Anti-LRP5 / 6 antibodies and methods of use

Abstract

To provide anti-LRP5 / 6 antibodies and methods of use.SOLUTION: The present invention provides anti-LRP5 / 6 monoclonal antibodies and related compositions, which may be used in any of a variety of therapeutic methods for treating diseases and disorders associated with Wnt pathway signaling. The present invention provides an isolated antibody, or an antigen-binding fragment thereof, that binds to one or more LRP5 or LRP6 receptor, comprising a sequence comprising: CDRH1, CDRH2 and CDRH3 sequences set forth for any of the antibodies of Table 1A; and / or CDRL1, CDRL2 and CDRL3 sequences set forth for any of the antibodies of Table 1A, or a variant of the antibody, or antigen-binding fragment thereof, comprising one or more amino acid modifications. 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 / 607,879, filed December 19, 2017, and U.S. Provisional Application No. 62 / 680,515, 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_005_02WO_ST25.txt. This text file is 181 KB, was created on December 19, 2018, and has been submitted electronically via EFS-Web.

[0003] The present invention relates generally to anti-LRP5 / 6 antibodies and compositions and methods of use employing anti-LRP5 / 6 antibodies. Such antibodies are useful, for example, in methods of 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;346:1table 1B8012). Modulation of the Wnt signaling pathway has potential for the treatment of degenerative diseases and tissue injury. One of the challenges of therapeutics modulating Wnt signaling is the existence of multiple Wnt ligands and Wnt receptors, Frizzled 1-10 (Fzd1-10), and many tissues express multiple overlapping Fzds. In addition to Fzds, canonical Wnt signaling also involves the co-receptors 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;346:1table 1B8012 Summary of the Invention [Means for solving the problem]

[0006] An isolated antibody or antigen-binding fragment thereof that binds to one or more LRP5 or LRP6 receptors, comprising the CDRH1, CDRH2, and CDRH3 sequences set forth for any antibody in Table 1A, and / or the CDRL1, CDRL2, and CDRL3 sequences set forth for any antibody in Table 1A, or a variant of such an antibody or antigen-binding fragment thereof comprising one or more amino acid modifications, wherein the variant comprises fewer than eight amino acid substitutions in the CDR sequences. In certain embodiments, the isolated 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: 1-24, or a heavy chain variable region comprising the amino acid sequence set forth in any of SEQ ID NOs: 1-24.

[0007] 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 VHH or sdAb. In certain embodiments, any antibody or antigen-binding fragment thereof is a Fab or Fab' fragment.

[0008] 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 one or more Fzd receptors. In certain embodiments, the polypeptide sequence that binds to one or more Fzd receptors is an antibody or antigen-binding fragment thereof that binds to one or more Fzd receptors.

[0009] In certain embodiments, any of the isolated antibodies or antigen-binding fragments thereof disclosed herein binds to LRP5, LRP6, or both LRP5 and LRP6.

[0010] 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 LRP5 or LRP6.

[0011] In certain embodiments, any antibody or antigen-binding fragment thereof binds to LRP5 or LRP6 with a KD of 50 μM or less.

[0012] 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.

[0013] 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.

[0014] 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.

[0015] 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 one or more Frizzled (Fzd) receptors.

[0016] 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.

[0017] 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, osteoarthritis, 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 function. Decline in vision, 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, muscular dystrophy, muscle atrophy in sarcopenia and cachexia, diseases affecting the blood-brain barrier (BBB), spinal cord injury, spinal cord diseases, oral mucositis, short bowel syndrome, inflammatory bowel disease (IBD), metabolic syndrome, diabetes, dyslipidemia, pancreatitis, extrapancreatic cancer Secretory 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 any other liver disorder or detection due to: 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 disorder or detection due to injury.

