage, coiled-coil, polypeptide zipper, biotin / avidin or streptavidin multimerization, etc. Wnt surrogates can also be linked to moieties such as PEG, Fc, etc., as known in the art, to enhance stability in vivo.

[0062] In certain embodiments, a Wnt surrogate directly activates canonical Wnt signaling by binding to one or more Fzd proteins and LRP5 / 6, particularly by binding to these proteins on the cell surface, e.g., the surface of a human cell. Direct activation of Wnt signaling by a Wnt surrogate is in contrast to enhanced Wnt signaling, which enhances activity only in the presence of native Wnt protein.

[0063] The Wnt surrogate activates Wnt signaling, for example, by mimicking the effect or activity of a Wnt protein bound to a Frizzled protein. The ability of the Wnt surrogate of the present invention to mimic the activity of a Wnt can be confirmed by multiple assays. A Wnt surrogate typically initiates a reaction or activity similar to or identical to that initiated by the receptor's natural ligand. Specifically, the Wnt surrogate of the present invention enhances the canonical 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 of the present invention.

[0064] In certain embodiments, the antibodies or antigen-binding fragments thereof disclosed herein inhibit Wnt pathway signaling. In certain embodiments, the anti-Fzd antibodies or antigen-binding fragments thereof block or inhibit the binding of endogenous Wnt to one or more Fzd receptors on the cell surface, thereby reducing or inhibiting Wnt signaling.

[0065] Various methods for measuring the level of Wnt / β-catenin signaling are known in the art. These methods include, but are not limited to, assays measuring Wnt / β-catenin target gene expression, TCF reporter gene expression, β-catenin stabilization, LRP phosphorylation, and axin translocation from the cytoplasm to the plasma membrane and its binding to LRP. The canonical Wnt / β-catenin signaling pathway ultimately leads to changes in gene expression via the transcription factors TCF7, TCF7L1, TCF7L2 (also known as TCF4), and LEF. The transcriptional response to Wnt activation has been characterized in multiple cells and tissues. Therefore, global transcriptional profiling using methods known in the art can be used to assess activation or inhibition of Wnt / β-catenin signaling.

[0066] Changes in Wnt-responsive gene expression are generally mediated by TCF and LEF transcription factors. The TCF reporter assay assesses changes in transcription of TCF / LEF-regulated genes to determine the level of Wnt / β-catenin signaling. The TCF reporter assay was first described by Korinek, V. et al., 1997. Also known as TOP / FOP, this method involves determining the transactivation activity of endogenous p-catenin / TCF4 using three copies of the optimal TCF motif CCTTTGATC or three copies of the mutant motif CCTTTGGCC upstream of a minimal c-Fos promoter driving luciferase expression (pTOPFl_ASH and pFOPFl_ASH, respectively). A higher ratio of the two reporter activities (TOP / FOP) indicates higher β-catenin / TCF4 activity, while a lower ratio indicates lower β-catenin / TCF4 activity.

[0067] A variety of other reporter transgenes that respond to Wnt signaling exist intact in animals, thus effectively reflecting endogenous Wnt signaling. These reporters are based on multimerized TCF binding sites that drive the expression of LacZ or GFP, and are easily detectable by methods known in the art. These reporter genes include TOP-GAL, BAT-GAL, ins-TOPEGFP, ins-TOPGAL, LEF-EGFP, Axin2-LacZ, Axin2-d2EGFP, Lgr5tm1(cre / ERT2), and TOPdGFP.

[0068] The recruitment of dephosphorylated β-catenin to the membrane, its stabilization and phosphorylation status, and its nuclear translocation (Klapholz-Brown Z et al., PLoS One. 2(9)e945, 2007) are, in some cases, mediated by complex formation with TCF transcription factors and TNIK and are key steps in the Wnt signaling pathway. Stabilization is mediated by Disheveled family proteins that inhibit the "destruction" complex, resulting in reduced degradation of intracellular β-catenin and subsequent nuclear translocation. Therefore, measuring the level and location of β-catenin in cells provides a good reflection of the level of Wnt / β-catenin signaling. One 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 a fluorescence microscope or HCS platform that allows visualization of the levels and distribution of EGFP-β-catenin.

[0069] Another way to inhibit the destruction complex is by axin removal, which recruits axin to the cytoplasmic tail of the Wnt co-receptor LRP. Axin has been shown to preferentially bind to the phosphorylated form of the LRP tail. Therefore, visualization of axin translocation (e.g., using a GFP-axin fusion protein) is another method for assessing the level of Wnt / β-catenin signaling.

[0070] In certain embodiments, a Wnt signaling pathway agonist enhances or increases canonical Wnt signaling (e.g., 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 a negative control, as measured in an assay described above, e.g., a TOPFlash assay. Such assays may include a negative control. In certain embodiments, a Wnt agonist can enhance β-catenin signaling by 2-fold, 5-fold, 10-fold, 100-fold, 1000-fold, 10,000-fold, or more compared to activity in the absence of the agonist, as measured in any of the assays described above, e.g., the TOPFlash assay, or other assays mentioned herein.

[0071] In certain embodiments, a Wnt signaling pathway antagonist or inhibitor inhibits or reduces canonical Wnt signaling (e.g., β-catenin signaling) by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, or 100% compared to β-catenin signaling observed in the presence of a neutral agent or negative control, as measured in an assay described above, e.g., a TOPFlash assay. Such assays may include a positive control.

[0072] "Wnt gene product" or "Wnt polypeptide," as used herein, encompasses native sequence Wnt polypeptides, Wnt polypeptide variants, Wnt polypeptide fragments, and chimeric Wnt polypeptides. In certain embodiments, the Wnt polypeptide is a native human full-length mature Wnt protein.

[0073] For example, human native sequence Wnt proteins of interest in the present application include the following: Wnt-1 (GenBank Accession No. NM_005430); Wnt-2 (GenBank Accession No. NM_003391); Wnt-2B (Wnt-13) (GenBank Accession Nos. NM_004185 (isoform 1), NM_024494.2 (isoform 2)), Wnt-3 (RefSeq.: NM_030753), Wnt3a (GenBank Accession No. NM_033131), Wnt-4 (GenBank Accession No. NM_030761), Wnt-5A (GenBank Accession No. NM_003392), Wnt-5B (GenBank Accession No. NM_032642), Wnt-6 (GenBank Accession No. NM_032642), Wnt-7 (GenBank Accession No. NM_032642), Wnt-8 (GenBank Accession No. NM_032642), Wnt-9 (GenBank Accession No. NM_032642), Wnt-10 (GenBank Accession No. NM_030753), Wnt-11 (GenBank Accession No. NM_030753), Wnt-12 (GenBank Accession No. NM_030753), Wnt-13a (GenBank Accession No. NM_033131), Wnt-14 (GenBank Accession No. NM_030761), Wnt-15 (GenBank Accession No. NM_030761), Wnt-16 (GenBank Accession No. NM_030761), Wnt-17 (GenBank Accession No. NM_030761), Wnt-18 (GenBank Accession No. NM_030761), Wnt-19 (GenBank _006522), Wnt-7A (GenBank accession no. NM_004625), Wnt-7B (GenBank accession no. NM_058238), Wnt-8A (GenBank accession no. NM_058244), Wnt-8B (GenBank accession no. NM_003393), Wnt-9A (Wnt-14) (GenBank accession no. NM_00339 5), Wnt-9B (Wnt-15) (GenBank accession no. NM_003396), Wnt-10A (GenBank accession no. NM_025216), Wnt-10B (GenBank accession no. NM_003394), Wnt-11 (GenBank accession no. NM_004626), and Wnt-16 (GenBank accession no. NM_016087)). Although individual members share varying degrees of sequence identity with the family, all encode small (i.e., 39-46 kD), secreted glycoproteins that are acylated, palmitoylated, and contain 23-24 conserved cysteine residues with highly conserved spacing (McMahon, AP et al., Trends Genet. 1992;8:236-242; Miller, JR. Genome Biol. 2002;3(1):3001.1-3001.15).Other native sequences of the Wnt polypeptide of interest include orthologs of the above from any mammal, including domestic and farm animals, as well as zoo, laboratory, or pet animals, such as dogs, cats, cows, horses, sheep, pigs, goats, rabbits, rats, mice, frogs, zebrafish, fruit flies, worms, etc.

[0074] "Wnt pathway signaling" or "Wnt signaling" is used herein to refer to the mechanism by which biologically active Wnt exerts its effect on cells to regulate cellular activity. Wnt proteins regulate cellular activity by binding to Wnt receptors, including proteins from the Frizzled (Fzd) family of proteins, proteins from the ROR family of proteins, proteins LRP5 and LRP6 from the LRP family of proteins, the protein FRL1 / crypto, and the protein Derailed / Ryk. Once activated by Wnt binding, a Wnt receptor(s) activates one or more intracellular signaling cascades. Such signaling cascades include the canonical Wnt signaling pathway; the Wnt / planar cell polarity (Wnt / PCP) pathway; the Wnt-calcium (Wnt / Ca) pathway; 2+ ) 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 as known in the art.

[0075] For example, activation of the canonical Wnt signaling pathway results in the inhibition of phosphorylation of the intracellular protein β-catenin, leading to its accumulation in the cytosol and subsequent translocation to the nucleus, where it interacts with transcription factors (e.g., TCF / LEF) to activate target genes. Activation of the Wnt / PCP pathway activates RhoA, c-Jun N-terminal kinase (JNK), and nemo-like kinase (NLK) signaling cascades to control biological processes such as tissue polarity and cell motility. For example, Wnt / Ca signaling via binding of Wnt-4, Wnt-5A, or Wnt-11. 2+ Activation of Wnt 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 pathways, the biological activity of an antibody or antigen-binding fragment thereof (e.g., a Wnt surrogate) can be readily determined.

[0076] In certain embodiments, the functional properties of anti-Fzd antibodies and antigen-binding fragments thereof can be assessed using a variety of methods known to those of skill in the art, including, for example, affinity / binding assays (e.g., surface plasmon resonance, competitive inhibition assays), cytotoxicity assays, cell viability assays, and cell proliferation or differentiation assays in response to Wnt, cancer cell, and / or tumor growth inhibition using in vitro or in vivo models (including, but not limited to, any of those described herein). Other assays may test the ability of the antibodies described herein to block normal Wnt / Fzd-mediated responses. The antibodies and antigen-binding fragments thereof described herein may also be tested for their effect on Fzd receptor internalization, in vitro and in vivo efficacy, etc. Such assays may be performed using well-established protocols known to those of skill in the art (e.g., Current Protocols in Molecular Biology(Greene Publ.Assoc.Inc.& John Wiley & Sons,Inc.,NY,NY);Current Protocols in Immunology (eds. John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M. Shevach, Warren Strober 2001 John Wiley & Sons, NY, NY) or can be performed using commercially available kits.

[0077] In certain embodiments, the Fzd-binding antibody or antigen-binding fragment thereof (e.g., a Wnt surrogate) comprises one or more CDRs of the antibodies described herein. In this regard, it has been shown that in some cases, transfer of only the VHCDR3 of an antibody can be performed while retaining the desired specific binding (Barbas et al., PNAS (1995) 92:2529-2533). See also McLane et al., PNAS (1995) 92:5214-5218, Barbas et al., J. Am. Chem. Soc. (1994) 116:2161-2162.

[0078] Marks et al. (Bio / Technology, 1992, 10:779-783) describe a method for producing a repertoire of antibody variable domains, in which a consensus primer directed toward or adjacent to the 5' end of the variable domain area is used together with a consensus primer for the third framework region of the human VH gene, resulting in a repertoire of VH variable domains lacking CDR3. Marks et al. further describe how this repertoire can be combined with the CDR3 of a particular antibody. Using similar techniques, CDR3-derived sequences of the antibodies described herein can be shuffled with a repertoire of VH or VL domains lacking CDR3, and the shuffled complete VH or VL domains can be combined with the cognate VL or VH domains to generate antibodies or antigen-binding fragments thereof that bind to one or more Fzd receptors. The repertoire can then be displayed in a suitable host system, such as the phage display system of WO92 / 01047, to allow selection of suitable antibodies or antigen-binding fragments thereof. The repertoire is at least about 10 4 can consist of distinct members of several orders of magnitude, e.g., about 10 6 From 10 8 Or 10 10 A shuffling or combinatorial fragment can consist of up to 10 or more members. Similar shuffling or combinatorial techniques have also been described by Stemmer (Nature, 1994, 370:389-391). Stemmer describes the technique in relation to β-lactamase genes, but states that this approach can be used to generate antibodies.

[0079] A further option is to use random mutagenesis of one or more selected VH and / or VL genes to generate mutations within the entire variable domain, thereby generating novel VH or VL regions bearing sequences derived from one or more CDRs of the invention embodiments described herein. Such a technique is described by Gram et al. (1992, Proc. Natl. Acad. Sci., USA, 89:3576-3580) using error-prone PCR. Another method that can be used is to direct mutagenesis to the CDR regions of the VH or VL gene. Such a technique is disclosed by Barbas et al. (1994, Proc. Natl. Acad. Sci., USA, 91:3809-3813) and Schier et al. (1996, J. Mol. Biol. 263:551-567).

[0080] In certain embodiments, a particular VH and / or VL of an antibody described herein can be used to screen libraries of complementary variable domains to identify antibodies with desirable properties (e.g., increased affinity for one or more Fzd receptors). Such methods are described, for example, in Portolano et al., J. Immunol. (1993) 150:880-887; Clarkson et al., Nature (1991) 352:624-628.

[0081] Other methods can also be used to mix and match CDRs to identify antibodies with desired binding activity (e.g., binding to one or more Fzd receptors). For example, Klimka et al., British Journal of Cancer (2000) 83:252-260, describe a screening process using a library of mouse VLs and human VHs in which CDR3 and FR4 were retained from the mouse VH. After obtaining antibodies, the VHs were screened against a human VL library to obtain antibodies that bind to the antigen. Beiboer et al., J. Mol. Biol. (2000) 296:833-849, describe a screening process using an entire mouse heavy chain and human light chain library. After obtaining antibodies, one VL was combined with a human VH library in which the mouse CDR3 was retained. Antibodies capable of binding to the antigen were obtained. Rader et al., PNAS (1998) 95:8910-8915, describe a process similar to that described by Beiboer et al.

[0082] The techniques described herein are per se known in the art, but one skilled in the art will be able to use such techniques to obtain antibodies or antigen-binding fragments thereof according to some embodiments of the invention described herein using methodology routine in the art.

[0083] Also disclosed herein is a method for obtaining an antibody or antigen-binding domain specific for a Fzd receptor, the method comprising providing a VH domain as set forth herein or a VH domain that is an amino acid sequence variant of the VH domain by adding, deleting, substituting, or inserting one or more amino acids in the amino acid sequence thereof, optionally combining the VH domain thus provided with one or more VL domains, and testing the VH domain or VH / VL combination(s) to identify a specific binding member of the antibody antigen-binding domain that is specific for one or more Fzd receptors and optionally has one or more desired properties. The VL domain can have an amino acid sequence substantially as set forth herein. Similar methods may be used in which one or more sequence variants of the VL domains disclosed herein are combined with one or more VH domains.

[0084] In certain embodiments, anti-Fzd antibodies and antigen-binding fragments thereof are water-soluble. "Water-soluble" refers to a composition that is soluble in aqueous buffer in the absence of detergent, typically at a concentration that provides a biologically effective dose of the polypeptide. A water-soluble composition forms a substantially homogeneous composition, having a specific activity that is at least about 5% of the starting material from which it is purified, usually at least about 10%, 20%, or 30%, more usually about 40%, 50%, or 60%, and sometimes about 50%, about 90%, or more. The anti-Fzd antibodies and antigen-binding fragments thereof (including Wnt surrogates) of the present invention typically form substantially homogeneous aqueous solutions at a concentration of at least 25 μM or greater, e.g., at least 25 μM, 40 μM, or 50 μM, usually at least 60 μM, 70 μM, 80 μM, or 90 μM, and sometimes as high as 100 μM, 120 μM, or 150 μM. In other words, the compositions of the present invention typically form substantially homogeneous aqueous solutions at concentrations of about 0.1 mg / ml, about 0.5 mg / ml, about 1 mg / ml or more.

[0085] The antigen or epitope to which an antibody or antigen-binding fragment thereof "specifically binds" or "preferentially binds" (used interchangeably herein) is a term well understood in the art, and methods for determining such specific or preferential binding are also well known in the art. A molecule is said to exhibit "specific binding" or "preferential binding" if it reacts or associates with a particular cell or substance more frequently, rapidly, for a longer duration, and / or with a higher affinity than with alternative cells or substances. An antibody "specifically binds" or "preferentially binds" to a target antigen (e.g., an Fzd receptor) if it binds with greater affinity, avidity, more readily, and / or with a longer duration than to other substances. For example, an antibody that specifically or preferentially binds to the Fzd1 receptor is one that binds to the Fzd1 receptor with greater affinity, avidity, more readily, and / or with a longer duration than to other Fzd receptors or non-Fzd proteins. It is also understood by reading this definition that, for example, an antibody (or portion or epitope) that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. Thus, "specific binding" or "preferential binding" does not necessarily require (although it can include) exclusive binding. Generally, but not necessarily, reference to binding means preferential binding.

[0086] In some embodiments, the anti-Fzd antibody or antigen-binding fragment thereof binds to one, two, three, four, five, or more different Frizzled proteins, e.g., one or more of the human Frizzled proteins Fzd1, Fzd2, Fzd3, Fzd4, Fzd5, Fzd6, Fzd7, Fzd8, Fzd9, and Fzd10. In some embodiments, the antibody-based signaling agonist binds to Fzd1, Fzd2, Fzd5, Fzd7, and Fzd8. In various embodiments, the anti-Fzd antibody or antigen-binding fragment thereof is selected from the group consisting of: (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; and (vi) Fzd1, Fzd2, Fzd9, Fzd11, Fzd2, Fzd12, Fzd13, Fzd14, Fzd15, Fzd16, Fzd17, Fzd18, Fzd19, Fzd19, Fzd19, Fzd116, Fzd117, Fzd118, Fzd119, Fzd120, Fzd121, Fzd122, Fzd123, Fzd124, Fzd125, Fzd126, Fzd127, Fzd128, Fzd129, Fzd130, Fzd131, Fzd132, Fzd133, Fzd134, Fzd135, Fzd136, Fzd137, Fzd138, Fzd139, Fzd140, Fzd141, Fzd142, Fzd143, Fzd144, Fzd145, Fzd146, Fzd147, Fzd148, Fzd149 ... (vii) Fzd4 and Fzd9; (viii) Fzd9 and Fzd10; (ix) Fzd5, Fzd8, and Fzd10; (x) Fzd4, Fzd5, and Fzd8; (xi) Fzd1, Fzd5, Fzd7, and Fzd8; or (xii) Fzd1, Fzd4, Fzd5, Fzd7, and Fzd8. In some embodiments, the Frizzled binding moiety is selective for one or more Frizzled proteins of interest, e.g., has at least 10-fold, 25-fold, 50-fold, 100-fold, 200-fold, or more specificity for one or more desired Frizzled proteins relative to other Frizzled proteins.

[0087] Immunological binding generally refers to the types of non-covalent interactions that occur between an immunoglobulin molecule and the antigen for which that immunoglobulin molecule is specific, including, by way of example and not limitation, electrostatic, ionic, hydrophilic and / or hydrophobic attractions or repulsions, steric forces, hydrogen bonding, 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 ) and K DA smaller K represents a 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 formation and dissociation of the antigen-binding site / antigen complex, which depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that affect the rates in both directions equally. Thus, the "on-rate constant" (K on ) and "off rate constant" (K off ) can be determined by calculating the concentrations and the actual rates of association and dissociation. off / K on The ratio of α to β allows the release of all parameters not related to affinity and therefore the dissociation constant K D See generally Davies et al. (1990) Annual Rev Biochem. 59:439-473. In certain embodiments, the anti-Fzd antibody binds to one or more Fzd receptors at a concentration of about 1 x 10 -4 M or less, approximately 1×10 -5 M or less, approximately 1×10 -6 M or less, approximately 1×10 -7 M or less, approximately 1×10 -8 M or less, approximately 1×10 -9 M or less, or approximately 1 x 10 -10 K below M D In certain embodiments, the anti-Fzd antibodies described herein bind to one or more Fzd receptors with a K of less than about 10,000 nM, less than about 1000 nM, less than about 100 nM, less than about 10 nM, less than about 1 nM, or less than about 0.1 nM. D In some embodiments, the antibodies may have even higher affinity for one or more Fzd receptors. In certain embodiments, the anti-Fzd antibodies described herein have a K of about 100, 150, 155, 160, 170, 175, 180, 185, 190, 191, 192, 193, 194, 195, 196, 197, 198, or 199 picomolar. D and in some embodiments, the antibody may have even higher affinity for one or more Fzd receptors.

[0088] Antibodies or antigen-binding fragments thereof according to certain embodiments include antibodies and antigen-binding fragments thereof that (i) specifically bind to one or more Fzd receptors and / or (ii) comprise a VH and / or VL domain (or VH and / or VL CDR set) disclosed herein, or (iii) compete for binding to one or more Fzd receptors with any of the antibodies described herein, or any variant thereof, that comprise a VH and / or VL domain (or VH and / or VL CDR set) disclosed herein, or (iii) comprise a VH CDR3 disclosed herein. Competition between antibodies can be readily assayed in vitro, for example, using ELISA and / or by tagging one antibody with a specific reporter molecule that can be detected in the presence of other, untagged antibodies, thereby making it possible to identify specific antibodies that bind to the same or overlapping epitopes. Accordingly, provided herein are specific antibodies or antigen-binding fragments thereof that comprise a human antibody antigen-binding site that competes with an antibody that binds to one or more Fzd receptors described herein.

[0089] In this regard, as used herein, the terms "compete with," "inhibit binding," and "block binding" (e.g., referring to the inhibition / blocking of Wnt binding to one or more Fzd receptors or the inhibition / blocking of anti-Fzd antibody binding to Fzd receptors) are used interchangeably and encompass partial and complete inhibition / blocking. Inhibition / blocking of Wnt binding to one or more Fzd receptors preferably reduces or alters the normal level or type of cell signaling that occurs when Wnt binds to an Fzd receptor without inhibition or blockage. Inhibition and blocking are also intended to include any measurable decrease in Wnt binding to a Fzd receptor when contacted with an anti-Fzd antibody disclosed herein compared to a ligand that has not been contacted with an anti-Fzd antibody, e.g., blocking of Wnt binding to a Fzd receptor by at least about 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0090] The constant regions of immunoglobulins exhibit less sequence diversity than the variable regions and are responsible for binding to multiple natural proteins and triggering important biochemical events. In humans, there are five different antibody classes, including IgA (including subclasses IgA1 and IgA2), IgD, IgE, IgG (including subclasses IgG1, IgG2, IgG3, and IgG4), and IgM. The distinguishing feature of these antibody classes is the constant region, although subtler differences may exist within the V regions.

[0091] The Fc region of an antibody interacts with multiple Fc receptors and ligands, conferring numerous important functional capabilities referred to as effector functions. In IgG, the Fc region comprises Ig domains CH2 and CH3, with an N-terminal hinge connecting CH2. An important family of Fc receptors in the IgG class are Fc gamma receptors (FcγRs). These receptors mediate communication between antibodies and the cellular arm of the immune system (Raghavan et al., 1996, Annu Rev Cell Dev Biol 12:181-220; Ravetch et al., 2001, Annu Rev Immunol 19:275-290). In humans, this protein family includes FcγRI (CD64) (including isoforms FcγRIa, FcγRIb, and FcγRIc); FcγRII (CD32) (including isoforms FcγRIIa (including allotypes H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc); and FcγRIII (CD16) (including isoforms FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2) (Jefferis et al., 2002, Immunol Lett 82:57-65). These receptors typically have an extracellular domain that mediates Fc binding, a transmembrane region, and an intracellular domain that may mediate some intracellular signaling event. These receptors are expressed in a variety of immune cells, including monocytes, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, platelets, B cells, large granular lymphocytes, Langerhans cells, natural killer (NK) cells, and T cells. Formation of the Fc / FcγR complex recruits these effector cells to the site of antigen binding, typically resulting in intracellular signaling events and subsequent important immune responses, such as the release of inflammatory mediators, B cell activation, endocytosis, phagocytosis, and cytotoxic attack.

[0092] The ability to mediate cytotoxic and phagocytic effector functions is a potent mechanism by which antibodies destroy target cells. The cell-mediated reaction in which nonspecific cytotoxic cells expressing FcγR recognize bound antibodies on target cells and subsequently cause lysis of the target cells is called antibody-dependent cell-mediated cytotoxicity (ADCC) (Raghavan et al., 1996, Annu Rev Cell Dev Biol 12:181-220; Ghetie et al., 2000, Annu Rev Immunol 18:739-766; Ravetch et al., 2001, Annu Rev Immunol 19:275-290). The cell-mediated reaction in which nonspecific cytotoxic cells expressing FcγR recognize bound antibodies on target cells and subsequently cause phagocytosis of the target cells is called antibody-dependent cell-mediated phagocytosis (ADCP). All FcγRs bind to the same region on Fc, at the N-terminus of the Cg2 (CH2) domain and immediately preceding the hinge. This interaction has been structurally well characterized (Sondermann et al., 2001, J Mol Biol 309:737-749), and several structures of human Fc bound to the extracellular domain of human FcγIIIb have been solved (pdb accession code 1E4K) (Sondermann et al., 2000, Nature 406:267-273) (pdb accession codes 1IIS and 1IIX) (Radaev et al., 2001, J Biol Chem 276:16469-16477).

[0093] Different IgG subclasses have different affinities for FcγRs, and typically IgG1 and IgG3 bind to the receptor substantially better than IgG2 and IgG4 (Jefferis et al., 2002, Immunol Lett 82:57-65). All FcγRs bind to the same region on the IgG Fc but with different affinities, with the high affinity binder FcγRI binding to IgG1 at 10 -8 M -1 K D whereas the low affinity receptors FcγRII and FcγRIII have approximately 10 -6 and 10-5 The extracellular domains of FcγRIIIa and FcγRIIIb are 96% identical, but FcγRIIIb lacks an intracellular signaling domain. Furthermore, FcγRI, FcγRIIa / c, and FcγRIIIa are positive regulators characterized by intracellular domains containing immunoreceptor tyrosine-based activation motifs (ITAMs), whereas FcγRIIb contains immunoreceptor tyrosine-based inhibitory motifs and is therefore inhibitory. Therefore, the former are called activating receptors, while FcγRIIb is called an inhibitory receptor. These receptors also differ in their expression patterns and levels on different immune cells. Another level of complexity is the existence of multiple FcγR polymorphisms in the human proteome. One polymorphism of particular clinical importance is the V158 / F158 FcγRIIIa. Human IgG1 binds to the V158 allotype with higher affinity than to the F158 allotype. This difference in affinity, and its putative effect on ADCC and / or ADCP, has been shown to be an important determinant of efficacy for the anti-CD20 antibody rituximab (Rituxan®, a registered trademark of IDEC Pharmaceuticals Corporation). Subjects with the V158 allotype respond favorably to rituximab treatment, whereas subjects with the low-affinity F158 allotype respond poorly (Cartron et al., 2002, Blood 99:754-758). Approximately 10-20% of humans are V158 / V158 homozygous, 45% are V158 / F158 heterozygous, and 35-45% are F158 / F158 homozygous (Lehrnbecher et al., 1999, Blood 94:4220-4232; Cartron et al., 2002, Blood 99:754-758). Therefore, 80-90% of humans are poor responders, i.e., they have at least one F158 FcγRIIIa allele.

[0094] The Fc region is also involved in activating the complement cascade. In the classical complement pathway, C1 binds via its C1q subunit to the Fc fragment of IgG or IgM complexed with antigen(s). In certain embodiments of the present invention, modifications to the Fc region alter (enhance or decrease) the ability of the Fzd-specific antibodies described herein to activate the complement system (see, e.g., U.S. Patent No. 7,740,847). To assess complement activation, a complement-dependent cytotoxicity (CDC) assay can be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods, 202:163 (1996)).

[0095] Thus, in certain embodiments, the invention provides anti-Fzd antibodies having modified Fc regions with altered functional properties (e.g., reduced or enhanced CDC, ADCC, or ADCP activity, or enhanced binding affinity for a particular FcγR, or increased serum half-life). Other modified Fc regions contemplated herein are described, for example, in issued U.S. Patent Nos. 7,317,091, 7,657,380, 7,662,925, 6,538,124, 6,528,624, 7,297,775, 7,364,731, published U.S. applications US2009092599, US20080131435, US20080138344, and published international applications WO2006 / 105338, WO2004 / 063351, WO2006 / 088494, WO2007 / 024249.

[0096] In certain embodiments, the Fc region can be derived from any of a variety of different Fc domains, including, but not limited to, wild-type or modified IgG1, IgG2, IgG3, IgG4, or other isotypes, such as wild-type or modified human IgG1, human IgG2, human IgG3, human IgG4, human IgG4Pro (containing a mutation in the core hinge region that prevents the formation of IgG4 half molecules), human IgA, human IgE, human IgM, or a modified IgG1 termed IgG1 LALAPG. The L235A, P329G (LALA-PG) variant has been shown in both mouse IgG2a and human IgG1 to ablate Fc-γ-dependent antibody-dependent cell-mediated cytotoxicity (ADCC) in addition to ablation of complement binding and fixation. In certain embodiments of any IgG disclosed herein, the IgG comprises one or more of the following amino acid substitutions: N297G, N297A, N297E, L234A, L235A, or P236G.

[0097] Thus, in certain embodiments, antibody variable domains with the desired binding specificities are fused to immunoglobulin constant domain sequences. In certain embodiments, the fusion comprises a hinge, C H 2, and C H The first heavy-chain constant region (C) containing the site necessary for light-chain binding is present in at least one of the fusions. H1). DNA encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain are inserted into separate expression vectors and co-transfected into a suitable host cell. This allows for greater flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments where unequal ratios of the three polypeptide chains are used in the construction to provide the optimal yield of the desired bispecific antibody. However, it is possible to insert the coding sequences for two or all three polypeptide chains into a single expression vector in cases where expression of at least two polypeptide chains in equal ratios provides high yields or where the ratio does not significantly affect the yield of the desired chain combination.

[0098] The antibodies of the invention (and antigen-binding fragments and variants thereof) can also be modified to include epitope tags or labels, e.g., for use in purification or diagnostic applications. Numerous linking groups for generating antibody conjugates are known in the art, and include those described, for example, in U.S. Pat. No. 5,208,020 or European Patent No. 0802064. 425 235 B1, and Chari et al., Cancer Research 52:127-131 (1992). Linking groups include disulfide groups, thioether groups, acid labile groups, photolabile groups, peptidase labile groups, or esterase labile groups, as disclosed in the above-identified patents, with disulfide groups and thioether groups being preferred.

[0099] In another contemplated embodiment, the Fzd-specific antibodies or antigen-binding fragments thereof described herein may be conjugated or operably linked to another therapeutic compound, referred to herein as a conjugate. The conjugate may be a cytotoxic agent, a chemotherapeutic agent, a cytokine, an anti-angiogenic agent, a tyrosine kinase inhibitor, a toxin, a radioisotope, or other therapeutically active agent. Chemotherapeutic agents, cytokines, anti-angiogenic agents, tyrosine kinase inhibitors, and other therapeutic agents are described above, and all of the aforementioned therapeutic agents can be used as antibody conjugates.

[0100] Immunoconjugates are prepared using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate, iminothiolane (IT), bifunctional derivatives of iminodiesters (e.g., dimethyl adipimidate HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azido compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). Specific coupling agents include N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) (Carlsson et al., Biochem. J. 173:723-737

[1978] ) and N-succinimidyl-4-(2-pyridylthio)pentanoate (SPP) to provide disulfide bonds. The linker may be a "cleavable linker" that facilitates the release of one or more cleavable components. For example, an acid-labile linker (Cancer Research 52:127-131 (1992); U.S. Patent No. 5,208,020) can be used.

[0101] In certain embodiments, the anti-LRP5 / 6 antibodies and antigen-binding fragments thereof are monoclonal antibodies. In certain embodiments, they are humanized.

[0102] The present invention further provides, in certain embodiments, isolated nucleic acids encoding the antibodies or antigen-binding fragments thereof described herein, e.g., nucleic acids encoding one or more CDRs or VH or VL domains described herein. Nucleic acids include DNA and RNA. These and related embodiments can include polynucleotides that encode antibodies that bind to one or more Fzd receptors described herein. As used herein, the term "isolated polynucleotide" is intended to mean a polynucleotide of genomic, cDNA, or synthetic origin, or any combination thereof, and based on its origin, an isolated polynucleotide (1) is not associated with all or a portion of polynucleotides when the isolated polynucleotide is found in nature, (2) is associated with polynucleotides with which it is not associated in nature, or (3) does not occur in nature as part of a larger sequence.

[0103] The term "operably linked" means that the components to which the term is applied are in a relationship allowing them to carry out their inherent functions under suitable conditions. For example, a transcriptional control sequence "operably linked" to a protein coding sequence is ligated with the protein coding sequence such that expression of the protein coding sequence is achieved under conditions compatible with the transcriptional activity of the control sequences.

[0104] As used herein, the term "control sequence" refers to a polynucleotide sequence capable of affecting the expression, processing, or subcellular localization of coding sequences to which it is linked or operably linked. The nature of such control sequences may depend on the host organism. In certain embodiments, a prokaryotic transcription control sequence may include a promoter, a ribosomal binding site, and a transcription termination sequence. In other specific embodiments, a eukaryotic transcription control sequence may include a promoter containing recognition sites for one or more transcription factors, a transcription enhancer sequence, a transcription termination sequence, and a polyadenylation sequence. In certain embodiments, a "control sequence" may include a leader sequence and / or a fusion partner sequence.

[0105] As used herein, the term "polynucleotide" refers to a single- or double-stranded nucleic acid polymer. In certain embodiments, the nucleotides comprising a polynucleotide can be ribonucleotides or deoxyribonucleotides, or modified forms of either type of nucleotide. Such modifications include base modifications (e.g., bromouridine), ribose modifications (e.g., arabinoside and 2',3'-dideoxyribose), and internucleotide linkage modifications (e.g., phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoraniladate, and phosphoroamidate). The term "polynucleotide" specifically includes single- and double-stranded forms of DNA.

[0106] The term "natural nucleotide" includes deoxyribonucleotides and ribonucleotides. The term "modified nucleotide" includes nucleotides with modified or substituted sugar groups, etc. The term "oligonucleotide linkage" includes oligonucleotide linkages such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoraniladate, phosphoramidate, etc. See, e.g., 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 See Uhlmann and Peyman, 1990, Chemical Reviews, 90:543, the disclosures of which are incorporated herein by reference for all purposes. The oligonucleotide can contain a detectable label to allow for detection of the oligonucleotide or its hybridization.

[0107] The term "vector" is used to refer to any molecule (e.g., nucleic acid, plasmid, or virus) used to transfer coding information to a host cell. The term "expression vector" refers to a vector that is suitable for transformation of a host cell and that contains nucleic acid sequences that direct and / or control the expression of inserted heterologous nucleic acid sequences. Expression includes processes such as, but are not limited to, transcription, translation, and, if introns are present, RNA splicing.

[0108] As will be understood by those skilled in the art, polynucleotides can include genomic sequences, extragenomic and plasmid coding sequences, and smaller engineered gene segments that express, or can be adapted to express, proteins, polypeptides, peptides, etc. Such segments can be naturally isolated or synthetically modified by one skilled in the art.

[0109] As will also be appreciated by 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 can include HnRNA molecules, which contain introns and correspond one-to-one to DNA molecules, and mRNA molecules, which do not contain introns. Additional coding or non-coding sequences can, but need not, be present within a polynucleotide in accordance with the present disclosure, and polynucleotides can, but need not, be linked to other molecules and / or supporting materials. Polynucleotides can include native sequences or can include sequences that encode variants or derivatives of such sequences.

[0110] Thus, in accordance with these and related embodiments, the present disclosure also provides polynucleotides encoding the anti-Fzd antibodies or antigen-binding fragments thereof described herein. In certain embodiments, polynucleotides are provided that include some or all of the polynucleotide sequences encoding the antibodies or antigen-binding fragments thereof described herein, as well as the complements of such polynucleotides.

[0111] 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 the antibodies described herein. Some of these polynucleotides have minimal sequence identity to the nucleotide sequence of a native or original polynucleotide sequence that encodes an antibody that binds to a Fzd receptor. Nevertheless, polynucleotides that differ by differences in codon usage are expressly contemplated by the present disclosure. In certain embodiments, sequences that are codon-optimized for mammalian expression are specifically contemplated.

[0112] Thus, in another embodiment of the invention, mutagenesis approaches (e.g., site-directed mutagenesis) can be used to prepare variants and / or derivatives of the antibodies described herein. By this approach, specific modifications in a polypeptide sequence can be made through mutagenesis of the underlying polynucleotides that encode them. These techniques introduce one or more nucleotide sequence changes into a polynucleotide, thereby providing a straightforward approach to preparing and testing sequence variants, for example, incorporating one or more of the above-mentioned considerations.

[0113] Site-directed mutagenesis allows for the production of variants by providing primer sequences of sufficient size and sequence complexity to form stable duplexes on either side of the deletion junction being traversed, using specific oligonucleotide sequences encoding the DNA sequence of the desired mutation and a sufficient number of flanking nucleotides. Mutations can be used in selected polynucleotide sequences to improve, alter, reduce, modify, or otherwise change the properties of the polynucleotide itself and / or to alter the properties, activity, composition, stability, or primary sequence of the encoded polypeptide.

[0114] In certain embodiments, the inventors contemplate mutagenizing a polynucleotide sequence encoding an antibody or antigen-binding fragment thereof disclosed herein to alter one or more properties of the encoded polypeptide (e.g., the binding affinity of the antibody or antigen-binding fragment thereof, or the function of a particular Fc region, or the affinity of the Fc region for a particular FcγR). The technique of site-directed mutagenesis is well known in the art and is widely used to create variants of both polypeptides and polynucleotides. For example, site-directed mutagenesis is often used to modify specific portions of DNA molecules. 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.

[0115] As those skilled in the art will understand, site-directed mutagenesis techniques often use phage vectors that exist in both single-stranded and double-stranded forms. Typical vectors useful for site-directed mutagenesis include vectors such as M13 phage. Such phages are readily commercially available, and their use is widely known to those skilled in the art. Double-stranded plasmids are also commonly used in site-directed mutagenesis, which eliminates the step of transferring the gene of interest from the plasmid to the phage.

[0116] The preparation of sequence variants of a DNA segment encoding a selected peptide using site-directed mutagenesis provides a means of generating potentially useful species and is not intended to be limiting, as there are other methods by which sequence variants of the peptides and DNA sequences encoding them can be obtained. For example, recombinant vectors encoding desired peptide sequences can be treated with mutagenic agents such as hydroxylamine to obtain sequence variants. Specific details regarding such 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, each of which is incorporated herein by reference for this purpose.

[0117] In many embodiments, nucleic acids encoding the subject monoclonal antibodies are directly introduced into host cells, and the cells are incubated under conditions sufficient to induce expression of the encoded antibody. Antibodies of the present disclosure are prepared using standard techniques well known to those of skill in the art, in combination with the polypeptide and nucleic acid sequences provided herein. The polypeptide sequences can be used to determine appropriate nucleic acid sequences encoding particular antibodies disclosed herein. Nucleic acid sequences can be optimized to reflect particular codon "preferences" in various expression systems, according to standard methods well known to those of skill in the art.

[0118] According to certain related embodiments, there are provided recombinant host cells comprising one or more constructs described herein and nucleic acids encoding any antibody, CDR, VH or VL domain, or antigen-binding fragment thereof, as well as methods of producing the encoded product, comprising expression from the encoding nucleic acid therefor. Expression can be conveniently achieved by culturing recombinant host cells containing the nucleic acid under appropriate conditions. Once produced by expression, the antibody or antigen-binding fragment thereof can be isolated and / or purified using any suitable technique and then used as desired.

[0119] Antibodies or antigen-binding fragments thereof, and encoding nucleic acid molecules and vectors provided herein can be isolated and / or purified, e.g., from their natural environment, in substantially pure or homogeneous form, or, in the case of nucleic acids, free or substantially free from nucleic acids or genes of origin other than the sequence encoding a polypeptide having a desired function. Nucleic acids can comprise DNA or RNA and can be wholly or partially synthetic. Reference to a nucleotide sequence set forth herein encompasses DNA molecules having the specified sequence, unless the context requires otherwise, and also encompasses RNA molecules having the specified sequence in which U is substituted for T.

[0120] Systems for cloning and expressing polypeptides in a variety of 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 Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells, NSO mouse melanoma cells, and many others. A common and preferred bacterial host is E. coli.

[0121] Expression of antibodies and antigen-binding fragments thereof in prokaryotic cells, such as E. coli, is well established in the art. For a review, see Pluckthun, A., Bio / Technology 9:545-551 (1991). Expression in eukaryotic cells in culture is also available to those skilled in the art as an option for producing antibodies or antigen-binding fragments thereof. For recent reviews, see, e.g., Ref, ME (1993) Curr. Opinion Biotech. 4:573-576; Trill JJ et al. (1995) Curr. Opinion Biotech 6:553-560.

[0122] Suitable vectors can be chosen or constructed, containing appropriate control sequences, including promoter sequences, termination sequences, polyadenylation sequences, enhancer sequences, marker genes, and other sequences as needed. Vectors can be plasmids, viruses (e.g., phage), or phagemids, as appropriate. For further details, see, for example, Molecular Cloning: a Laboratory Manual: 2nd Edition. See, Current Protocols in Molecular Biology, Second Edition, Ausubel et al., eds., John Wiley & Sons, 1992, or subsequent revisions. For the manipulation of nucleic acids, e.g., in preparing nucleic acid constructs, mutagenesis, sequencing, introducing DNA into cells and expressing genes, and analyzing proteins, many known techniques and protocols are described in detail in Current Protocols in Molecular Biology, Second Edition, Ausubel et al., eds., John Wiley & Sons, 1992, or subsequent revisions.