[0018] 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. 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 LRP5 or LRP6 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 the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 sequences set forth for any antibody 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 an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 24. (Item 3) 3. 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 24. (Item 4) 4. The isolated antibody or antigen-binding fragment thereof according to any of items 1 to 3, wherein the antibody or antigen-binding fragment thereof is humanized. (Item 5) 6. The isolated antibody or antigen-binding fragment thereof according to any one of items 1 to 5, 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 6) 6. The isolated antibody or antigen-binding fragment thereof of item 5, wherein the antibody or antigen-binding fragment thereof is a VHH VHH or an sdAb. (Item 7) 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 8) 8. The isolated antibody or antigen-binding fragment thereof according to any one of items 1 to 7, wherein the antibody or antigen-binding fragment thereof is a fusion protein. (Item 9) 9. The isolated antibody or antigen-binding fragment thereof of item 8, wherein the antibody or antigen-binding fragment thereof is fused to a polypeptide sequence that binds to one or more Frizzled (Fzd) receptors. (Item 10) 10. The isolated antibody or antigen-binding fragment thereof of item 9, wherein the polypeptide sequence that binds to one or more Fzd receptors is an antibody or antigen-binding fragment thereof that binds to one or more Fzd receptors. (Item 11) 11. The isolated antibody or antigen-binding fragment thereof according to any of items 1 to 10, wherein the antibody or antigen-binding fragment thereof binds to LRP5. (Item 12) 12. The isolated antibody or antigen-binding fragment thereof of item 11, wherein the antibody or antigen-binding fragment thereof binds to LRP6. (Item 13) 13. The isolated antibody or antigen-binding fragment thereof of item 12, wherein the antibody or antigen-binding fragment thereof binds to LRP5 and LRP6. (Item 14) 14. An isolated antibody or antigen-binding fragment thereof that competes with the antibody of any of items 1 to 13 for binding to human LRP5 or LRP6. (Item 15) 15. The isolated antibody or antigen-binding fragment thereof according to any one of items 1 to 14, which binds to LRP5 or LRP6 with a KD of 50 μM or less. (Item 16) 16. The isolated antibody or antigen-binding fragment thereof of any of paragraphs 1 to 15, which modulates the Wnt signaling pathway in a cell, optionally in a mammalian cell. (Item 17) 17. The isolated antibody or antigen-binding fragment thereof of item 16, which increases signaling through the Wnt signaling pathway in the cell. (Item 18) 17. The isolated antibody or antigen-binding fragment thereof of item 16, which reduces signaling through the Wnt signaling pathway in the cell. (Item 19) 19. The isolated antibody or antigen-binding fragment thereof according to any of items 16 to 18, wherein the Wnt signaling pathway is the canonical Wnt signaling pathway. (Item 20) 19. The isolated antibody or antigen-binding fragment thereof according to any one of items 16 to 18, wherein the Wnt signaling pathway is a non-canonical Wnt signaling pathway. (Item 21) 21. An isolated polynucleotide encoding the isolated antibody or antigen-binding fragment thereof according to any one of items 1 to 20. (Item 22) 23. An expression vector comprising the isolated polynucleotide of item 22. (Item 23) 23. An isolated host cell comprising the expression vector of item 22. (Item 24) 34. 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 20 or 33. (Item 25) 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 described in item 17. (Item 26) 25. The method of claim 24, wherein the antibody or antigen-binding fragment thereof is a fusion protein comprising a polypeptide sequence that binds to one or more Frizzled (Fzd) receptors. (Item 27) 20. A method for inhibiting the Wnt signaling pathway in a cell, comprising contacting the cell with the isolated antibody or antigen-binding fragment thereof described in paragraph 18. (Item 28) 25. 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 24, wherein the isolated antibody or antigen-binding fragment thereof is an agonist of the Wnt signaling pathway. (Item 29) The disease or disorder is selected from the group consisting of fractures, stress fractures, vertebral compression fractures, osteoarthritis, 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, 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, muscular dystrophy, muscle atrophy in sarcopenia and cachexia, diseases affecting the blood-brain barrier (BBB), spinal cord injury, spinal cord diseases, oral mucositis, short bowel syndrome, inflammatory bowel disease (IBD), metabolic syndrome, diabetes, dyslipidemia, pancreatitis, exocrine pancreatic insufficiency , 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 29. The method of item 28, 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 30) 25. 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 item 24, wherein the isolated antibody or antigen-binding fragment thereof is an inhibitor of the Wnt signaling pathway. (Item 31) 30. The method of item 29, wherein the disease or disorder is selected from the group consisting of tumors and cancers, degenerative disorders, fibrosis of any organ or tissue, heart failure, coronary artery disease, heterotopic ossification, osteoporosis, and congenital disorders of high bone mass. (Item 32) An isolated antibody or antigen-binding fragment thereof that binds to the LRP6 receptor, wherein the isolated antibody or antigen-binding fragment thereof binds to the E3 β-propeller region of the LRP6 receptor or the corresponding region of the LRP5 receptor. (Item 33) An isolated antibody or antigen-binding fragment thereof that binds to the LRP6 receptor, wherein the antibody or antigen-binding fragment thereof binds to one or more epitopes within a region of the LRP6 receptor that includes or consists of amino acid residues 637 to 878, or the corresponding region of the LRP5 receptor. (Item 34) An isolated antibody or antigen-binding fragment thereof that binds to one or more LRP receptors, wherein the antibody or antigen-binding fragment thereof contacts the LRP receptor at a distance of less than 5 angstroms at any set of amino acid residues shown in Table 3. [Brief explanation of the drawings]