[0123] The term "host cell" is used to refer to a cell into which a nucleic acid sequence encoding one or more of the antibodies 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 of the antibodies described herein). The term includes progeny of the parent cell, regardless of whether such progeny is identical in morphology or genetic make-up to the original parent, so long as the selected gene is present. Thus, methods involving introducing such nucleic acids into a host cell are also contemplated. This introduction can use any available technique. For eukaryotic cells, suitable techniques include calcium phosphate transfection, DEAE-dextran, electroporation, liposome-mediated transfection, and transduction using retroviruses or other viruses (e.g., vaccinia virus, or, in the case of insect cells, baculovirus). For bacterial cells, suitable techniques include calcium chloride transformation, electroporation, and transfection using bacteriophage. After introduction, expression from the nucleic acid can be caused or permitted, for example, by culturing the host cells under conditions for gene expression. In one embodiment, the nucleic acid is integrated into the genome (e.g., chromosome) of the host cell. Integration can be facilitated by the inclusion of sequences that facilitate recombination with the genome, according to standard techniques.

[0124] The present invention also provides, in certain embodiments, methods that involve using constructs as described above in an expression system to express a particular polypeptide, such as a Fzd-specific antibody as described herein. The term "transduction" is used to refer to the transfer of genes from one bacterium to another, usually by phage. "Transduction" also refers to the acquisition and transfer of eukaryotic sequences by retroviruses. The term "transfection" refers to the uptake of exogenous or foreign DNA by a cell; a cell is "transfected" when the foreign DNA is introduced inside the cell membrane. Several transfection techniques are known in the art and are disclosed herein. See, e.g., Graham et al., 1973, Virology 52:456; Sambrook et al., 2001, MOLECULAR CLONING, A LABORATORY MANUAL, Cold Spring Harbor Laboratories; Davis et al. See, e.g., J. Am. Chem. Soc. 1999, 14:101-104, 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 foreign DNA moieties into a suitable host cell.

[0125] As used herein, the term "transformation" refers to a change in the genetic characteristics of a cell; 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 transforming DNA may recombine with the cell's DNA by physically integrating into the cell's chromosome, may be maintained transiently as an episomal element without replication, or may replicate independently as a plasmid. A cell is considered stably transformed if the DNA replicates with cell division. The terms "natural" or "native," when used in conjunction with biological material such as nucleic acid molecules, polypeptides, host cells, etc., refer to material found in nature and not manipulated by humans. Similarly, as used herein, the terms "non-natural" or "non-native" refer to material not found in nature and structurally modified or synthesized by humans.

[0126] The terms "polypeptide," "protein," and "peptide," as well as "glycoprotein," are used interchangeably and refer to a polymer of amino acids, not limited to any particular length. The terms do not exclude modifications such as myristylation, sulfation, glycosylation, phosphorylation, and the addition or deletion of a signal sequence. The term "polypeptide" or "protein" refers to one or more chains of amino acids, each chain comprising amino acids covalently linked by peptide bonds. The polypeptide or protein may comprise multiple chains non-covalently and / or covalently linked together by peptide bonds, having the sequence of a native protein, i.e., a protein produced by a naturally occurring, specifically non-recombinant, cell, and may include molecules having the amino acid sequence of a native protein or molecules having deletions from, additions to, and / or substitutions of one or more amino acids from the native sequence. The terms "polypeptide" and "protein" specifically encompass antibodies that bind to Fzd receptors of the present disclosure, or sequences having deletions from, additions to, and / or substitutions of one or more amino acids from an anti-Fzd antibody. Thus, a "polypeptide" or "protein" can include either a single amino acid chain (referred to as a "monomer") or multiple amino acid chains (referred to as a "multimer").

[0127] The term "isolated protein" or "isolated antibody," as used herein, means that the subject protein or antibody (1) is free from at least some other proteins with which it would typically be found in nature, (2) is essentially free from other proteins from the same source (e.g., from the same species), (3) is expressed by cells from a different species, (4) is separated from at least about 50% of the polynucleotides, lipids, carbohydrates, or other materials with which it is naturally associated, (5) is not associated (by covalent or noncovalent interactions) with portions of proteins with which it is naturally associated, (6) is operably associated (by covalent or noncovalent interactions) with polypeptides with which it is not naturally associated, or (7) is not naturally occurring. Such isolated proteins can be encoded by genomic DNA, cDNA, mRNA, or other RNA, or can be of synthetic origin, or any combination thereof. In certain embodiments, an isolated protein is substantially free of proteins or polypeptides or other contaminants found in its natural environment that would interfere with its use (therapeutic, diagnostic, prophylactic, research, or other use).

[0128] Amino acid sequence modification(s) of the antibodies described herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. For example, amino acid sequence variants of an antibody can be prepared by introducing appropriate nucleotide changes into a polynucleotide encoding the antibody or a chain thereof, or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into, and / or substitutions of, residues within the amino acid sequence of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final antibody, provided that the final construct possesses the desired characteristics (e.g., high-affinity binding to one or more Fzd receptors). Amino acid changes may also alter post-translational processing of the antibody, for example, resulting in changes in the number or location of glycosylation sites. Any of the variations and modifications described above for the polypeptides of the invention can be included in the antibodies of the present invention.

[0129] The present disclosure provides variants of the antibodies and antigen-binding fragments thereof disclosed herein. In certain embodiments, such variant antibodies, or antigen-binding fragments or CDRs thereof, bind to one or more Fzd receptors with at least about 50%, at least about 70%, and in certain embodiments, at least about 90% of the affinity of the antibody sequences specifically described herein. In further embodiments, such variant antibodies, or antigen-binding fragments or CDRs thereof, bind to one or more Fzd receptors with higher affinity than the antibodies described herein, e.g., at least about 105%, 106%, 107%, 108%, 109%, or 110% of the affinity of the antibody sequences specifically described herein.

[0130] In certain embodiments, the antibody or antigen-binding fragment thereof, e.g., Fab, scFv, VHH, or sdAb, or Wnt surrogate, can comprise: a) a heavy chain variable region comprising: i. a CDR1 region having an amino acid sequence identical to that of the heavy chain CDR1 region of a selected antibody described herein; ii. a CDR2 region having an amino acid sequence identical to that of the heavy chain CDR2 region of the selected antibody; and iii. a CDR3 region having an amino acid sequence identical to that of the heavy chain CDR3 region of the selected antibody; and / or b) a light chain variable region comprising: i. a CDR1 region having an amino acid sequence identical to that of the light chain CDR1 region of the selected antibody; ii. a CDR2 region having an amino acid sequence identical to that of the light chain CDR2 region of the selected antibody; and iii. a CDR3 region having an amino acid sequence identical to that of 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). In further embodiments, the antibody or antigen-binding fragment thereof is a variant antibody or antigen-binding fragment thereof, which comprises heavy and light chains identical to the selected antibody except for up to 8, 9, 10, 11, 12, 13, 14, 15, or more amino acid substitutions within the CDR regions of the VH and VL regions. In this regard, there may be 1, 2, 3, 4, 5, 6, 7, 8, or in certain embodiments, 9, 10, 11, 12, 13, 14, 15, or more amino acid substitutions within the CDR regions of the selected antibody. The substitutions may be within the CDRs of either the VH and / or VL regions (see, e.g., Muller, 1998, Structure 6:1153-1167).

[0131] In certain embodiments, a subject antibody or antigen-binding fragment thereof (e.g., a Fab, scFv, VHH, or sdAb, or a Wnt surrogate) may have: a) a heavy chain variable region having an amino acid sequence at least 80% identical, at least 95% identical, at least 90%, at least 95%, or at least 98% or 99% identical to that of an anti-Fzd antibody or antigen-binding fragment thereof described herein; and / or b) a light chain variable region having an amino acid sequence at least 80% identical, at least 85%, at least 90%, at least 95%, or at least 98% or 99% identical to that of an anti-Fzd antibody or antigen-binding fragment thereof described herein. Exemplary amino acid sequences of antigen-binding fragments thereof are set forth in SEQ ID NOs: 1-65.

[0132] In certain embodiments, an antibody or antigen-binding fragment thereof (e.g., a Fab, scFv, VHH or sdAb, or a Wnt surrogate) may comprise one or more, two or more, three or more, four or more, five or more, or six of the CDRs identified in Table 1A for any particular antibody. In certain embodiments, the antibody or antigen-binding fragment thereof comprises a CDRH1 comprising or consisting of any of SEQ ID NOs: 72-312 or 1327-1347; a CDRH2 comprising or consisting of any of SEQ ID NOs: 313-574 or 1348-1360; a CDRH3 comprising or consisting of any of SEQ ID NOs: 575-930, 1361-1387, or 1436-1443; a CDRL1 comprising or consisting of any of SEQ ID NOs: 931-1060 or 1388-1406; a CDRL2 comprising or consisting of any of SEQ ID NOs: 1061-1158 or 1407-1419; and / or a CDRL3 comprising or consisting of any of SEQ ID NOs: 1159-1326, 1420-1435, or 1444-1453.

[0133] A polypeptide has a certain percentage of "sequence identity" to another polypeptide, meaning that when aligned, the percentage of amino acids in the two sequences are the same. Sequence similarity can be determined in several different ways. To determine sequence identity, sequences can be aligned using methods and computer programs, such as 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 Oxford Molecular Group, Inc., a wholly owned subsidiary of Oxford Molecular Group, Inc., Madison, Wis., USA. Other alignment techniques 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. The Smith-Waterman algorithm is one type of algorithm that allows gaps in sequence alignments. See Meth. Mol. Biol. 70:173-187 (1997). The GAP program, which uses the Needleman and Wunsch alignment method, can also be used to align sequences. See J. Mol. Biol. 48:443-453 (1970).

[0134] Of interest is the BestFit program, which determines sequence identity using the local homology algorithm of Smith and Waterman (Advances in Applied Mathematics 2:482-489 (1981)). The gap creation penalty generally ranges from 1 to 5, usually from 2 to 4, and in many embodiments is 3. The gap extension penalty generally ranges from about 0.01 to 0.20, and in many cases is 0.10. The program has default parameters determined by the input sequences to be compared. Sequence identity is preferably determined using the default parameters determined by the program. This program is also available from the Genetics Computing Group (GCG) package, Madison, Wis., USA.

[0135] 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. Percent sequence identity is calculated by FastDB based on the following parameters: Mismatch penalty: 1.00; gap penalty: 1.00; gap size penalty: 0.33; and ligation penalty: 30.0.

[0136] In certain embodiments, the antibody can comprise: a) a heavy chain variable region comprising: i. a CDR1 region having an amino acid sequence identical to the heavy chain CDR1 region of a selected antibody described herein; ii. a CDR2 region having an amino acid sequence identical to the heavy chain CDR2 region of the selected antibody; and iii. a CDR3 region having an amino acid sequence identical to the heavy chain CDR3 region of the selected antibody; and b) a light chain variable region comprising: i. a CDR1 region having an amino acid sequence identical to the light chain CDR1 region of the selected antibody; ii. a CDR2 region having an amino acid sequence identical to the light chain CDR2 region of the selected antibody; and iii. a CDR3 region having an amino acid sequence identical to the light chain CDR3 region of the selected antibody, wherein the antibody specifically binds to a selected target (e.g., a Fzd receptor such as Fzd1). In further embodiments, the antibody or antigen-binding fragment thereof is a variant antibody, wherein the variant comprises heavy and light chains identical to the selected antibody except for up to 8, 9, 10, 11, 12, 13, 14, 15, or more amino acid substitutions within the CDR regions of the VH and VL regions. In this regard, there may be 1, 2, 3, 4, 5, 6, 7, 8, or in certain embodiments, 9, 10, 11, 12, 13, 14, 15, or more amino acid substitutions within the CDR regions of the selected antibody. The substitutions may be within the CDRs of either the VH and / or VL regions (see, e.g., Muller, 1998, Structure 6:1153-1167).

[0137] Determination of the three-dimensional structure of a representative polypeptide (e.g., a variant Fzd-specific antibody provided herein, e.g., an antibody protein having an antigen-binding fragment provided herein) can be performed using conventional methodologies such that one or more amino acid substitutions, additions, deletions, or insertions with selected natural or unnatural amino acids can be virtually modeled for the purpose of determining whether the structural variants so derived retain the space-filling properties of the species disclosed herein. See, e.g., 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. See, e.g., Dodson 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). Some additional non-limiting examples of computer algorithms that may be used in these and related embodiments, e.g., for the rational design of Fzd-specific antibodies, antigen-binding fragments thereof, provided herein, include VMD, a molecular visualization program for displaying, animating, and analyzing large biomolecular systems using 3D graphics and built-in scripts (see the website of the Theoretical and Computational Biophysics Group, University of Illinois at Urbana-Champagne: ks.uiuc.edu / Research / vmd / ). Numerous other computer programs that allow for the determination of atomic dimensions (van der Waals radii) from space-filling models of energy-minimized conformations are known in the art and available to those skilled in the art, including GRID, which determines regions of high affinity for different chemical groups and attempts to enhance binding; Monte Carlo studies, which calculate mathematical alignments; CHARMM (Brooks et al. (1983) J. Comput. Chem. 4:187-217); and AMBER (Weiner et al. (1981) J. Comput. Chem. 106:765), which evaluate force field calculations and analysis (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 ... (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).A variety of suitable computational computer programs are also commercially available (eg, Schrodinger, Munich, Germany).

[0138] In certain embodiments, the disclosure provides antibodies or antigen-binding fragments thereof that bind to one or more Fzd receptors in the region set forth in Table 3. In certain embodiments, they bind to a region of LRP6 that includes or consists of amino acid residues 637-878, where the amino acid sequence and numbering correspond to those set forth in the Examples. In certain embodiments, they bind to an epitope within a region of LRP6 that includes amino acids 637-878. In certain embodiments, the antibodies or antigen-binding fragments thereof contact LRP6 at any or all of the contact points disclosed in Table 3. In one embodiment, the core interaction site or epitope on LRP6 (atomic distance between Lrp6E3E4 and VHH26 of 5.0 Å or less) includes Arg639, Ala640, Lys622, Glu663, Ile681, Ser682, Lys684, Asp705, Tyr706, Glu708, Thr724, Gly725, Arg751, Try767, Gly768, Gly769, Arg792, Leu810, Asp811, His834, Phe836, Trp850, Ser851, Arg853, Asp874, Tyr875, and Met877 of LRP6. In another embodiment, the core interaction site (atomic distance between Lrp6E3E4 and VHH36 of 5.0 Å or less) includes Glu663, Ser665, Ile681, Tyr706, Glu708, Thr724, Ser749, Arg751, Trp767, Gly768, Arg792, Leu810, Asn813, Pro833, His834, Phe836, Trp850, Ser851, Arg853, Asp874, Try875, and Met877 of LRP6.

[0139] The present disclosure also includes antibodies and antigen-binding fragments thereof that bind to one or more Fzd receptors at specific contact points, including any of the contact points disclosed in Table 3, which shows specific sets of contact points for binding of various anti-Fzd antibodies or fragments thereof.

[0140] In another embodiment of the present invention, anti-Fzd antibodies and humanized versions thereof are derived from rabbit monoclonal antibodies, particularly those produced using RabMAb® technology. Such antibodies are advantageous because they require minimal sequence modifications, thereby facilitating retention of functional properties after humanization using mutational lineage-guided (MLG) humanization techniques (see, e.g., U.S. Pat. No. 7,462,697). Accordingly, exemplary methods for producing anti-Fzd antibodies of the present disclosure include the RabMAb® rabbit monoclonal antibody technology described, for example, in U.S. Pat. Nos. 5,675,063 and 7,429,487. In this regard, in certain embodiments, anti-Fzd antibodies of the present disclosure are produced in rabbits. In certain embodiments, rabbit-derived immortalized B lymphocytes capable of fusing with rabbit splenocytes are used to generate antibody-producing hybrid cells. Immortal B lymphocytes do not detectably express endogenous immunoglobulin heavy chains and, in certain embodiments, can comprise an altered immunoglobulin heavy chain-encoding gene.

[0141] composition Also disclosed are pharmaceutical compositions comprising an anti-Fzd antibody or antigen-binding fragment thereof (e.g., a Wnt surrogate) described herein and one or more pharmaceutically acceptable diluents, carriers, or excipients. In certain embodiments, the pharmaceutical composition further comprises one or more Wnt or Norrin polypeptides.

[0142] In further embodiments, pharmaceutical compositions are also disclosed comprising a polynucleotide comprising a nucleic acid encoding an anti-Fzd antibody or antigen-binding fragment thereof (e.g., a Wnt surrogate) described herein and one or more pharmaceutically acceptable diluents, carriers, or excipients. In certain embodiments, the pharmaceutical composition further comprises one or more polynucleotides comprising a nucleic acid sequence encoding a Wnt polypeptide or a Norrin polypeptide. In certain embodiments, the polynucleotide is DNA or mRNA (e.g., a modified mRNA). In certain embodiments, the polynucleotide is a modified mRNA further comprising a 5' cap sequence and / or a 3' tailing sequence (e.g., a polyA tail). In other embodiments, the polynucleotide is an expression cassette comprising a promoter operably linked to the coding sequence. In certain embodiments, the nucleic acid sequence encoding the anti-Fzd antibody or antigen-binding fragment thereof (e.g., a Wnt surrogate) and the nucleic acid sequence encoding the Wnt polypeptide or Norrin polypeptide are present within the same polynucleotide.

[0143] In further embodiments, pharmaceutical compositions are also disclosed that include an expression vector (e.g., a viral vector) comprising a polynucleotide that includes a nucleic acid encoding an anti-Fzd antibody or antigen-binding fragment thereof (e.g., a Wnt surrogate) described herein, and one or more pharmaceutically acceptable diluents, carriers, or excipients. In certain embodiments, the pharmaceutical composition further comprises an expression vector (e.g., a viral vector) that includes a polynucleotide that includes a nucleic acid sequence encoding a Wnt polypeptide or a Norrin polypeptide. In certain embodiments, the nucleic acid sequence encoding the anti-Fzd antibody or antigen-binding fragment thereof (e.g., a Wnt surrogate) and the nucleic acid sequence encoding the Wnt polypeptide or Norrin polypeptide are present within the same polynucleotide (e.g., expression cassette).

[0144] The present invention further contemplates pharmaceutical compositions comprising a cell containing an expression vector comprising a polynucleotide comprising a promoter operably linked to a nucleic acid encoding an anti-Fzd antibody or antigen-binding fragment thereof described herein, and one or more pharmaceutically acceptable diluents, carriers, or excipients. In certain embodiments, the pharmaceutical composition further comprises a cell containing an expression vector comprising a polynucleotide comprising 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 anti-Fzd antibody or antigen-binding fragment thereof (e.g., a Wnt surrogate) and the nucleic acid sequence encoding the Wnt polypeptide or Norrin polypeptide are present within the same polynucleotide (e.g., expression cassette) and / or within the same cell. In certain embodiments, the cells are xenogeneic cells or autologous cells obtained from the subject to be treated. In certain embodiments, the cells are stem cells (e.g., adipose-derived stem cells or hematopoietic stem cells).

[0145] The present disclosure contemplates a pharmaceutical composition comprising a first molecule for delivering an anti-Fzd antibody or antigen-binding fragment thereof (e.g., a Wnt surrogate) as a first active agent, and a second molecule for delivering a Wnt polypeptide or a Norrin polypeptide. The first and second molecules may be the same type of molecule or different types of molecules. For example, in certain embodiments, the first and second molecules may each be independently selected from the following types of molecules: polypeptides, small organic molecules, nucleic acids (optionally DNA or mRNA, optionally modified RNA) encoding the first and second active agents, vectors (optionally expression vectors or viral vectors) comprising a nucleic acid sequence encoding the first or second active agent, and cells (optionally expression cassettes) comprising a nucleic acid sequence encoding the first or second active agent.

[0146] The subject molecules, alone or in combination, can be combined with generally safe, non-toxic, and pharmaceutically acceptable carriers, diluents, excipients, and reagents useful in preparing desired formulations, including excipients acceptable for use in mammals (e.g., humans or primates). Such excipients can be solid, liquid, semisolid, or, in the case of aerosol compositions, gaseous. Examples of such carriers, diluents, and excipients include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Additional active compounds can also be incorporated into the formulation. The solution or suspension used in the formulation may include sterile diluents such as water for injection, saline, fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antibacterial compounds such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating compounds such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetic acid, citric acid, or phosphoric acid; surfactants for preventing aggregation such as Tween 20; and compounds for adjusting osmotic pressure such as sodium chloride or dextrose. pH can be adjusted using acids or bases such as hydrochloric acid or sodium hydroxide. In certain embodiments, the pharmaceutical composition is sterile.

[0147] Pharmaceutical compositions may also include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. 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 incorporated into a syringe and delivered to a subject via syringe. In certain embodiments, the composition is stable under the conditions of manufacture and storage, preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be, for example, a solvent or dispersion medium containing, for example, water, ethanol, polyol (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 the maintenance of the required particle size in dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents in the composition, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride, etc. Prolonged absorption of the internal composition can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

[0148] Sterile solutions can be prepared by incorporating the required amount of an anti-Fzd antibody or antigen-binding fragment thereof (or an encoding polynucleotide or cells containing the same) in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, preparation methods include vacuum drying and freeze-drying, which yield a powder containing the active ingredient and any additional desired ingredient from a previously sterile-filtered solution thereof.

[0149] In one embodiment, pharmaceutical compositions are prepared with carriers that protect the antibody or antigen-binding fragment thereof against rapid elimination from the body (e.g., controlled-release formulations, including implants and microencapsulated delivery systems). Biodegradable, biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid, can be used. Methods for preparing such formulations will be apparent to those skilled in the art. Materials are also commercially available. Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art.

[0150] It may be advantageous to formulate pharmaceutical compositions in dosage unit form for ease of administration and uniformity of dosage. As used herein, dosage unit form refers to physically discrete units suitable for unitary dosage of the subject to be treated, each unit containing a predetermined amount of active antibody or antigen-binding fragment thereof calculated to produce the desired effect together with the required pharmaceutical carrier. The specifications for the dosage unit form are determined by and directly depend on the characteristics unique to the antibody or antigen-binding fragment thereof, the particular therapeutic effect to be achieved, and the constraints inherent in the technical field of formulating such active antibody or antigen-binding fragment thereof for the treatment of individuals.

[0151] The pharmaceutical compositions can be included in a container, pack, or dispenser, eg, a syringe, eg, a pre-filled syringe, together with instructions for administration.

[0152] The pharmaceutical compositions of the present invention include any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compounds that are capable of providing (directly or indirectly) biologically active antibodies or antigen-binding fragments thereof when administered to an animal, including a human.

[0153] The present invention includes pharmaceutically acceptable salts of the anti-Fzd antibodies or antigen-binding fragments thereof (e.g., Wnt surrogates) described herein. The term "pharmaceutically acceptable salt" refers to a physiologically and pharmaceutically acceptable salt of a compound of the present invention, i.e., a salt that retains the desired biological activity of the parent compound and does not impart undesired toxicological effects thereto. 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). See also "Handbook of Pharmaceutical Salts: Properties, Selection, and Use" by Stahl and Wermuth (Wiley-VCH, 2002) for a review of suitable salts.

[0154] Pharmaceutically acceptable base addition salts are formed with metals or amines, such as alkali and alkaline earth metals, or organic amines. Metals used as cations include sodium, potassium, magnesium, calcium, and the like. 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 aforementioned acidic compounds are prepared in a 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 a conventional manner by contacting the salt form with an acid and isolating the free acid. The free acid forms may differ somewhat from their respective salt forms in certain physical properties (e.g., solubility in polar solvents), but otherwise the salts are equivalent to their respective free acids for purposes of this invention.

[0155] In some embodiments, the pharmaceutical compositions provided herein comprise a therapeutically effective amount of an anti-Fzd antibody or antigen-binding fragment thereof (e.g., a Wnt surrogate) described herein in admixture with a pharmaceutically acceptable carrier, diluent, and / or excipient (e.g., saline, phosphate-buffered saline, phosphate, 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, citrate, acetate, Ringer's solution, Hank's 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.

[0156] In some embodiments, the pharmaceutical compositions provided herein comprise a buffer, such as phosphate-buffered saline (PBS) or sodium phosphate / sodium sulfate, Tris buffer, glycine buffer, sterile water, and other buffers known to those of skill in the art, such as those described by Good et al. (1966) Biochemistry 5:467. The pH of the buffer can be within the range of 6.5 to 7.75, preferably 7 to 7.5, and most preferably 7.2 to 7.4.

[0157] How to use The present disclosure also provides methods for using the Fzd-specific antibodies or antigen-binding fragments thereof (e.g., Wnt surrogates) disclosed herein, e.g., to modulate the Wnt signaling pathway, e.g., to increase or decrease Wnt signaling, as well as methods for administering Fzd-specific antibodies or antigen-binding fragments thereof and Wnt surrogates in various therapeutic settings. Provided herein are therapeutic methods using antibodies or antigen-binding fragments thereof that bind to one or more Fzd receptors. In one embodiment, an antibody or antigen-binding fragment thereof of the present invention is provided to a subject having a disease associated with inappropriate or deregulated Wnt signaling, e.g., increased or decreased Wnt signaling.

[0158] Increasing the Wnt signaling pathway and related therapeutic methods In certain embodiments, anti-Fzd antibodies or antigen-binding fragments thereof (e.g., Wnt surrogates) can be used to increase Wnt signaling in tissues or cells. Accordingly, in some aspects, the present invention provides methods for increasing or enhancing Wnt signaling in tissues or cells, comprising contacting the tissue or cells with an effective amount of an anti-Fzd antibody or antigen-binding fragment thereof (e.g., a Wnt surrogate) disclosed herein, wherein the anti-Fzd antibody or antigen-binding fragment thereof is a Wnt signaling pathway agonist. In some embodiments, the contacting is performed in vitro, ex vivo, or in vivo. In certain embodiments, the cells are cultured cells and the contacting 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.

[0159] In a related aspect, the present invention provides a method for increasing Wnt signaling in a tissue or cell, comprising contacting the tissue or cell with an effective amount of a polynucleotide comprising an anti-Fzd antibody or antigen-binding fragment thereof (e.g., a Wnt surrogate) of the present invention. 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., a modified mRNA). In certain embodiments, the polynucleotide is a modified mRNA further comprising a 5' cap sequence and / or a 3' tailing sequence (e.g., a polyA tail). In other embodiments, the polynucleotide is an expression cassette comprising a promoter operably linked to the coding sequence. In certain embodiments, the nucleic acid sequence encoding the anti-Fzd antibody or antigen-binding fragment thereof (e.g., a Wnt surrogate) and the nucleic acid sequence encoding the Wnt polypeptide or Norrin polypeptide are present within the same polynucleotide.

[0160] In a related aspect, the present invention provides a method for increasing Wnt signaling in a tissue or cell, comprising contacting the tissue or cell with an effective amount of a vector comprising a nucleic acid sequence encoding an anti-Fzd antibody or antigen-binding fragment thereof (e.g., a Wnt surrogate). In certain embodiments, the tissue or cell is also contacted with a vector comprising a nucleic acid sequence encoding a Wnt polypeptide or a Norrin polypeptide. In certain embodiments, the vector is an expression vector and may comprise 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 anti-Fzd antibody or antigen-binding fragment thereof (e.g., a Wnt surrogate) and the nucleic acid sequence encoding the Wnt polypeptide or a Norrin polypeptide are present in the same vector, e.g., the same expression cassette.

[0161] In a related aspect, the present invention provides a method for increasing Wnt signaling in a tissue, comprising contacting the tissue with an effective amount of cells comprising a nucleic acid sequence encoding an anti-Fzd antibody or antigen-binding fragment thereof (e.g., a Wnt surrogate) of the present invention. In certain embodiments, the tissue is also contacted with cells comprising a nucleic acid sequence encoding a Wnt polypeptide or a Norrin polypeptide. In certain embodiments, the nucleic acid sequence encoding the anti-Fzd antibody or antigen-binding fragment thereof (e.g., a Wnt surrogate) and the nucleic acid sequence encoding the Wnt polypeptide or a Norrin polypeptide are present in the same cell. In certain embodiments, the cells are xenogeneic cells or autologous cells obtained from the subject to be treated. In certain embodiments, the cells are transduced with a vector comprising an expression cassette encoding an anti-Fzd antibody or antigen-binding fragment thereof (e.g., a Wnt surrogate) or encoding 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).

[0162] Anti-Fzd antibodies or antigen-binding fragments thereof (e.g., Wnt surrogates) can be used to treat diseases, disorders, or conditions, e.g., by increasing Wnt signaling in targeted cells, tissues, or organs. Accordingly, in some embodiments, the present invention provides methods for treating a disease or condition in a subject in need thereof, e.g., a disease or disorder associated with reduced Wnt signaling or a disease or disorder in which increased Wnt signaling is believed to provide therapeutic benefit, comprising contacting the subject with an effective amount of a composition of the present disclosure. In certain embodiments, the composition is a pharmaceutical composition comprising any of the following: an anti-Fzd antibody or antigen-binding fragment thereof (e.g., a Wnt surrogate); a polynucleotide, e.g., DNA or mRNA, optionally modified mRNA, comprising a nucleic acid sequence encoding an anti-Fzd antibody or antigen-binding fragment thereof (e.g., a Wnt surrogate); a vector, e.g., an expression vector or viral vector, comprising a nucleic acid sequence encoding an anti-Fzd antibody or antigen-binding fragment thereof (e.g., a Wnt surrogate); or a cell comprising a nucleic acid sequence encoding an anti-Fzd antibody or antigen-binding fragment thereof (e.g., a Wnt surrogate), e.g., a cell transduced with an expression vector or viral vector encoding an anti-Fzd antibody or antigen-binding fragment thereof (e.g., a Wnt surrogate). In certain embodiments, the disease or condition is a pathological disease or disorder, or injury (e.g., 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 poor tissue repair, healing, or regeneration, or would benefit from increased tissue repair, healing, or regeneration. In some embodiments, the contacting occurs in vivo, ie, a subject composition is administered to a subject.

[0163] In certain embodiments, the method further includes contacting the subject with a pharmaceutical composition comprising one or more Wnt or Norrin polypeptides. The present disclosure includes contacting the subject with a first molecule for delivering an anti-Fzd antibody or antigen-binding fragment thereof (e.g., a Wnt surrogate) as a first active agent and a second molecule for delivering a Wnt or Norrin polypeptide. The first and second molecules may be the same type of molecule or different types of molecules. For example, in certain embodiments, the first and second molecules may each be independently selected from the following types of molecules: polypeptides, small organic molecules, nucleic acids (optionally DNA or mRNA, optionally modified RNA) encoding the first and second active agents, vectors (optionally expression vectors or viral vectors) comprising a nucleic acid sequence encoding the first or second active agent, and cells (optionally expression cassettes) comprising a nucleic acid sequence encoding the first or second active agent.

[0164] In a related aspect, the present invention provides a method for treating a disease or condition, e.g., a disease or disorder associated with reduced Wnt signaling or a disease or disorder in which increased Wnt signaling is believed to provide therapeutic benefit, comprising contacting a subject in need thereof with a pharmaceutical composition comprising an effective amount of a polynucleotide comprising a nucleic acid sequence encoding an anti-Fzd antibody or antigen-binding fragment thereof (e.g., a Wnt surrogate) disclosed herein. In certain embodiments, the subject is also contacted with a pharmaceutical composition comprising an effective amount of 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 a modified mRNA further comprising a 5' cap sequence and / or a 3' tailing sequence (e.g., a polyA tail). In other embodiments, the polynucleotide is an expression cassette comprising a promoter operably linked to a coding sequence. In certain embodiments, the nucleic acid sequence encoding the anti-Fzd antibody or antigen-binding fragment thereof (eg, a Wnt surrogate) and the nucleic acid sequence encoding the Wnt polypeptide or Norrin polypeptide are present within the same polynucleotide.

[0165] In a related aspect, the present invention provides a method for treating a disease or condition, e.g., a disease or disorder associated with reduced Wnt signaling or a disease or disorder for which increased Wnt signaling is believed to provide therapeutic benefit, comprising contacting a subject in need thereof with a pharmaceutical composition comprising an effective amount of a vector comprising a nucleic acid sequence encoding an anti-Fzd antibody or antigen-binding fragment thereof (e.g., a Wnt surrogate). In certain embodiments, the subject is also contacted with a pharmaceutical composition comprising an effective amount of a vector comprising a nucleic acid sequence encoding a Wnt polypeptide or a Norrin polypeptide. In certain embodiments, the vector is an expression vector and may comprise 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 anti-Fzd antibody or antigen-binding fragment thereof (e.g., a Wnt surrogate) and the nucleic acid sequence encoding the Wnt polypeptide or Norrin polypeptide are present in the same vector, e.g., in the same expression cassette.

[0166] In a related aspect, the present invention provides a method for treating a disease or condition, e.g., a disease or disorder associated with reduced Wnt signaling or a disease or disorder for which increased Wnt signaling is believed to provide therapeutic benefit, comprising contacting a subject in need thereof with a pharmaceutical composition comprising an effective amount of cells comprising a nucleic acid sequence encoding an anti-Fzd antibody or antigen-binding fragment thereof (e.g., a Wnt surrogate). In certain embodiments, the subject is also contacted with cells comprising a nucleic acid sequence encoding a Wnt polypeptide or a Norrin polypeptide. In certain embodiments, the nucleic acid sequence encoding the anti-Fzd antibody or antigen-binding fragment thereof (e.g., a Wnt surrogate) and the nucleic acid sequence encoding the Wnt polypeptide or Norrin polypeptide are present in the same cell. In certain embodiments, the cells are xenogeneic cells or autologous cells obtained from the subject to be treated. In certain embodiments, the cells are transduced with a vector comprising an expression cassette encoding an anti-Fzd antibody or antigen-binding fragment thereof (e.g., a Wnt surrogate), or encoding a Wnt polypeptide or a Norrin polypeptide. In certain embodiments, the cells are stem cells (eg, adipose-derived stem cells or hematopoietic stem cells).

[0167] Wnt signaling plays a critical role in the developmental process and maintenance of stem cells. Reactivation of Wnt signaling is associated with the regeneration and repair of most tissues after injury and disease. Anti-Fzd antibodies or antigen-binding fragments thereof (e.g., 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, infection and inflammation, traumatic injury, toxin / metabolism-induced toxicity, or other pathological conditions. Wnt signaling and enhancers of Wnt signaling have been shown to activate adult tissue-resident stem cells. In some embodiments, compounds of the present invention are administered for use in the treatment of diseased or damaged tissue, for use in tissue regeneration, for use in cell growth and proliferation, and / or for use in tissue engineering.

[0168] Human diseases associated with mutations in the Wnt pathway provide strong evidence for enhancing Wnt signaling in the treatment and prevention of disease. Preclinical in vivo and in vitro studies have provided further evidence that Wnt signaling is involved in many disease states, further supporting the use of anti-Fzd antibodies or antigen-binding fragments thereof (e.g., Wnt surrogates) in various human diseases.

[0169] Human diseases associated with mutations in the Wnt pathway provide strong evidence for the enhancement of Wnt signaling in the treatment and prevention of disease.Preclinical in vivo and in vitro studies have provided further evidence that Wnt signaling is involved 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 enhance bone growth or regeneration, bone grafting, fracture healing, osteoporosis and osteoporotic fractures, spinal fusion, spinal cord injury including vertebral compression fractures, preoperative spinal surgery optimization, orthopedic device osseointegration, tendon-bone integration, tooth growth and regeneration, dental implants, periodontal disease, maxillofacial reconstruction, and the treatment of osteonecrosis of the jaw. The compositions of the present invention may also be used in the treatment of alopecia; enhancing the regeneration of sensory organs, for example, the treatment of hearing loss (including inner and outer hair cell regeneration, treatment of vestibular hypofunction), the treatment of macular degeneration, the treatment of retinopathies (including vitreoretinopathy, diabetic retinopathy, and other retinal degenerative diseases), Fuchs' dystrophy, other corneal diseases, and the like; the treatment of stroke, traumatic brain injury, Alzheimer's disease, multiple sclerosis, muscular dystrophy, muscle atrophy as a result of sarcopenia or cachexia, and other conditions affecting the degeneration or integrity of the blood-brain barrier. The compositions of the present invention may also be used in the treatment of oral mucositis, short bowel syndrome, inflammatory bowel disease (IBD) (including Crohn's disease (CD) and ulcerative colitis (UC), especially CD associated with fistulization), and other gastrointestinal disorders; the treatment of metabolic syndrome, dyslipidemia, the treatment of diabetes, pancreatitis, and conditions in which extrapancreatic and endocrine pancreatic tissues are damaged; conditions in which enhanced epidermal regeneration is desirable, e.g., epidermal wound healing, diabetic foot ulcers, and syndromes involving hypoplasia of the teeth, nails, or skin, and other conditions in which angiogenesis is beneficial; the treatment of myocardial infarction, coronary artery disease, and heart failure; the treatment of enhanced hematopoietic cell growth, e.g., enhancing hematopoietic stem cell transplantation from bone marrow or mobilized peripheral blood, and the treatment of immunodeficiency, graft-versus-host disease, and the like; the treatment of acute kidney injury and chronic kidney disease; the treatment of pulmonary disease, chronic obstructive pulmonary disease (COPD), pulmonary fibrosis (including idiopathic pulmonary fibrosis), and enhanced regeneration of lung tissue.The compositions of the present invention can also be used to enhance hepatocyte regeneration (e.g., liver regeneration), treat cirrhosis, enhance liver transplantation, treat acute liver failure, treat chronic liver disease associated with hepatitis C or B virus infection or following antiviral drug therapy, alcoholic liver disease, alcoholic hepatitis, non-alcoholic liver disease associated with hepatic steatosis or steatohepatitis, etc. The compositions of the present invention can treat diseases and disorders, including, but not limited to, conditions in which regenerative cell growth is desired.

[0170] Human genetics, with loss-of-function or gain-of-function mutations in Wnt signaling components, provides strong evidence supporting enhanced Wnt signaling for bone growth. Conditions in which enhanced bone growth is desirable include, but are not limited to, fractures, grafts, periprosthetic bone growth, osteoporosis, osteoporotic fractures, spinal fusion, vertebral compression fractures, preoperative optimization for spinal surgery, osteonecrosis of the jaw, dental implants, periodontal disease, maxillofacial reconstruction, etc. Anti-Fzd antibodies or antigen-binding fragments thereof (e.g., Wnt surrogates) enhance and promote Wnt signaling, which is crucial for promoting bone regeneration. Methods for regenerating bone tissue benefit from the administration of compounds of the present invention, which may be administered systemically or locally. In some embodiments, bone marrow cells are exposed to a molecule of the present invention, resulting in the activation of stem cells within the bone marrow cells.

[0171] In some embodiments, bone regeneration is enhanced by contacting a responsive cell population (e.g., bone marrow, osteoprogenitor cells, osteoprogenitor cells, etc.) with an effective dose of an anti-Fzd antibody or antigen-binding fragment thereof (e.g., a Wnt surrogate disclosed herein). Methods for regenerating bone tissue benefit from the administration of an anti-Fzd antibody or antigen-binding fragment thereof (e.g., a Wnt surrogate) disclosed herein, which administration may be systemic or localized. In some such embodiments, the contacting is performed in vivo. In other such embodiments, the contacting is performed ex vivo. Molecules can be localized to the site of action, for example, by loading onto a matrix that is optionally biodegradable and optionally provides sustained release of the active agent. Matrix carriers include, but are not limited to, absorbable collagen sponges, ceramics, hydrogels, polymer microspheres, nanoparticles, bone cement, etc.

[0172] Compositions comprising one or more anti-Fzd antibodies or antigen-binding fragments thereof (e.g., Wnt surrogates) disclosed herein can be used in the in vivo treatment of skeletal tissue defects. A "skeletal tissue defect" refers to a defect in bone or other bone-connective tissue at any site where bone or connective tissue restoration is desired, regardless of how the defect occurred, for example, as a result of surgical intervention, tumor removal, ulcer, transplant, fracture, or other traumatic or degenerative condition. The compositions of the present invention can be used as part of a regimen for restoring cartilage function to connective tissue, repairing abnormalities or lesions of cartilage tissue, for example, those caused by degenerative wear and arthritis, trauma to the tissue, meniscus tear replacement, meniscectomy, joint dislocation due to ligament tear, joint malalignment, fracture, or genetic disease.

[0173] Anti-Fzd antibodies or antigen-binding fragments thereof (e.g., Wnt surrogates) can also be used to treat periodontal disease. Periodontal disease is a leading cause of tooth loss and is associated with multiple systemic conditions. In some embodiments, contacting a responsive cell population enhances tooth or basal bone regeneration. In some such embodiments, the contacting is performed in vivo. In other such embodiments, the contacting is performed ex vivo, followed by transplantation of activated stem or progenitor cells. Molecules can be localized to the site of action, for example, by loading onto a matrix that is optionally biodegradable and optionally provides sustained release of the active agent. Matrix carriers include, but are not limited to, absorbable collagen sponges, ceramics, hydrogels, bone cements, polymer microspheres, nanoparticles, and the like.

[0174] Multiple studies have shown that Wnt signaling and R-spondin biology can promote the regeneration of sensory hair cells in the inner ear after injury, aging, or degeneration. Sensory hair cell loss in the inner ear associated with hearing loss or vestibular dysfunction can also benefit from the compositions of the present invention. In the inner ear, the auditory organ contains mechanosensitive hair cells required to convert sound vibrations into electrical impulses. The vestibular organ, consisting of the semicircular canals (SSCs), utricle, and saccule, also contains sensory hair cells to detect 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 local application to the ear, to enhance hearing regeneration.

[0175] Anti-Fzd antibodies or antigen-binding fragments thereof (e.g., Wnt surrogates) can also be used to regenerate retinal tissue. In the adult mammalian retina, Müller glia cells can regenerate retinal cells, including photoreceptors, after, for example, neurotoxic injury in vivo. Wnt signaling and enhancers of Wnt signaling can promote the proliferation of Müller glia-derived retinal progenitor cells after injury or during degeneration. The compositions of the present invention can also be used to regenerate tissues and other cell types within the eye. For example, age-related macular degeneration (AMD), other retinal degenerative diseases, corneal diseases, Fuchs' dystrophy, vitreoretinopathy, genetic disorders, and the like can benefit from the compositions of the present invention. AMD is characterized by a progressive loss of central vision and visual acuity. Fuchs' dystrophy is characterized by a progressive loss of corneal endothelial cells. Enhancement of Wnt signaling and Wnt signaling 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 retinal regeneration and treatment of macular degeneration, for example, by injection, in a matrix or other depot system, or other topical application to the eye.

[0176] Lineage tracing studies have identified specific proliferating cell populations involved in homeostatic hepatocyte regeneration, such as Axin 2-positive cells in the pericentral region. Lineage tracing studies have also identified additional potential hepatic progenitor cells, including, but not limited to, Lgr-positive cells. Self-renewing hepatocytes, as well as other potential progenitor cell populations, including Lgr5-positive and Axin 2-positive cells, have been shown to be capable of regeneration after injury in response to Wnt signaling and / or R-spondin. Numerous preclinical models of acute liver injury and chronic liver disease have demonstrated that hepatocyte recovery and regeneration benefit from Wnt signaling. The compositions of the present invention can be used to treat acute liver failure, acute alcoholic liver injury, chronic liver disease associated with hepatitis C or B virus infection or following antiviral drug therapy, chronic alcoholic liver disease, alcoholic hepatitis, nonalcoholic fatty liver disease, and nonalcoholic steatohepatitis (NASH), cirrhosis, and chronic liver disease of all causes, as well as to enhance hepatocyte regeneration. Methods for regenerating liver tissue benefit from the administration of the compounds of the present invention, which may be systemic or localized, including, but not limited to, systemic and localized administration, such as by injection into liver tissue, by injection into veins or blood vessels leading to the liver, by placement of sustained release formulations, and the like.

[0177] Wnt signaling plays an important 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 rapid division of epithelial cells lining the gastrointestinal tract is disrupted, leaving the mucosal tissue susceptible to ulcers and infection. The epithelial lining lining the mouth, called 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, particularly 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 related to hypoplasia of teeth, nails, or skin. The molecules of the present invention can be used in all such conditions, where regenerative cells are contacted with the compounds of the present invention. Methods for regenerating epithelial tissue benefit from the administration of the compounds of the present invention, which may be systemic or localized. Contacting can be, for example, topical (including intradermal, subcutaneous), applied to the target site, etc. in a gel, lotion, cream, etc.

[0178] Wnt signaling and its enhancement and promotion play an important role in the repair and regeneration of tissues, including the skin and gastrointestinal tract, as well as the pancreas, kidney, and lung in preclinical models. Anti-Fzd antibodies or antigen-binding fragments thereof (e.g., Wnt surrogates) may be beneficial in various disease states involving the extrapancreatic and endocrine pancreatic regions, kidney, or lung. Anti-Fzd antibodies or antigen-binding fragments thereof can be used in the treatment of metabolic syndrome, diabetes, acute or chronic pancreatitis, exocrine pancreatic insufficiency, acute kidney injury, chronic kidney disease, and lung diseases, including, but not limited to, chronic obstructive pulmonary disease (COPD) and other conditions that cause loss of lung epithelial tissue. These tissue regeneration methods benefit from the administration of compounds of the present invention, which may be administered systemically or locally.

[0179] Epidermal Wnt signaling, in cooperation with signaling via other developmental factors, is crucial for adult hair follicle regeneration. Hair loss is a common problem, and androgenetic 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 a molecule of the present invention. In some such embodiments, contacting is performed in vivo. In other such embodiments, contacting is performed ex vivo. The molecule can be localized to the site of action, for example, by topical lotion, gel, cream, etc.

[0180] Stroke, traumatic brain injury, Alzheimer's disease, multiple sclerosis, and other conditions affecting the blood-brain barrier (BBB) can be treated using anti-Fzd antibodies or antigen-binding fragments thereof (e.g., Wnt surrogates). 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 because nervous tissue is sensitive 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-neuronal tissues in that they express specific transporters. Wnt signaling regulates angiogenesis and / or function in the central nervous system. Conditions in which the BBB is compromised can benefit from the administration of compounds of the present invention, which can be systemic or localized, 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 neuroprotection after injury. The compositions of the present invention can also be used to treat spinal cord injury, other spinal cord diseases, stroke, traumatic brain injury, and the like.

[0181] Wnt signaling also plays a role in angiogenesis. Anti-Fzd antibodies or antigen-binding fragments thereof (e.g., Wnt surrogates) may be beneficial in conditions where angiogenesis is beneficial, such as the treatment of myocardial infarction, coronary artery disease, heart failure, diabetic retinopathy, and conditions resulting from genetic diseases. These tissue regeneration methods benefit from the administration of compounds of the invention, which may be administered systemically or locally.

[0182] In certain embodiments, the methods of the present invention promote tissue regeneration, for example, in tissues that have been damaged or have suffered a reduction or loss of tissue or cells. Loss or damage can be any event that reduces the number of cells, including disease or injury. For example, an accident, an autoimmune disorder, a side effect of treatment, or a disease state would be considered to constitute trauma. Tissue regeneration increases the number of cells in the tissue, preferably allowing connections between cells in the tissue to be reestablished, and more preferably allowing the functionality of the tissue to be restored.

[0183] Reduction of the Wnt signaling pathway and related therapeutic methods In certain embodiments, anti-Fzd antibodies or antigen-binding fragments thereof can be used to reduce or inhibit Wnt signaling in tissues or cells. Accordingly, in some aspects, the present invention provides methods for reducing or inhibiting Wnt signaling in tissues or cells, comprising contacting the tissue or cells with an effective amount of an anti-Fzd antibody or antigen-binding fragment thereof disclosed herein, wherein the anti-Fzd antibody or antigen-binding fragment thereof is a Wnt signaling pathway antagonist or inhibitor. In some embodiments, the contacting is performed in vitro, ex vivo, or in vivo. In certain embodiments, the cells are cultured cells, and the contacting is performed in vitro.

[0184] In a related aspect, the present invention provides a method for reducing or inhibiting Wnt signaling in a tissue or cell, comprising contacting the tissue or cell with an effective amount of a polynucleotide comprising an anti-Fzd antibody or antigen-binding fragment thereof of the present invention, wherein the anti-Fzd antibody or antigen-binding fragment thereof is a Wnt signaling pathway antagonist or inhibitor. 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 polyA tail). In other embodiments, the polynucleotide is an expression cassette comprising a promoter operably linked to a coding sequence.

[0185] In a related aspect, the present invention provides a method for reducing or inhibiting Wnt signaling in a tissue or cell, comprising contacting the tissue or cell with an effective amount of a vector comprising a nucleic acid sequence encoding an anti-Fzd antibody or antigen-binding fragment thereof, wherein the anti-Fzd antibody or antigen-binding fragment thereof is a Wnt signaling pathway antagonist or inhibitor. In certain embodiments, the vector is an expression vector and can include a promoter operably linked to the nucleic acid sequence. In certain embodiments, the vector is a viral vector.

[0186] In a related aspect, the present invention provides a method for reducing or inhibiting Wnt signaling in a tissue, comprising contacting the tissue with an effective amount of cells comprising a nucleic acid sequence encoding an anti-Fzd antibody or antigen-binding fragment thereof, wherein the anti-Fzd antibody or antigen-binding fragment thereof is a Wnt signaling pathway antagonist or inhibitor. In certain embodiments, the cells are heterologous cells or autologous cells obtained from the subject to be treated. In certain embodiments, the cells are transduced with a vector comprising an expression cassette encoding an anti-Fzd antibody or antigen-binding fragment thereof, wherein the anti-Fzd antibody or antigen-binding fragment thereof is a Wnt signaling pathway antagonist or inhibitor. In certain embodiments, the cells are stem cells (e.g., adipose-derived stem cells or hematopoietic stem cells).

[0187] Anti-Fzd antibodies and antigen-binding fragments thereof, wherein the anti-Fzd antibody or antigen-binding fragment thereof is a Wnt signaling pathway antagonist or inhibitor, can be used to treat a disease, disorder, or condition, e.g., by reducing or inhibiting Wnt signaling in a cell, tissue, or organ. Accordingly, in some embodiments, the present invention provides a method for treating a disease or condition in a subject in need thereof, e.g., a disease or disorder associated with increased or deregulated Wnt signaling, or a disease or disorder in which decreased Wnt signaling is believed to provide a therapeutic benefit, comprising contacting the subject with an effective amount of a composition comprising an anti-Fzd antibody or antigen-binding fragment thereof, wherein the anti-Fzd antibody or antigen-binding fragment thereof is a Wnt signaling pathway antagonist or inhibitor. In certain embodiments, the composition is a pharmaceutical composition comprising any of the following: an anti-Fzd antibody or antigen-binding fragment thereof; a polynucleotide, e.g., DNA or mRNA, optionally modified mRNA, comprising a nucleic acid sequence encoding the anti-Fzd antibody or antigen-binding fragment thereof; a vector, e.g., an expression vector or viral vector, comprising a nucleic acid sequence encoding the anti-Fzd antibody or antigen-binding fragment thereof; or a cell comprising a nucleic acid sequence encoding the anti-Fzd antibody or antigen-binding fragment thereof, e.g., a cell transduced with an expression vector or viral vector encoding the anti-Fzd antibody or antigen-binding fragment thereof. In certain embodiments, the disease or condition is a pathological disease or disorder, or injury. In some embodiments, the contacting occurs in vivo, i.e., the subject composition is administered to a subject.

[0188] In a related aspect, the present invention provides a method for treating a disease or condition, e.g., a disease or disorder associated with increased Wnt signaling or a disease or disorder in which reduced Wnt signaling is believed to provide therapeutic benefit, comprising contacting a subject in need thereof with a pharmaceutical composition comprising an effective amount of a polynucleotide comprising a nucleic acid sequence encoding an anti-Fzd antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof is a Wnt signaling pathway antagonist or inhibitor disclosed herein. In certain embodiments, the polynucleotide is DNA or mRNA (e.g., modified mRNA). In certain embodiments, the polynucleotide is a modified mRNA further comprising a 5' cap sequence and / or a 3' tailing sequence (e.g., a polyA tail). In other embodiments, the polynucleotide is an expression cassette comprising a promoter operably linked to the coding sequence.

[0189] In a related aspect, the present invention provides a method for treating a disease or condition, such as a disease or disorder associated with increased Wnt signaling or a disease or disorder for which decreased Wnt signaling is believed to provide therapeutic benefit, comprising contacting a subject in need thereof with a pharmaceutical composition comprising an effective amount of a vector comprising a nucleic acid sequence encoding an anti-Fzd antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof is a Wnt signaling pathway antagonist or inhibitor. In certain embodiments, the vector is an expression vector and may comprise a promoter operably linked to the nucleic acid sequence. In certain embodiments, the vector is a viral vector.

[0190] In a related aspect, the present invention provides a method for treating a disease or condition, e.g., a disease or disorder associated with increased Wnt signaling or a disease or disorder for which decreased Wnt signaling is believed to provide therapeutic benefit, comprising contacting a subject in need thereof with a pharmaceutical composition comprising an effective amount of cells comprising a nucleic acid sequence encoding an anti-Fzd antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof is a Wnt signaling pathway antagonist or inhibitor. In certain embodiments, the cells are heterologous cells or autologous cells obtained from the subject to be treated. In certain embodiments, the cells are transduced with a vector comprising an expression cassette encoding an anti-Fzd antibody or antigen-binding fragment thereof. In certain embodiments, the cells are stem cells (e.g., adipose-derived stem cells or hematopoietic stem cells).

[0191] In certain embodiments, the method of treating a disease or disorder in a subject in need thereof by providing to the subject an effective amount of an anti-Fzd antibody or antigen-binding fragment thereof that is an inhibitor of the Wnt signaling pathway can be used to treat cancer or tumors (e.g., solid tumors or liquid tumors). Examples of cancers and tumors that can be treated include, but are not limited to, colon tumors (e.g., colon cancer or colon adenoma), gastric tumors (e.g., gastric cancer), small intestine tumors (e.g., small intestine cancer), liver tumors (e.g., liver cancer), pancreatic tumors (e.g., pancreatic cancer), lung tumors (e.g., lung cancer), ovarian tumors (e.g., ovarian cancer), kidney (e.g., kidney cancer), brain tumors (e.g., brain cancer), spinal cord tumors (e.g., spinal cord cancer), skin tumors (e.g., skin cancer or melanoma), head and neck tumors (e.g., head and neck cancer), gastrointestinal tract tumors (e.g., gastrointestinal tract cancer, esophageal cancer, oral mucosal cancer, tongue cancer, stomach cancer, intestinal cancer, colon cancer), breast tumors (e.g., breast cancer), prostate tumors (e.g., prostate cancer), bone tumors (e.g., bone cancer), vascular tumors, Wilms' tumor, leukemia / lymphoma, soft tissue tumors (e.g., soft tissue sarcoma or synovial sarcoma), and metastatic cancer.

[0192] In certain embodiments, the method of treating a disease or disorder in a subject in need thereof by providing to the subject an effective amount of an anti-Fzd antibody or antigen-binding fragment thereof that is an inhibitor of the Wnt signaling pathway can be used to treat degenerative diseases. Examples of degenerative diseases that can be treated include, but are not limited to, osteoarthritis, cartilage degeneration, sports injuries (e.g., cartilage damage), retinopathy, atherosclerosis, neurodegenerative disorders, and vascular disorders, e.g., vasculitis, conditions involving abnormal angiogenesis.

[0193] In certain embodiments, the method of treating a disease or disorder in a subject in need thereof by providing to the subject an effective amount of an anti-Fzd antibody or antigen-binding fragment thereof that is an inhibitor of the Wnt signaling pathway can be used to treat fibrosis. Examples of fibrosis that can be treated include, but are not limited to, pulmonary fibrosis (including, but not limited to, COPD and idiopathic pulmonary fibrosis), renal fibrosis (e.g., end-stage renal failure), hepatic fibrosis, congenital hepatic storage disease, and cardiac fibrosis.

[0194] In certain embodiments, the method of treating a disease or disorder in a subject in need thereof by providing to the subject an effective amount of an anti-Fzd antibody or antigen-binding fragment thereof that is an inhibitor of the Wnt signaling pathway can be used to treat heart failure, e.g., congestive heart failure, systolic heart failure, heart failure with preserved ejection fraction, or coronary artery disease.

[0195] In certain embodiments, the method of treating a disease or disorder in a subject in need thereof by providing to the subject an effective amount of an anti-Fzd antibody or antigen-binding fragment thereof that is an inhibitor of the Wnt signaling pathway can be used to treat heterotopic ossification, osteoporosis, or congenital high bone mass disorders.

[0196] The terms "administering" or "introducing" or "providing," as used herein, refer to delivering a composition to a cell, cells, tissue, and / or organ of a subject, or to a subject. Such administration or introduction can occur in vivo, in vitro, or ex vivo.

[0197] In certain embodiments, the pharmaceutical composition is administered parenterally (e.g., intravenously), orally, rectally, or by injection. In some embodiments, the pharmaceutical composition is administered locally (e.g., topically or intramuscularly). In some embodiments, the composition is administered to a target tissue (e.g., bone, joint, ear tissue, ocular tissue, gastrointestinal tract, skin, wound site, or spinal cord). The methods of the present invention can be performed in vivo or ex vivo. In some embodiments, contacting a target cell or tissue with a tissue-specific Wnt signal-enhancing molecule is performed ex vivo, followed by transplantation of the cell or tissue (e.g., activated stem or progenitor cells) into a subject. One skilled in the art can determine the appropriate site and route of administration based on the disease or disorder to be treated.

[0198] The dosage and dosing regimen can depend on a variety of factors readily determined by a physician, such as the nature of the disease or disorder, the subject's characteristics, and the subject's medical history. In certain embodiments, the amount of an anti-Fzd antibody or antigen-binding fragment thereof (e.g., a Wnt surrogate) administered or provided to a subject is within the range of about 0.01 mg / kg to about 50 mg / kg, 0.1 mg / kg to about 500 mg / kg, or 0.1 mg / kg to about 50 mg / kg of the subject's body weight.

[0199] The terms "treatment," "treating," and the like are used herein broadly to mean achieving a desired pharmacological and / or physiological effect. The effect may be prophylactic, in terms of completely or partially preventing a disease or its symptoms (e.g., reducing the likelihood that a disease or its symptoms will occur in a subject), and / or therapeutic, in terms of partially or completely curing a disease or adverse effects that may result from a disease. As used herein, "treatment" encompasses any treatment of a disease in a mammal, including (a) preventing a disease from occurring in a subject who may be susceptible to the disease but has not yet been diagnosed with it; (b) inhibiting a disease, i.e., arresting its development; or (c) alleviating a disease, i.e., causing regression of a disease. A therapeutic agent (e.g., an anti-Fzd antibody or antigen-binding fragment thereof) can be administered before, during, or after the onset of a disease or injury. Treatment of an ongoing disease is of particular interest if the treatment stabilizes or reduces undesirable clinical symptoms in the patient. Such treatment is desirably performed before the affected tissue loses all function. The subject therapy is administered during the symptomatic stage of the disease, and in some cases, desirably after the symptomatic stage of the disease. In some embodiments, the subject method provides a therapeutic benefit (e.g., preventing the onset of a disorder, halting the progression of a disorder, reversing the progression of a disorder, etc.). In some embodiments, the subject method includes a step of detecting that a therapeutic benefit has been achieved. Those skilled in the art will understand that such measures of therapeutic efficacy will be applicable to the particular disease being modified, and will recognize appropriate detection methods to use to measure therapeutic efficacy.

[0200] Promoting cell, tissue, and organoid growth and related methods Another embodiment is, in part, the use of the Wnt surrogate 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 surrogates, optionally in combination with Norrin or Rspondin polypeptide.In certain embodiments, cells or tissues are contacted ex vivo, in vitro, or in vivo.Such methods can be used to generate cells, tissues, or organoids for therapeutic use, for example, transplanted or grafted into subjects.Such methods can also be used to generate cells, tissues, or organoids for research use.Wnt surrogate molecules are widely applied in non-therapeutic methods, for example, in vitro research methods.

[0201] The present invention provides methods for tissue regeneration of damaged tissue (e.g., the tissues discussed above), comprising administering a Wnt surrogate molecule to cells. The Wnt surrogate molecule may be administered directly to cells in vivo, orally, intravenously, or by other methods known in the art to a subject, or to ex vivo cells. In some embodiments in which a Wnt surrogate molecule is administered to ex vivo cells, such cells may be transplanted into a subject before, after, or during administration of the Wnt surrogate molecule.

[0202] Wnt signaling is a key component of stem cell culture.For example, stem cell culture medium as described in WO2010 / 090513, WO2012 / 014076, Sato et al., 2011 (GASTROENTEROLOGY 2011;141:1762-1772) and Sato et al., 2009 (Nature 459,262-5).The Wnt surrogate molecule disclosed herein is suitable for use in such stem cell culture medium as a substitute for Rspondin, or can be combined with Rspondin.

[0203] Therefore, in one embodiment, the present disclosure provides a method for enhancing the proliferation of stem cells, comprising contacting stem cells with one or more Wnt surrogate molecules disclosed herein.In one embodiment, the present disclosure provides a cell culture medium comprising the Wnt surrogate molecules disclosed herein.In some embodiments, the cell culture medium can be any cell culture medium that is already known in the art, which usually contains Wnt or Rspondin, and wherein Wnt or Rspondin is replaced (fully or partially) or supplemented with the Wnt surrogate molecule(s) disclosed herein. For example, the medium may be as 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 entireties.

[0204] Stem cell culture media often contain additional growth factors. Therefore, the method may additionally include supplying growth factors to stem cells. Growth factors commonly used in cell culture media include epidermal growth factor (EGF, (Peprotech)), transforming growth factor (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 mitogen for a variety of cultured ectodermal and mesodermal cells and has a pronounced effect on the differentiation of certain cells in vivo and in vitro, as well as on the differentiation of some fibroblasts in cell culture. The EGF precursor exists as a membrane-bound molecule that is proteolytically cleaved to generate a 53-amino acid peptide hormone that stimulates cells. EGF or other mitogenic growth factors can be supplied to stem cells in this manner. During stem cell culture, mitogenic growth factors may be added to the medium every two days, while the medium is preferably changed every four days. Generally, the mitogenic 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 present disclosure includes a stem cell culture medium comprising a Wnt surrogate molecule disclosed herein, optionally in combination with one or more growth factors or combinations thereof described herein.

[0205] These methods of 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).

[0206] In some embodiments, Wnt surrogate 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 derived 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.

[0207] Diagnostic and Related Methods 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. Accordingly, the present disclosure provides methods for detecting one or more Fzd receptors in a sample, e.g., methods for detecting cells or tissues expressing Fzd1. Such methods can be applied to a variety of known detection formats, 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-linked immunoassay (ELISA). In certain embodiments, the method includes contacting tissue or cells (e.g., obtained from a subject) with an anti-Fzd antibody or antigen-binding fragment thereof disclosed herein, quantifying the amount of antibody or antigen-binding fragment thereof bound to the tissue or cells, and then quantifying the presence or amount of Fzd receptor(s) in the tissue or cells.

[0208] ISH is a type of hybridization that uses labeled complementary DNA or RNA strands (i.e., primary binders) to localize specific DNA or RNA sequences within a portion or section of a cell or tissue (in situ), or within the entire tissue (whole mount) if the tissue is small enough. Those skilled in the art will understand that this ISH differs from immunohistochemistry, which uses antibodies as primary binders to localize proteins within tissue sections. DNA ISH can be used on genomic DNA to determine chromosome structure. Fluorescent DNA ISH (FISH) can be used in medical diagnostics, for example, to assess chromosomal integrity. RNA ISH (hybridization histochemistry) is used to measure and localize mRNA and other transcripts within tissue sections or whole mounts.

[0209] In various embodiments, the antibodies and antigen-binding fragments thereof described herein are conjugated to a detectable label that can be detected directly or indirectly. In this regard, an antibody "conjugate" refers to an anti-Fzd antibody or antigen-binding fragment thereof covalently linked to a detectable label. In the present invention, DNA probes, RNA probes, monoclonal antibodies, antigen-binding fragments thereof, 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 itself be detected without the need for the addition of a second antibody (secondary antibody).

[0210] A "detectable label" is a molecule or substance that can produce a detectable signal (e.g., visually, electrically, or otherwise) that indicates the presence and / or concentration of the label in a sample. When conjugated to an antibody, a detectable label can be used to localize and / or quantify the target to which a particular antibody is directed. The presence and / or concentration of the target in a sample can thereby be detected by detecting the signal produced 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 to detect one or more targets.

[0211] Examples of detectable labels that can be directly detected 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 a primary antibody. Thus, detection is performed by detecting the binding of the secondary antibody or binder to the detectable primary antibody. Examples of detectable primary binders or antibodies that require the addition of a secondary binder or antibody include detectable enzyme binders and detectable hapten binders.

[0212] In some embodiments, the detectable label is conjugated to a nucleic acid polymer that comprises a first binding agent (e.g., in an ISH, WISH, or FISH process), while in other embodiments, the detectable label is conjugated to an antibody that comprises a first binding agent (e.g., in an IHC process).

[0213] Examples of detectable labels that can be conjugated to antibodies 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.

[0214] Examples of fluorescent labels include 5-(and 6)-carboxyfluorescein, 5- or 6-carboxyfluorescein, 6-(fluorescein)-5-(and 6)-carboxamidohexanoic acid, fluorescein isothiocyanate, rhodamine, tetramethylrhodamine, and dyes such as Cy2, Cy3, and Cy5, optionally substituted coumarins (including AMCA), PerCP, phycobiliproteins (including R-phycoerythrin (RPE) and allophycoerythrin (APC)), Texas Red, Princeton Red, green fluorescent protein (GFP) and its analogs, and conjugates of R-phycoerythrin or allophycoerythrin, inorganic fluorescent labels, e.g., particles based on semiconductor materials such as coated CdSe nanocrystallites.

[0215] Examples of polymer particle labels include polystyrene, PMMA, or silica microparticles or latex particles into which fluorescent dyes can be embedded, or polymer micelles or capsules containing dyes, enzymes, or substrates.

[0216] Examples of metal particles 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 labels include horseradish peroxidase (HRP), alkaline phosphatase (ALP or AP), β-galactosidase (GAL), glucose-6-phosphate dehydrogenase, β-N-acetylglucosaminidase, β-glucuronidase, invertase, xanthine oxidase, firefly luciferase, and glucose oxidase (GO). Examples of commonly used horseradish peroxidase substrates include 3,3'-diaminobenzidine (DAB), diaminobenzidine with nickel enhancement, 3-amino-9-ethylcarbazole (AEC), benzidine dihydrochloride (BDHC), Hanker-Yates reagent (HYR), Indophane blue (IB), tetramethylbenzidine (TMB), 4-chloro-1-naphthol (CN), and alpha-naphthol. These include pyronin (alpha-NP), o-dianisidine (OD), 5-bromo-4-chloro-3-indolyl phosphate (BCIP), nitroblue tetrazolium (NBT), 2-(p-iodophenyl)-3-p-nitrophenyl-5-phenyl tetrazolium chloride (INT), tetranitroblue tetrazolium (TNBT), and 5-bromo-4-chloro-3-indoxyl-beta-D-galactoside / iron(II) ferricyanide (BCIG / FF).

[0217] Examples of commonly used horseradish peroxidase substrates and alkaline phosphatase substrates include naphthol-AS-B1-phosphate / Fast Red TR (NABP / FR), naphthol-AS-MX-phosphate / Fast Red TR (NAMP / FR), naphthol-AS-B1-phosphate / Fast Red TR (NABP / FR), naphthol-AS-MX-phosphate / Fast Red TR (NAMP / FR), naphthol-AS-B1-phosphate / New Fuchsin (NABP / NF), bromochloroindolyl phosphate / nitroblue tetrazolium (BCIP / NBT), and 5-bromo-4-chloro-3-indolyl-bd-galactopyranoside (BCIG).

[0218] Examples of luminescent labels include luminol, isoluminol, acridinium esters, 1,2-dioxetanes, and pyridopyridazines. Examples of electrochemiluminescent labels include ruthenium derivatives. Examples of radioactive labels include radioactive isotopes of iodide, cobalt, selenium, tritium, carbon, sulfur, and phosphorus.

[0219] The detectable label may be conjugated to an antibody described herein or to any other molecule (e.g., an antibody, a nucleic acid probe, or a polymer) that specifically binds to the biological marker of interest. Furthermore, those skilled in the art will understand that the detectable label may be conjugated to a second, third, 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 serves as a signal amplification step. Biological markers can be visually detected, for example, using a light microscope, a fluorescence microscope, or an electron microscope, when the detectable label is, for example, a dye, a colloidal gold particle, or a luminescent reagent. Visually detectable substances bound to biological markers can also be detected using a spectrophotometer. When the detectable substance is a radioisotope, detection can be visually performed by autoradiography or non-visually performed using a scintillation counter. See, e.g., 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).

[0220] The present invention further provides kits for detecting one or more Fzd receptors or cells or tissues expressing one or more Fzd receptors in a sample, comprising at least one antibody, polypeptide, polynucleotide, vector, or host cell described herein. In certain embodiments, the kits can include buffers, enzymes, labels, substrates, beads or other surfaces to which the antibodies of the invention can be attached, and instructions for use.

[0221] All of the above U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications referenced herein and / or listed in the Application Data Sheet are hereby incorporated by reference in their entirety.

[0222] From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not to be limited except as by the appended claims. [Example]

[0223] Example 1 Characterization of anti-Fzd antibodies The antibody Fab, scFv, VHH or sdAb fragments disclosed herein were sequenced and subcloned into mammalian expression vectors for expression, purification, and binding affinity characterization for various Fzd receptors.

[0224] Soluble recombinant proteins were prepared by transfecting each expression vector into Expi293F cells (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer's instructions. Briefly, 4 days after transfection, cell culture medium was collected after cell pellet sedimentation. The medium was incubated with either Protein A resin (REPLIGEN, Waltham, MA) to collect proteins containing human IgG-Fc moieties, or nickel affinity resin (Roche, Basel, Switzerland) to collect proteins conjugated with His tags. Proteins were eluted from the Protein A resin with 10 mM glycine, pH 3.5, or from the nickel affinity resin with 150 mM indazole, pH 7.4, respectively.

[0225] The protein eluate was subsequently fractionated and further purified by size-exclusion chromatography (SEC). SEC was performed by fast protein liquid chromatography using a Superdex 200 Increase 10 / 300 GL (GE Healthcare, Pittsburgh, PA) in HBS buffer (10 mM HEPES, 150 mM NaCl, pH 7.4). Each protein was injected onto the column in a volume of 475 μl or 500 μl. Absorbance at 280 nm was monitored, and 500 μl fractions of the total eluate were collected. Each collected fraction near the main peak was further analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) to confirm its contents. SDS-PAGE was performed under both non-reducing and reducing conditions using Tris-HCl 4-15% gels (Bio-Rad, Hercules, CA). Samples were prepared in Laemmli sample buffer and heated at 100°C for 5 minutes.

[0226] Protein concentrations were quantified by the direct UV A280 method using a NanoDrop spectrophotometer (Thermo Scientific). The relationship of absorbance to protein concentration is linear based on the Beer-Lambert equation, A = εlc, where A is the absorbance value, ε is the wavelength-dependent extinction coefficient, l is the path length in centimeters, and c is the protein concentration. Experimental extinction coefficients for all produced proteins were estimated by their amino acid sequences.

[0227] The binding kinetics of antibody fragments to various Fzd cysteine-rich domain (CRD) protein targets (Fzd1, Fzd2, Fzd3, Fzd4, Fzd5, Fzd6, Fzd7, Fzd8, Fzd9, and / or Fzd10 CRDs) were determined by biolayer interferometry (BLI) using an Octet Red 96 (PALL ForteBio, Fremont, CA) instrument with a streptavidin (SA) biosensor at 30°C and 1000 rpm. C-terminally biotinylated Fzd CRD recombinant proteins were diluted to 20 nM in running buffer (PBS, 0.05% Tween-20, 0.5% BSA, pH 7.2) and captured on the SA biosensor until the coupling length reached 0.2 nm. After capture of the Fzd CRDs, the captured biotinylated Fzds were The SA biosensor with CRD was immersed in wells containing seven different concentrations of the relevant antibody fragment in running buffer (0, 1.37, 4.12, 12.4, 37, 111.1, 333.3, and 1000 nM) and running buffer alone as a reference channel. K was calculated using a 1:1 binding model by global fitting according to the manufacturer's recommended settings. D It was decided that:

[0228] Table 1A shows the heavy chain CDRs (CDRH1, CDRH2, and CDRH3) and light chain CDRs (CDRL1, CDRL2, and CDRL3) for the indicated antibody clones. The specificity-determining regions (SDRs) shown below were mapped using Distributed Bio's Abgenesis software, which includes the Kabat definition of CDRs (Padlan et al. FASEB J. 9, 133-139 (1995)).

[0229] If a light chain CDR is not shown, this means that the antibody fragment did not contain a light chain (e.g., the antibody was a Fab or VHH or sdAb). Table 1A also shows the initial Fzd receptor to which the antibody fragment was shown to bind. The Fzd receptor to which each clone was initially identified as binding was determined by detecting phage-displayed antibody fragments bound to target antigens immobilized on Nunc Maxisorb microtiter plates (Thermo Fisher Scientific, Waltham, MA) using single-dose or dose-dependent ELISA. Detection of bound phage was determined calorimetrically by the turnover of TMB substrate (Thermo Fisher Scientific, Waltham, MA) at 415 nm with an anti-M13-HRP antibody (GE Healthcare, Pittsburgh, PA). A clone was identified as binding to an Fzd receptor if its OD at 450 nm over background was greater than a threshold level. [Table 1A-1] [Table 1A-2] [Table 1A-3] [Table 1A-4] [Table 1A-5] [Table 1A-6] [Table 1A-7] [Table 1A-8] [Table 1A-9]

Table 1A-10

Table 1A-11

Table 1A-12

Table 1A-13

Table 1A-14

Table 1A-15

Table 1A-16

Table 1A-17

Table 1A-18

Table 1A-19

Table 1A-20

Table 1A-21

Table 1A-22

Table 1A-23

Table 1A-24

Table 1A-25

Table 1A-26

[0230] Table 1B lists the clone IDs and, if present, the sequence identification numbers of the antibody heavy and / or light chain fragments in exemplary clones. In certain embodiments, the Fzd-binding domain is a Fab or is derived from a Fab, such that the heavy chains in Table 1B contain VH and CH1 sequences but no CH2 or CH3 sequences. In certain embodiments, the Fzd-binding domain is a VHH or sdAb® or is derived from a VHH or sdAb, such that Table 1B contains a VHH domain. Table 1B also shows data demonstrating binding to various Fzd receptors in these clones. Kd values were determined by BLI as described above. Blank cells indicate that binding to a specific Fzd receptor has not yet been determined. The designation "nb" indicates no binding. As shown in Table 1B, a subset of antibody fragments showed specificity for a single Fzd or a subfamily of Fzds, as determined by binding affinity in Octet BLI. [Table 1B-1] [Table 1B-2] [Table 1B-3] [Table 1B-4] [Table 1B-5]

[0231] Example 2 Alanine scanning mutagenesis of anti-Fzd antibody fragments One antibody fragment, clone 001S-A04, was selected for alanine scanning mutagenesis of the CDRs, and the Fzd1-binding affinities of various mutants were determined by Octet BLI as described in Example 1. As shown in Table 2, many mutants bound to Fzd1 with affinities similar to the wild-type antibody fragment, demonstrating that Fzd1 antibodies and their antigen-binding fragments can tolerate amino acid modifications within the CDRs. [Table 2-1] [Table 2-2]

[0232] Example 3 Crystal structure of an anti-Fzd antibody fragment bound to the Fzd extracellular domain Fzds are a class of GPCRs in which an extracellular Cys-rich domain (CRD) is connected to its seven-transmembrane helix domain and cytoplasmic tail through a linker region. Fzds have either one or two predicted NxS / T-glycosylation motifs within their extracellular domains. To enable high-resolution structures, the Fzd extracellular domains containing the two glycosylation motifs were truncated before the second predicted NxS / T-glycosylation motif, resulting in a construct designated CRD-Xtal. The sequences of the ten Fzd CRD-Xtals, each containing an eight-histidine motif at the C-terminus, are as follows: hFzd1_Q9UP38_101-230 QYNGERGISVPDHGYCQPISIPLCTDIAYNQTIMPNLLGHTNQEDAGLEVHQFYPLVKVQCSAELKFFLCSMYAPVCTVLEQALPPCRSLCERARQGCEALMNKFGFQWPDTLKCEKFPVHGAGELCVGQGSHHHHHHHH (SEQ ID NO: 1454) hFzd2_Q14332_24-153 QFHGEKGISIPDHGFCQPISIPLCTDIAYNQTIMPNLLGHTNQEDAGLEVHQFYPLVKVQCSPELRFFLCSMYAPVCTVLEQAIPPCRSICERARQGCEALMNKFGFQWPERLRCEHFPRHGAEQICVGQHHHHHHHH (SEQ ID NO: 1455) hFzd3_Q9NPG1_23-148 HSLFSCEPITLRMCQDLPYNTTFMPNLLNHYDQQTAALAMEPFHPMVNLDCSRDFRPFLCALYAPICMEYGRVTLPCRRLCQRAYSECSKLMEMFGVPWPEDMECSRFPDCDEPYPRLVDLNLAGEHHHHHHHH (SEQ ID NO: 1456) hFzd4_ Q9ULV1_38-167 GDEEERRCDPIRISMCQNLGYNVTKMPNLVGHELQTDAELQLTTFTPLIQYGCSSQLQFFLCSVYVPMCTEKINIPIGPCGGMCLSVKRRCEPVLKEFGFAWPESLNCSKFPPQNDHNHMCMEGPGDEEVHHHHHHHH (SEQ ID NO: 1457) hFzd5_ Q13467_27-152 ASKAPVCQEITVPMCRGIGYNLTHMPNQFNHDTQDEAGLEVHQFWPLVEIQCSPDLRFFLCSMYTPICLPDYHKPLPPCRSVCERAKAGCSPLMRQYGFAWPERMSCDRLPVLGRDAEVLCMDYNRHHHHHHHH (SEQ ID NO: 1458) hFzd6_ O60353_18-145 HSLFTCEPITVPRCMKMAYNMTFFPNLMGHYDQSIAAVEMEHFLPLANLECSPNIETFLCKAFVPTCIEQIHVVPPCRKLCEKVYSDCKKLIDTFGIRWPEELECDRLQYCDETVPVTFDPHTEFLGHHHHHHHH (SEQ ID NO: 1459) hFzd7_O75084_36-165 HGEKGISVPDHGFCQPISIPLCTDIAYNQTILPNLLGHTNQEDAGLEVHQFYPLVKVQCSPELRFFLCSMYAPVCTVLDQAIPPCRSLCERARQGCEALMNKFGFQWPERLRCENFPVHGAGEICVGQNTHHHHHHHH (SEQ ID NO: 1460) hFzd8_Q9H461_28-153 ASAKELACQEITVPLCKGIGYNYTYMPNQFNHDTQDEAGLEVHQFWPLVEIQCSPDLKFFLCSMYTPICLEDYKKPLPPCRSVCERAKAGCAPLMRQYGFAWPDRMRCDRLPEQGNPDTLCMDYNRHHHHHHHH (SEQ ID NO: 1461) hFzd9_O00144_23-159 LEIGRFDPERGRGAAPCQAVEIPMCRGIGYNLTRMPNLLGHTSQGEAAAELAEFAPLVQYGCHSHLRFFLCSLYAPMCTDQVSTPIPACRPMCEQARLRCAPIMEQFNFGWPDSLDCARLPTRNDPHALCMEAPENAHHHHHHHH (SEQ ID NO: 1462) hFzd10_Q9ULW2_21-154 SSMDMERPGDGKCQPIEIPMCKDIGYNMTRMPNLMGHENQREAAIQLHEFAPLVEYGCHGHLRFFLCSLYAPMCTEQVSTPIPACRVMCEQARLKCSPIMEQFNFKWPDSLDCRKLPNKNDPNYLCMEAPNNGHHHHHHHH (SEQ ID NO: 1463)

[0233] Example 4 Expression and purification of Fzd CRD_Xtal constructs Lentiviral technology was used to generate FreeStyle™ 293-F cells (Thermofisher) stably expressing all Fzd CRD_Xtal protein constructs. For large-scale expression, a frozen vial of FreeStyle™ 293-F cells expressing Fzd CRD_Xtal was thawed into 20 mL of FreeStyle (Thermofisher) medium in the presence of 10 U of penicillin and 10 μg of streptomycin (Lonza) per mL. On another day, the cells were grown at approximately 3.0 × 10 in the desired volume (typically 6–10 L). 6The cells were expanded until they reached 100 cells / mL. At this stage, cells were grown continuously to high density, and the medium was harvested by centrifugation at approximately 70% viability. Fzd CRD_Xtal protein was purified from the medium by incubation with Ni-NTA resin (1 mL per L of medium; Qiagen) pre-equilibrated in HBS (20 mM HEPES pH 7.4, 150 mM NaCl) and eluted with 500 mM imidazole in HBS. The Ni-NTA eluate was concentrated to 5 mL and further polished on a HiLoad 16 / 600 Superdex 200 pg column (GE Life Sciences) pre-equilibrated with HBS. Fractions around the main peak were further analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) to confirm their contents. SDS-PAGE was performed under both reducing and non-reducing conditions using Tris-HCl 4-15% gels (Bio-Rad, Hercules, CA). Samples were prepared in Laemmli sample buffer and heated at 100°C for 5 min. Fractions containing Fzd CRD_Xtal were concentrated to approximately 2 mg / mL and frozen at -80°C in the presence of 10% glycerol for storage until further use. Protein concentrations were quantified by the direct UV A280 method using a NanoDrop spectrophotometer (Thermo Scientific). The relationship of absorbance to protein concentration is linear based on the Beer-Lambert equation, A = εlc (where A is the absorbance value, ε is the wavelength-dependent extinction coefficient, l is the path length in centimeters, and c is the protein concentration). The extinction coefficients of all produced proteins were estimated by their amino acid sequences.

[0234] Example 5 Expression and purification of Fab binders Plasmids expressing the light and heavy chains of the Fab binder of Fzd CRD_Xtal (containing a hexahistidine at its C-terminus) were co-transfected into Expi293 cells for co-expression at a typical 1000 mL scale, according to the standard protocol from the manufacturer (Thermofisher). After 4 days of continuous cell growth, the medium was collected by centrifugation, bound to complete His resin (2.5 mL per 1 L of medium; Roche) pre-equilibrated in PBS, and eluted under gravity flow with 250 mM imidazole in PBS. The eluate containing the Fab binder was concentrated to approximately 5 mL and further polished on a HiLoad 16 / 600 Superdex 200 pg column (GE Life Sciences) pre-equilibrated with HBS. Fractions around the main peak were further analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) to confirm their contents. SDS-PAGE was performed under both reducing and non-reducing conditions using Tris-HCl 4–15% gels (Bio-Rad, Hercules, CA). Samples were prepared in Laemmli sample buffer and heated at 100°C for 5 min. Fractions containing Fzd CRD_Xtal were concentrated to approximately 3 mg / mL and frozen at -80°C in the presence of 10% glycerol for storage until further use. Protein concentration was quantified by the direct UV A280 method using a NanoDrop spectrophotometer (Thermo Scientific). The relationship of absorbance to protein concentration is linear based on the Beer-Lambert equation, A = εlc (where A is the absorbance value, ε is the wavelength-dependent extinction coefficient, l is the path length in centimeters, and c is the protein concentration). The extinction coefficients of all produced proteins were estimated by their amino acid sequences.

[0235] Example 6 Fzd:Fab complex formation, crystallization, and structure determination Purified Fzd CRD_Xtal and Fab-binding material were mixed in a 1.1:1 molar ratio (a slight excess of the smaller protein) and incubated overnight at 4°C with carboxypeptidases A and B at a 100:1 w / w ratio. Complex formation was confirmed by observing a single major peak on a Superdex S200 Increase (10 / 300GL) column pre-equilibrated in HBS. Fractions containing the complex were further checked by SDS-PAGE and concentrated to a range of 10–55 mg / mL for crystallization screening. Initial crystallization screening was performed using commercially available MCSG1, MCSG2, MCSG3, MCSG4, PACT (Molecular Dimensions), PEG I, and PEG II (Qiagen) screens, either as a grid screen or as a microseed matrix screen [MMS; Microseed matrix screening for optimization in protein Crystallization: what have we learned? D'Arcy, A., Bergfors, T., Cowan-Jacob SW, and Marshd, M. Acta Cryst. F70, 1117-1126 (2014)] was optimized using a Mosquito (TTP LabTech) pipettor equilibrated at 18°C in an EchoTherm incubator (Torrey Pines Scientific). 96-well plate crystal screening experiments were monitored periodically by hand using a Discovery V20 stereomicroscope (Zeiss), and crystals were frozen for data collection by immersion in liquid nitrogen in the presence of various cryoprotectants (typically 15-30% v / v glycerol or ethylene glycol). X-ray diffraction datasets were collected from the Berkeley Center for Structural Biology at the Advanced Light Source. The data were collected at ALS (Berkeley, CA) and processed using the XDS [Kabsch, W. XDS. Acta Cryst. D66, 125-132 (2010)] and xdsme [Legrand, P. XDSME: XDS Made Easier (2017) GitHub repository, https: / / github.com / legrandp / xdsme DOI 10.5281 / zenodo.837885] programs. The structure of the Fzd:Fab complex was constructed using molecular replacement (using the constant and variable domains of related Fabs as templates) in Phaser [Phaser crystallographic software. A.J. McCoy, R.W. Grosse-Kunstleve, P.D.A. Dams, M.D. Winn, L.C. Storoni, and R.J. Read. J Appl Crystallogr 40, 658-674 (2007)], followed by refinement and validation with MolProbity as implemented in Phenix [PHENIX: a comprehensive Python-based system for macromolecular structure solution. P.D.A. Dams, P.V.A. Fonine, G. Buncoczi, V.B.Chen, I.W.Davis, N. Echols, J.J. Head, L.W. Hung, G.J. Kapral, R.W. Grosse-Kunstleve, A.J. McCoy, N.W. Moriarty, R. Oeffner, R.J. Read, D.C. Richardson, J.S. Richardson, T.C. Terwilliger, and P.H. Zwart. Acta Cryst. D66, 213-221 (2010); MolProbity: all-atom structure validation for macromolecular crystallography. VB Chen, WB Arendall, JJ Headd, DAKeedy, RMI Immormino, GJ Kapral, LW Murray, JSR Richardson, and DCRichardson. Acta Cryst. D66, 12-21 (2010)].Crystallographic models were manually inspected and constructed using COOT [Features and development of Coot. P. Emsley, B. Lohkamp, W.G. Scott, and K. Cowtan. Acta Cryst. D66, 486-501 (2010)]. Analysis and imaging of refined crystal structures was performed using MOE (CCG) and PyMol (Schrodinger).

[0236] Example 7 Structure of the Fzd1:1RC07 complex: 1RC07(001S-B03) Fab sequence: 1RC07_L chain SYVLTQPPSVSVSPGQTASITCSGDKVGHKYASWYQQKPGQSPVLVIYEDSQRPSGIPVRFSGSNSGNTATLTISGTQAMDEADYYCQAWDSSTDVVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 1464) 1RC07_H chain QVQLQQWGAGLLKPSETLSLTCAVSGASFSGHYWTWIRQPPGKGLEWIGEIDHTGSTNYEPSLRSRVTISVDTSKNQFSLNLKSVTAADTAVYYCARGGQGGYDWGHYHGLDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCGSGSGHHHHHH (SEQ ID NO: 1465) Diffraction-quality crystals (concentration = 28 mg / mL) of the Fzd1:1RC07 complex were grown under crystallization conditions containing 0.1 M lithium chloride, 0.1 M HEPES:NaOH, pH 7.5, and 25% (w / v) PEG6000. Crystals were cryoprotected using 20% glycerol in the well solution. The Fzd1:1RC07 complex crystallized with one complex molecule per asymmetric unit in the P21212 space group (a = 65.79, b = 192.21, c = 44.79). The structure of the Fzd1:1RC07 complex was determined at 2.10 Å resolution and R 結晶 Factors and R 遊離 The refinement factors were 19.9% and 24.8%, respectively. In the crystal structure, residues 101-114, 179-189, 203-207, and 217-230 of Fzd1 could not be modeled due to irregularities in the electron density map.

[0237] The overall structure of the Fzd1:1RC07 complex is shown in Figure 3.1 (A and B), which reveals that the heavy chain CDR3 of 1RC07 binds more tightly to the lipid-binding site, as observed in the Fzd8:Wnt8a complex [PDB Code: 4F0A; Janda, CY, Waghray, D., Levin, AM, Thomas, C., Garcia, KC (2012) Science 337:59-64]. The crystallographic model supports strong mF o -DF c During the construction of the difference map, >10 electron densities were observed at the interface between the antigen Fzd1 and antibody 1RC07. This strong positive difference density map could be fully explained by modeling zinc ions and was further confirmed by >15 strong anomalous difference maps calculated at 3.5 Å resolution (Figure 3.1(C)). This Zn2+ ion bound His107 (2.04 Å) and His109 (2.01 Å) from the CDR H3 loop of 1RC07, as well as Glu49 (2.03 and 2.76 Å) from the CDR L2 loop, and His151 (2.00 Å) of Fzd1 (conserved within the sequences of Fzd2, Fzd7, Fzd5, Fzd8, and Fzd10).

[0238] From the structure of the complex, the epitope of Fzd1 for 1RC07 can be identified, with the following residues defining the core interaction site on Fzd1 (5 Å cutoff): Pro122, Leu148, His151, Gln152, Tryptophan 154, Pro155, Leu156, Lys158, and Gln160.

[0239] In addition, the following residues on Fzd1 could be identified as direct interaction sites of 1RC07 (interaction distance >5.0 Å and <=8.0 Å): Ser120, Ile121, Leu123, Cys124, Thr125, Asp126, Glu144, Gly147, Glu149, Val150, Phe153, Val157, Val159, Cys161, Cys198, Leu201, and Met202.

[0240] Additionally, from the structure of the Fzd1:1RC07 complex, we can identify residues on 1RC07 that are within 5.0 Å of any atom in Fzd1: 1RC07 heavy chain: Tyr103, Trp105, Gly106, His107, and His109. 1RC07 light chain: Val27, Gly28, His29, Lys30, Tyr31, Ala32, Tyr48, Glu49, Asp50, Ser51, Gln52, and Asn65.

[0241] Furthermore, the Fzd1:1RC07 complex reveals that the following residues on 1RC07 are direct interaction sites for Fzd1 with interaction distances >=5.0 Å and <=8.0 Å: 1RC07 heavy chain: Gln100, Gly101, and Tyr108. 1RC07 light chain: Lsy26, Ser33, Ile47, Arg53, Val59, Ser62, Gly63, Ser64, Asn65, Ser66, Gly67, Thr69, Ala70, Trp90, and Ser92.

[0242] Example 8 Structure of the Fzd1:R2M9 complex: R2M9(003S-E07) Fab sequence: R2M9_L chain DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYRTPFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 1466) R2M9_H chain QVQLVQSGAEVKKPGSSVKVSCKASGYTFTNNFMHWVRQAPGQGLEWMGWINPNSGGTKYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARSVGEVGATMLGIGVWYWFDPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCGSGSGHHHHHH (SEQ ID NO: 1467) Diffraction-quality crystals (concentration = 32 mg / mL) of the Fzd1:R2M9 complex were grown under crystallization conditions containing 0.1 M sodium formate and 11% (w / v) PEG3350. Crystals were cryoprotected using 26% glycerol in the well solution. The Fzd1:R2M9 complex crystallized with two complex molecules per asymmetric unit in the P21 space group (a = 50.57, b = 160.60, c = 88.97 Å, and β = 95.5°). The structure of the Fzd1:R2M9 complex was determined at 2.60 Å resolution, and R 結晶 Factors and R 遊離 The factors were refined to 23.1% and 26.3%, respectively.

[0243] The overall structure of the Fzd1:R2M9 complex is shown in Figure 3.1 (A and B), revealing that R2M9 recognizes Fzd1 from the opposite direction of the lipid-binding site, as observed for the Fzd8:Wnt8a complex [PDB Code: 4F0A; Janda, CY, Waghray, D., Levin, AM, Thomas, C., Garcia, KC (2012) Science 337:59-64]. From the structure of the complex, the epitope of Fzd1 for R2M9 can be identified, with the following residues defining the core interaction site on Fzd1 (5 Å cutoff): Tyr115, Ala128, Tyr129, Phe167, Val176, Thr178, Val179, Leu180, Glu181, Gln182, Leu184, Gly224, Leu226, Cys227, and Val228.

[0244] In addition, the following residues on Fzd1 could be identified as direct interaction sites of R2M9 (interaction distance >=5.0 Å and <=8.0 Å): Cys116, Ile127, Asn130, Gln131, Ser171, Cys177, Ala183, Pro185, Cys187, His221, Ala223, Glu225, Gly229, and Gln230.

[0245] The crystal structure of the Fzd1:R2M9 complex reveals that a glycerol molecule used as a cryoprotectant during sample preparation interacts with Ser95 of the R2M9 CDR H3 loop at a distance less than the 5 Å cutoff from Leu180, Gln182, Leu184, and Leu226 of Fzd1. Another glycerol molecule bound to Val108 of the R2M9 CDR H3 loop was less than 5 Å from Val176, Cys177, Thr178, and Val179 of Fzd1. Such interactions can be exploited for structure-guided engineering to optimize the properties of R2M9.

[0246] Additionally, from the structure of the Fzd1:R2M9 complex, we can identify residues on R2M9 that are within 5.0 Å of any atom in Fzd1: R2M9 heavy chain: Asn31, Phe33, His35, Trp50, Asn52, Lys58, Ser95, Gly97, Glu98, Val99, Leu104, Gly105, Ile106, Val108, and Tyr110. R2M9 light chain: Ser91, Tyr92, Arg93, Thr94, and Phe96.

[0247] Furthermore, the structure of the Fzd1:R2M9 complex reveals that the following residues on R2M9 are direct interaction sites for Fzd1 with interaction distances >5.0 Å and <=8.0 Å: R2M9 heavy chain: Thr30, Asn32, Met34, Trp47, Ile51, Asn53, Ser54, Gly56, Thr57, Tyr59, Val96, Gly100, Ala101, Thr102, Met103, Gly107, Trp109, Trp111, and Phe112. R2M9 light chain: Try32, Gln89, Gln90, and Pro95.

[0248] Example 9 Structure of the Fzd4:003S-D10 complex: 003S-D10 Fab sequence: 003S-D10_L chain DIQMTQSPSSLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIYAASNLLGGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYSTPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 1468) 003S-D10_H chain EVQLVESGGGLVKPGGSLRLSCAASGFNFGIYSMTWVRQAPGKGLEWISYISGDSGYTNYADSVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCARVGPGGWFDPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCGSGSGHHHHHH (SEQ ID NO: 1469) Diffraction-quality crystals of Fzd4:003S-D10 (concentration = 23 mg / mL) were obtained by MMS in an MCSG1 screen under H4 conditions containing 0.1 M sodium citrate:HCl, pH 5.6, 20% (v / v) PEG4000, and 20% (v / v) isopropanol. Crystals were cryoprotected using 20% glycerol in the well solution. Fzd4:3SD10 crystallized with one complex molecule per asymmetric unit in the P3121 space group (a = b = 149.4 Å and c = 91.9 Å). The structure of the Fzd4:003S-D10 complex was determined at 2.10 Å resolution and analyzed by R. 結晶 Factors and R 遊離 The factors were refined to 18.5% and 21.6%, respectively.

[0249] The overall structure of the Fzd4:003S-D10 complex is shown in Figure 4.1(A), revealing that the heavy chain CDR3 of 003S-D10 binds at the lipid-binding site (Figure 4.1(B)), as observed for the Fzd8:Wnt8a complex [PDB Code: 4F0A; Janda, CY, Waghray, D., Levin, AM, Thomas, C., Garcia, KC (2012) Science 337:59-64]. From the structure of the complex, the epitope of Fzd4 for 003S-D10 can be identified, with the following residues defining the core interaction site on Fzd4 (5 Å cutoff), shown in dark color in Figure 4.1(C): Val67, Gly68, His69, Thr73, Asp74, Glu76, Leu77, Gln78, Thr80, Thr81, Phe82, Thr83, Pro84, Leu85, Gln87, Tyr88, Tyr102, Leu132, Phe135, Gly136, Phe137, Ala138, and Ser142.

[0250] In addition, the following residues on Fzd4 were identified as direct interaction sites (interaction distances >=5.0 Å and <=8.0 Å; light green surface in Figure 4.1(C)): Ile50, Ser51, Met52, Pro64, Asn65, Leu66, Glu70, Leu71, Gln72, Ala75, Leu79, Ile86, Gly89, Leu94, Gln95, Leu98, Val101, Tyr102, Val131, Lys133, Glu134, Trp139, Pro140, Glu141, Leu143, and Lys147.

[0251] Additionally, from the structure of Fzd4:003S-D10, we can identify residues on 003S-D10 that are within 5.0 Å of any atom in Fzd4: 003S-D10 heavy chain: Gly30, Ile31, Tyr32, Ser33, Tyr50, Ser52, Gly53, Asp54, Tyr57, Asn59, Arg98, Val99, Gly100, Pro101, Gly102, Gly103, Trp104, and Asp106. 003S-D10 light chain: Ser30, Tyr32, Leu46, Try49, Asn53, Leu55, Gly56, Thr91, Tyr92, Ser93, Thr94, and Trp96.

[0252] Furthermore, the structure of Fzd4:003S-D10 reveals that the following residues on 003S-D10 are direct interaction sites for Fzd4 with interaction distances >=5.0 Å and <=8.0 Å: 003S-D10 heavy chain: Val2, Phe27, Asn28, Phe29, Met34, Trp47, Ile51, Ser55, Gly56, Thr58, Tyr60, Arg72, Asp74, Phe105, and Pro107. 003S-D10 light chain: Ile2, Gln27, Gly28, Ile29, Ser31, Tyr36, Ile48, Ala50, Leu54, Ser67, Gln90, Pro95, and Thr97.

[0253] Example 10 Structure of the Fzd5:R2M3 complex R2M3(001S-A04) Fab sequence: >R2M3_L chain QAVVLQEPSLSVSPGGTVTLTCGLSSGSVSTNYYPSWYQQTPGQAPRTLIYYTNTRSSDVPERFSGSIVGNKAALTITGAQPDDESVYFCLLYLGRGIWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 1470) >R2M3_H chain EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCASSKEKATYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCGSGSGHHHHHH (SEQ ID NO: 1471)

[0254] Diffraction-quality crystals (concentration = 28 mg / mL) of the Fzd5:R2M3 complex were grown under crystallization conditions containing 0.1 M lithium chloride, 0.1 M HEPES:NaOH, pH 7.5, and 25% (w / v) PEG6000. Crystals were cryoprotected using 20% glycerol in the well solution. The Fzd5:R2M3 complex crystallized with two complex molecules per asymmetric unit in the P212121 space group (a = 59.61 Å, b = 69.29 Å, c = 284.54 Å). The structure of the Fzd5:R2M3 complex was determined at 2.0 Å resolution, and R 結晶 Factors and R 遊離 The factors were refined to 21.9% and 23.8%, respectively.

[0255] The overall structure of the Fzd5:R2M3 complex is shown in Figure 3.4 (A and B), which reveals that R2M3 binds more tightly to the lipid-binding site, as observed for the Fzd8:Wnt8a complex [PDB Code: 4F0A; Janda, CY, Waghray, D., Levin, AM, Thomas, C., Garcia, KC (2012) Science 337:59-64]. R2M3 binds with broader specificity to both the Fzd1, 2, 7 and Fzd5, 8 subfamilies of Fzds.

[0256] From the structure of the complex, the epitope of Fzd5 for R2M3 can be identified, with the following residues defining the core interaction site on Fzd5 (5 Å cutoff): Thr37, Val38, Pro39, Arg42, Asn56, His57, Asp58, Gln60, Asp61, Glu62, Gly64, Leu65, Glu66, His68, Gln69, Trp71, Pro72, Try123, and Gly124.

[0257] In addition, the following residues on Fzd5 could be identified as direct interaction sites of R2M3 (interaction distance >5.0 Å and <=8.0 Å): Gln34, Glu35, Ile36, Met40, Cys41, Pro52, Asn53, Phe55, Thr59, Ala63, Val67, Phe70, Glu75, Tyr90, Met120, Arg121, Gln122, Phe125, Ala126, Pro128, and Glu129.

[0258] Additionally, from the structure of the Fzd5:R2M3 complex, we can identify residues on R2M3 that are within 5.0 Å of any atom in Fzd5: R2M3 heavy chain: Ser31, Trp50, Tyr54, Asn55, Asn57, Lys102, Ile103, Thr104, Tyr105, and Tyr106. R2M3 light chain: Thr31, Asn32, Tyr34, Tyr52, Asn54, Thr55, Tyr93, Gly95, Arg96, Gly97, and Trp99.

[0259] Furthermore, the structure of the Fzd5:R2M3 complex reveals that the following residues on R2M3 are direct interaction sites for Fzd5 with interaction distances >5.0 Å and <=8.0 Å: R2M3 heavy chain: Phe29, Thr30, Tyr32, Gly33, Ile51, Ser52, Gly56, Thr58, Asn59, Glu101, Tyr107, and Gly108. R2M3 light chain: Ser30, Tyr33, Pro35, Tyr51, Thr53, Gly66, Ser67, Ile68, Leu94, and Ile98.

[0260] Example 11 Structure of the Fzd8:005S-H05 complex 005S-H05 Fab sequence: >005S-H05_L chain DIQMTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYSMPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 1472) >005S-H05_H chain QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGRINPNSGGTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARVPDFWSGYLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCGSGSGHHHHHH (SEQ ID NO: 1473) Diffraction-quality crystals (concentration = 38 mg / mL) of the Fzd8:005S-H05 complex were grown under crystallization conditions containing 0.2 M ammonium sulfate, 0.1 M HEPES:NaOH, pH 7.5, and 25% (w / v) PEG3350. Crystals were cryoprotected using 26% glycerol in the well solution. The Fzd8:005S-H05 complex crystallized with two complex molecules per asymmetric unit in the C2 space group (a = 92.63 Å, b = 60.38 Å, c = 110.35 Å, β = 97.5°). The structure of the Fzd8:005S-H05 complex was determined at 1.65 Å resolution and analyzed by R. 結晶 Factors and R 遊離 The factors were refined to 16.8% and 18.6%, respectively.

[0261] The overall structure of the Fzd8:005S-H05 complex is shown in Figure 3.5 (A and B), which reveals that the heavy chain CDR3 of 005S-H05 is inserted into the lipid-binding site, as observed in the Fzd8:Wnt8a complex [PDB Code: 4F0A; Janda, CY, Waghray, D., Levin, AM, Thomas, C., Garcia, KC (2012) Science 337:59-64]. 005S-H05 binds to both Fzd5 and Fzd8 with subfamily specificity.

[0262] From the structure of the complex, the epitope of Fzd8 for 005S-H05 can be identified, with the following residues defining the core interaction site on Fzd8 (5 Å cutoff): Phe57, Asn58, Glu64, Leu67, Glu68, His70, Gln71, Phe72, Trp73, Pro74, Glu77, Try92, Arg123, Gln124, Try125, Gly126, Phe127, Ala128, Trp129, Pro130, Arg132, and Met133.

[0263] In addition, the following residues on Fzd8 could be identified as direct interaction sites of 005S-H05 (interaction distance >5.0 Å and <=8.0 Å): Asn55, Gln56, His59, Asp60, Gly66, Val69, Leu75, Ile78, Leu88, Leu121, Met122, Asp131, and Arg137.

[0264] Additionally, from the structure of the Fzd8:005S-H05 complex, we can identify residues on 005S-H05 that are within 5.0 Å of any atom in Fzd8: 005S-H05 heavy chain: Gly26, Try27, Thr28, Ser31, Tyr32, Pro100, Asp101, Phe102, Trp103, Ser104, Gly105, Tyr106, and Asp108. 005S-H05 light chain: Ile29, Ser30, Ser31, Ala32, Tyr49, Ala50, Ser52, Ser53, Leu54, Gln55, Ser56, Thr91, Tyr92, and Ser93.

[0265] Furthermore, the structure of the Fzd8:005S-H05 complex reveals that the following residues on R2M3 are direct interaction sites for Fzd5 with interaction distances >5.0 Å and <=8.0 Å: 005S-H05 heavy chain: Gln1, Val2, Phe29, Thr30, Tyr33, Arg50, Asn52, Asn54, Arg98, Val99, Leu107, and Tyr109. 005S-H05 light chain: Ile2, Gly28, Leu33, Ala34, Leu46, Ala51, Gly57, Gly66, Ser67, Gly68, Phe71, Gln90, and Met94.

[0266] Example 12 Structure of the Fzd5:004S-E05 complex 004S-E05 Fab sequence: >004S-E05_L chain DIQMTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGKAPKLLIYAASALQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYSTPRTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 1474) >004S-E05_H chain EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYEMNWVRQAPGKGLEWVSGVSWNGSRTHYVDSVKGRFTISRDNSKNTLYLQLNSLRAEDTAVYYCARGQSEKWWSGLYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCGSGSGHHHHHH (SEQ ID NO: 1475) Diffraction-quality crystals (concentration = 30 mg / mL) of the Fzd5:004S-E05 complex were grown under crystallization conditions containing 0.1 M Bis-Tris pH 5.7 and 24% (w / v) PEG3350. Crystals were cryoprotected using 33% ethylene glycol in the well solution. The Fzd5:004S-E05 complex crystallized with two complex molecules per asymmetric unit in the P21212 space group (a = 72.87 Å, b = 192.35 Å, c = 90.25 Å). The structure of the Fzd5:004S-E05 complex was determined at 1.70 Å resolution and analyzed by R. 結晶 Factors and R 遊離 The factors were refined to 18.5% and 20.7%, respectively.

[0267] The overall structure of the Fzd5:004S-E05 complex is shown in Figure 3.4 (A and B), revealing that the CDR-H3 of 004S-E05 binds at the lipid-binding site, as observed for the Fzd8:Wnt8a complex [PDB Code: 4F0A; Janda, CY, Waghray, D., Levin, AM, Thomas, C., Garcia, KC (2012) Science 337:59-64]. 004S-E050 binds both Fzd5 and Fzd8, with a bias toward the former. Ethylene glycol, used as a cryoprotectant and bound to Glu75 of Fzd5, interacts with Ala50 and Ala53 of the light chain of 004S-E05.

[0268] From the structure of the complex, the epitope of Fzd5 for 004S-E05 can be identified, with the following residues defining the core interaction site on Fzd5 (5 Å cutoff): Gln69, Phe70, Trp71, Pro72, Leu73, Glu75, Ile76, Gln77, Cys78, Gly115, Pro118, Leu119, Met120, Arg121, Gln122, Try123, Gly124, and Phe125.

[0269] In addition, the following residues on Fzd5 could be identified as direct interaction sites of 004S-E05 (interaction distance >5.0 Å and <=8.0 Å): Leu65, Glu66, Val67, His68, Val74, Ser79, Leu82, Cys116, Ser117, Ala126, and Pro128.

[0270] Additionally, from the structure of the Fzd5:004S-E05 complex, we can identify residues on 004S-E05 that are within 5.0 Å of any atom in Fzd5: 004S-E05 heavy chain: Arg57, His59, Ser101, Trp104, Tryp105, Ser106, Gly107, Leu108, and Tyr109. 004S-E05 light chain: Ser30, Ser31, Ala32, Tyr49, Ala50, Ala53, Ser67, Thr91, Tyr92, Ser93, Thr94, and Arg96.

[0271] Furthermore, the structure of the Fzd5:004S-E05 complex reveals that the following residues on R2M3 are direct interaction sites for Fzd5 with interaction distances >5.0 Å and <=8.0 Å: 004S-E05 heavy chain: Glu33, Ser52, Trp53, Ser56, His59, Gln100, Glu102, Lys103, and Gly110. 004S-E05 light chain: Ile2, Gln27, Gly28, Ile29, Leu33, Tyr49, Ala51, Ser52, Leu54, Gly66, Gly68, Phe71, Gln89, Gln90, Pro95, and Arg96.

[0272] Example 13 Structure of the Fzd5:4A12 complex 4A12 Fab sequence: >4A12_L chain DIVMTQSHKFMSTSVGDRVSITCKASQDVGTAVAWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTINNVQSEDLADYFCQQYSTYPLTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 1476) >4A12_H chain QVQLQQSGPELVKPGASVKLSCKASGYTFTNYDINWVKQRPGQGLEWIGWIYPRDGSTKYNEKFKGKATLTVDTSSSTAYMELHSLTSEDSAVYFCVRSAWGFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCGSGSGHHHHHH (SEQ ID NO: 1477) Diffraction-quality crystals (concentration = 10 mg / mL) of the Fzd5:4A12 complex obtained by MMS were grown under crystallization conditions containing 0.2 M sodium chloride, 0.1 M NaHPO:citric acid, pH 4.2, and 20% (w / v) PEG8000. Crystals were cryoprotected using 25% ethylene glycol in the well solution. The Fzd5:4A12 complex crystallized with two complex molecules per asymmetric unit in the C2 space group (a = 93.84 Å, b = 60.07 Å, c = 110.80 Å, β = 104.7°). The structure of the Fzd5:4A12 complex was determined at 1.75 Å resolution and analyzed by R. 結晶 Factors and R 遊離 The factors were refined to 17.7% and 21.6%, respectively.

[0273] The overall structure of the Fzd5:4A12 complex is shown in Figure 3.4 (A and B), revealing that 4A12 binds opposite the lipid-binding site and recognizes the C-terminal region of Fzd5, as observed for the Fzd8:Wnt8a complex [PDB Code: 4F0A; Janda, CY, Waghray, D., Levin, AM, Thomas, C., Garcia, KC (2012) Science 337:59-64]. Electron density maps revealed that a chloride ion binds at the interface of the Fzd5:4A12 complex, interacting with Arg111 of Fzd5 and the main-chain amide nitrogen of Thr28 and the side-chain amide group of Asn31 of the heavy chain of 4A12.

[0274] From the structure of the complex, the epitope of Fzd5 for 4A12 can be identified, with the following residues defining the core interaction site on Fzd5 (5 Å cutoff): Cys105, Arg106, Ser107, Glu110, Arg111, Cys133, Asp134, Val138, Leu139, Gly140, Arg141, Asp142, Ala143, Val145, Leu146, Cys147, and Asp149.

[0275] In addition, the following residues on Fzd5 could be identified as direct interaction sites of 4A12 (interaction distance >5.0 Å and <=8.0 Å): Asp81, Tyr98, Leu102, Pro103, Val108, Cys109, Ala112, Ala114, Ser132, Arg135, Leu136, Pro137, Glu144, and Met148.

[0276] Additionally, from the structure of the Fzd5:4A12 complex, we can identify residues on 4A12 that are within 5.0 Å of any atom in Fzd5: 4A12 heavy chain: Asn31, Tyr32, Asp33, Trp50, Tyr52, Arg54, Ser99, Ala100, and Trp101. 4A12 light chain: Ala32, Trp50, Tyr91, Ser92, and Tyr94.

[0277] Furthermore, the structure of the Fzd5:4A12 complex reveals that the following residues on 4A12 are direct interaction sites for Fzd5 with interaction distances >5.0 Å and <=8.0 Å: 4A12 heavy chain: Gly26, Tyr27, Thr28, Thr30, Ile34, Asp35, Trp47, Ile51, Pro53, Asp55, Ser57, Thr58, Lys59, Arg98, Gly102, Phe103, Ala104, and Tyr105. 4A12 light chain: Val29, Thr31, Tyr49, Gln89, Gln90, Thr93, and Leu96.

[0278] Example 14 Structure of the Fzd9:014S-B06 complex: 014S-B06 Fab sequence: 014S-B06_L chain DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 1478) 014S-B06_H chain EVQLVQSGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSYIENDGSITTYADSVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCARAPYYYGSGSLFRLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCGSGSGHHHHHH (SEQ ID NO: 1479) Diffraction-quality crystals of the Fzd9:014S-B06 complex (concentration = 31 mg / mL) were obtained at 4% Crystallization conditions included PEG3350, 0.1 M HEPES pH 7.5, and 0.2 M lithium chloride. Crystals were cryoprotected using 27% glycerol in the well solution. Fzd9:014S-B06 crystallized with one complex molecule per asymmetric unit in the P212121 space group (a = 63.8 Å, b = 81.4 Å, and c = 160.5 Å). The structure of the Fzd9:014S-B06 complex was determined at 1.95 Å resolution and analyzed by R. 結晶 Factors and R 遊離 The factors were refined to 19.8% and 22.6%, respectively.

[0279] The overall structure of the Fzd9:014S-B06 complex is shown in Figure 3.7 (A and B), which reveals that the heavy chain CDR3 of 014S-B06 binds away from the lipid-binding site and recognizes a region closer to the C-terminus of Fzd9, as observed for the Fzd8:Wnt8a complex [PDB Code: 4F0A; Janda, CY, Waghray, D., Levin, AM, Thomas, C., Garcia, KC (2012) Science 337:59-64].

[0280] From the structure of the complex, the epitope of Fzd9 for O14S-B06 can be identified, with the following residues defining the core interaction site on Fzd9 (5 Å cutoff): Leu60, Leu61, Leu95, Thr106, Pro107, Pro109, Arg112, Arg119, Asp135, Ser136, Leu137, Asp138, Ala140, Arg141, Leu142, Pro143, Thr144, Asp147, Pro148, His149, and Ala150.

[0281] In addition, the following residues on Fzd9 could be identified as direct interaction sites of 014S-B06 (interaction distance >=5.0 Å and <=8.0 Å): Asn59, Gly62, Phe91, Ser94, Pro98, Ser105, Ile108, Ala110, Trp133, Pro134, Asp138, Cys139, Arg145, Asn146, Leu151, and Cys152.

[0282] Additionally, from the structure of the Fzd9:014S-B06 complex, we can identify residues on 3SD10 that are within 5.0 Å of any atom in Fzd4: 014S-B06 heavy chain: Thr28, Ser30, Ser31, Tyr32, Asn53, Tyr101, Tyr102, Try103, Gly104, Ser105, Leu108, and Arg110. 014S-B06 light chain: Ser31, Try32, Try49, Ala50, Ser53, and Ser91.

[0283] Furthermore, the structure of the Fzd9:014S-B06 complex reveals that the following residues on 014S-B06 are direct interaction sites for Fzd9 with interaction distances >=5.0 Å and <=8.0 Å: 014S-B06 heavy chain: Phe27, Phe29, Asp54, Arg72, Asp74, Asn77, Arg98, Ala99, Pro100, Gly106, Ser107, Phe109, and Asp112. 014S-B06 light chain: Ile29, Ser30, Leu33, Asn34, Ala51, Ser52, and Tyr92.

[0284] Example 15 Structure of the Fzd10:005S-A07 complex 005S-A07 Fab sequence: 005S-A07_L chain EIVLTQSPATLSVSPGERATLSCRASQSVSRNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQRSNWPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 1480) 005S-A07_H chain QVQLVQSGAEVKKPGSSVKVSCKASGFTFTGSAVQWVRQAPGQGLEWVGGILPIYGTTKYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGARLYGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCGSGSGHHHHHH (SEQ ID NO: 1481) Diffraction-quality crystals of the Fzd10:005S-A07 complex (concentration = 37 mg / mL) were grown under crystallization conditions containing 0.2 M ammonium sulfate, 0.1 M sodium citrate:HCl, pH 5.6, and 25% (w / v) PEG4000. Crystals were cryoprotected using 20% glycerol in the well solution. The Fzd10:005S-A07 complex crystallized with one complex molecule per asymmetric unit in the H32 space group (a = b = 138.2 Å and c = 190.6 Å). The structure of the Fzd10:005S-A07 complex was determined at 2.40 Å resolution and analyzed by R. 結晶 Factors and R 遊離 The factors were refined to 20.6% and 25.0%, respectively.

[0285] The overall structure of the Fzd10:005S-A07 complex is shown in Figure 3.8 (A and B), revealing that 005S-A07 binds to Fzd10 at the lipid-binding site, as observed for the Fzd8:Wnt8a complex [PDB Code: 4F0A; Janda, CY, Waghray, D., Levin, AM, Thomas, C., Garcia, KC (2012) Science 337:59-64]. Interestingly, the crystal structure of Fzd10:005S-A07 reveals potential binding sites for two sulfate ions (SO4 2-) at the antigen-Fab interface, which may be useful for further structure-guided engineering to optimize the properties of 005S-A07. From the complex structure, the epitope of Fzd10 for 005S-A07 could be identified, with the following residues defining the core interaction site on Fzd10 (5 Å cutoff): Pro40, Met41, Ile66, Gln67, His69, Glu70, Phe71, Ala72, Pro73, Val75, Glu76, Tyr77, Arg84, Met121, Glu122, Gln123, Phe124, Asn125, Phe126, Lys127, Pro129, and Asp130.

[0286] In addition, the following residues on Fzd10 could be identified as direct interaction sites of 005S-A07 (interaction distance >=5.0 Å and <=8.0 Å): Ile39, Cys42, Lys43, Arg62, Glu63, Ala65, Ile66, Leu68, Leu74, Gly78, Cys88, Tyr91, Ser118, Pro119, Ile120, Trp128, Ser131, and Leu132.

[0287] Additionally, from the structure of the Fzd10:005S-A07 complex, we can identify residues on 005S-A07 that are within 5.0 Å of any atom in Fzd4: 005S-A07 heavy chain: Thr28, Thr30, Gly31, Ser32, Leu52, Ile54, Tyr55, Thr57, Lys59, Arg98, Ala100, Arg101, Leu102, Tyr103, Gly104, and Asp106. 005S-A07 light chain: Arg33, Asn34, Leu48, Tyr51, Gly52, Ala57, Thr58, and Trp96.

[0288] Furthermore, the structure of the Fzd10:005S-A07 complex reveals that the following residues on 005S-A07 are direct interaction sites for Fzd9 with interaction distances >=5.0 Å and <=8.0 Å: 005S-A07 heavy chain: Phe27, Phe29, Ala33, Gln35, Pro53, Gly56, Thr58, Gly99, Phe105, and Tyr107. 005S-A07 light chain: Val31, Ser32, Leu35, Leu49, Ile50, Ala53, Thr55, Arg56, Gly59, Ile60, Arg93, Ser94, Asn95, and Ile98.

[0289] Example 16 Structure of the Fzd10:005S-E12 complex: 005S-E12 Fab sequence: 005S-E12_L chain DIQMTQSPSSLSASVGDRVTITCRASQSVGRWMAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANTFPFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 1482) 005S-E12_H chain QVQLVQSGAEVKKPGASVKVSCKASGYIFTDYYMHWVRQAPGQGLEWMGVIFPVYPTPDYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGSTGYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCGSGSGHHHHHH (SEQ ID NO: 1483) Diffraction-quality crystals (concentration = 34 mg / mL) of the Fzd10:005S-E12 complex were obtained by MSS under crystallization conditions containing 0.1 M bis-trispropane:HCl, pH 7, and 2.5 M ammonium sulfate. Crystals were cryoprotected using 1.7 M sodium malonate, pH 7.0, in the well solution. The Fzd10:005S-E12 complex crystallized with one complex molecule per asymmetric unit in the P312 space group (a = b = 90.4 Å and c = 185.1 Å). The structure of the Fzd10:005S-E12 complex was determined at 2.50 Å resolution and analyzed by R. 結晶 Factors and R 遊離 The factors were refined to 20.1% and 24.4%, respectively.

[0290] The overall structure of the Fzd10:005S-E12 complex (Figure 3.9(A and B)) reveals that 005S-E12 binds to Fzd10 adjacent to the lipid-binding site and recognizes the tail and helix of the N-terminal region of Fzd10, as observed for the Fzd8:Wnt8a complex [PDB Code: 4F0A; Janda, CY, Waghray, D., Levin, AM, Thomas, C., Garcia, KC (2012) Science 337:59-64]. From the complex structure, the epitope of Fzd10 for 005S-E12 can be identified, with the following residues defining the core interaction site on Fzd10 (5 Å cutoff): Ile37, Glu38, Ile39, Pro40, Met41, Cys42, Lys43, Asp44, Ile45, Gly46, Asn48, Gln61, Arg62, Glu63, Ala65, Ile66, Leu68, His69, Ala72, Pro73, Val75, Glu76, and Arg84.

[0291] In addition, the following residues on Fzd10 could be identified as direct interaction sites of 005S-E12 (interaction distance >=5.0 Å and <=8.0 Å): Glu35, Pro36, Tyr47, Asn60, Gln61, Ala64, Gln67, Glu70, Phe71, Leu74, Tyr77, and Phe124.

[0292] Additionally, from the structure of the Fzd10:005S-E12 complex, we can identify residues on 005S-E12 that are within 5.0 Å of any atom in Fzd4: 005S-E12 heavy chain: Tyr33, Phe52, Pro53, Val54, Tyr55, Thr57, Asp59, Gly100, Ser101, Thr102, Gly103, Tyr104, and Tyr105. 005S-E12 light chain: Ile2, Gln27, Ser28, Val29, Gly30, Arg31, Trp32, Ala50, Ala91, Asn92, Thr93, Phe94, and Phe96.

[0293] Furthermore, the structure of the Fzd10:005S-E12 complex reveals that the following residues on 005S-E12 are direct interaction sites for Fzd9 with interaction distances >=5.0 Å and <=8.0 Å: 005S-E12 heavy chain: Thr30, Asp31, Tyr32, His35, Trp47, Val50, Ile51, Pro56, Pro58, Tyr60, Gln62, Arg72, Gly99, and Gly106. 005S-E12 light chain: Asp1, Ala25, Ser26, Met33, Tyr49, Ala51, Ser52, Ser53, Ser67, Gly68, Thr69, Gln90, and Pro95.

[0294] Example 17 Structure of the Fzd3:029S-E03 complex 029S-E03 Fab sequence: >029S-E03_L chain DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSFRLPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 1484) >029S-E03_H chain QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGNTGYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSYYGVIDAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCGSGSGHHHHHH (SEQ ID NO: 1485) Diffraction-quality crystals of the Fzd3:029S-E03 complex (concentration = 25 mg / mL) were grown under crystallization conditions containing 0.2 M trimethylamine N-oxide, 0.1 M Tris:HCl, pH 8.5, and 20% (w / v) PEG2000 MME. Crystals were cryoprotected using 20% glycerol in the well solution. Fzd3:029S-E03 crystallized with one complex molecule per asymmetric unit in the P21 space group (a = 69.60 Å, b = 145.50 Å, and c = 104.31 Å, β = 101.8°). The structure of the Fzd3:029S-E03 complex was determined at 2.40 Å resolution. Refinement was performed using R 結晶 Factors and R 遊離 The factors were ongoing in 24.4% and 31.8%, respectively.

[0295] 029S-E03 is a monospecific binder of Fzd3 and shows no detectable binding to other Fzds. The overall structure of the Fzd3:029S-E03 complex is shown in Figure 11 (A and B), which reveals that the heavy chain CDR3 of 029S-E03 binds adjacent to the lipid-binding site and recognizes a region closer to the N-terminus of Fzd3, as observed for the Fzd8:Wnt8a complex [PDB Code: 4F0A; Janda, CY, Waghray, D., Levin, AM, Thomas, C., Garcia, KC (2012) Science 337:59-64].

[0296] From the structure of the complex, the epitope of Fzd3 for 029S-E03 can be identified, with the following residues defining the core interaction site on Fzd3 (5 Å cutoff): Pro30, Ile31, Thr32, Leu33, Arg34, Gln37, Asp38, Leu39, Gln55, Gln66, Ala59, Leu60, Glu63, His66, and Asn70.

[0297] In addition, the following residues on Fzd3 could be identified as direct interaction sites of 029S-E03 (interaction distance >=5.0 Å and <=8.0 Å): Gln29, Met35, Cys36, Pro40, Tyr41, Asn42, Thr43, Asp54, Thr57, Ala58, Ala61, Met62, Pro67, Val69, Leu71, Asp72, and Arg78.

[0298] Additionally, from the structure of the Fzd3:029S-E03 complex, we can identify residues on 3SD10 that are within 5.0 Å of any atom in Fzd3: 029S-E03 heavy chain: Tyr33, Trp50, Asn52, Ser55, Asn57, Gln62, Tyr101, Val103, Ile104, and Asp105. 029S-E03 light chain: Ile2, Gln27, Ser28, Ser30, Tyr32, Ser91, Phe92, Arg93, and Leu94.

[0299] Furthermore, the structure of the Fzd3:029S-E03 complex reveals that the following residues on 029S-E03 are direct interaction sites for Fzd3 with interaction distances >=5.0 Å and <=8.0 Å: 029S-E03 heavy chain: Trp47, Ile51, Pro53, Asn54, Gly56, Thr58, Gly59, Tyr60, Gln65, Ser99, Tyr100, Gly102, and Ala06. 029S-E03 light chain: Asp1, Ser26, Ile29, Ser31, Leu33, Asn34, Gly68, Gln90, Pro95, and Leu96. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4]

[0300] The various embodiments described above can be combined to provide further embodiments. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications referenced herein and / or listed in the Application Data Sheets are incorporated herein by reference in their entirety. Aspects of the embodiments can be modified as necessary to employ concepts from the various patents, applications, and publications to provide still further embodiments.

[0301] These and other changes can be made to the embodiments in light of the above detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but rather such terms should be construed to include all possible embodiments, along with the full range of equivalents to which the claims are entitled. Accordingly, the claims are not limited by this disclosure.

Claims

[Claim 1] The invention described in the specification.

Images