[0019] [Figure 1] Figure 1 shows a schematic representation of the crystal structure of the LRP6E3E4:VHH26 binding complex. LRP6E3E4 is shown in clear gray, and VHH26 is shown in dark gray. The positions of the CDR loops of VHH26 are marked. Glycans on the surface of LRP6E3E4 are shown in stick representation. [Figure 2]Figure 2 shows a schematic representation of the crystal structure of the LRP6E3E4:VHH36 binding complex. LRP6E3E4 is shown in clear gray, and VHH36 is shown in dark gray. The positions of the CDR loops of VHH36 are marked. Glycans on the surface of LRP6E3E4 are shown in stick representation. [Figure 3] Figure 3A shows Wnt activation using the A375 Wnt reporter assay. Figure 3B shows Wnt reporter activation in HEK293 cells with Rspo. The clones tested in both assays are Wnt surrogate molecules containing the Fzd binder 18R5 scFv in combination with LRP VHH or sdAb binders. 18R5:LRP surrogate molecules were tagged with FLAG and His tags. DETAILED DESCRIPTION OF THE INVENTION

[0020] The present disclosure relates to antibodies and antigen-binding fragments thereof that specifically bind to LRP5 and / or LRP6, particularly antibodies with particular LRP receptor specificity and functional properties. One embodiment of the present invention encompasses certain humanized antibodies and fragments thereof that bind to LRP5 and / or LRP6 and can modulate downstream Wnt pathway signaling and biological effects. For convenience, the term "anti-LRP5 / 6" is used to collectively refer to antibodies and antigen-binding fragments thereof that bind to either or both LRP5 and / or LRP6.

[0021] Embodiments of the present invention relate to the use of anti-LRP5 / 6 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 are used in the treatment or prevention of diseases and disorders associated with aberrant (e.g., increased or decreased) Wnt signaling or in which decreased or increased Wnt signaling is believed to provide therapeutic benefit.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] Each embodiment herein is intended to apply mutatis mutandis to every other embodiment unless otherwise stated.

[0026] 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 patients.

[0027] Embodiments of the present invention relate to antibodies that bind to LRP5 and / or LRP6. Exemplary antibodies, or antigen-binding fragments or complementarity-determining regions (CDRs) thereof, are set forth in SEQ ID NOs: 1-24.

[0028] As is well known in the art, an antibody is an immunoglobulin molecule that can specifically bind 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 portion having an antigen-binding fragment with the required specificity, humanized antibodies, chimeric antibodies, and any other modified configuration of an immunoglobulin molecule that contains 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 See, et al., Cancer Res., 56, 3055-3061, 1996.

[0029] The term "antigen-binding fragment," as used herein, refers to a polypeptide fragment containing at least one CDR of an immunoglobulin heavy and / or light chain that binds to an antigen of interest, particularly LRP5 and / or LRP6. In this regard, antigen-binding fragments of the antibodies described herein can contain one, two, three, four, five, or all six CDRs of the VH and VL sequences described herein from an antibody that binds to LRP5 and / or LRP6. Antigen-binding fragments of the LRP5 / 6-specific antibodies described herein can bind to LRP5 and / or LRP6. In certain embodiments, the antigen-binding fragment or an antibody comprising the antigen-binding fragment increases a Wnt signaling event. In certain embodiments, the antibody or antigen-binding fragment specifically binds to or modulates the biological activity of the human Wnt signaling pathway. In certain embodiments, the antibody or antigen-binding fragment thereof increases or decreases Wnt signaling.

[0030] 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. An antigen can have one or more epitopes. 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 -10 It can be M.

[0031] The term "epitope" includes any determinant, preferably a polypeptide determinant, capable of specific binding to an immunoglobulin or T-cell receptor. An epitope is a region of an antigen to which an antibody binds. In certain embodiments, epitopic determinants include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl groups, and in certain embodiments, can have specific three-dimensional structural characteristics and / or specific charge characteristics. 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. An antibody is said to specifically bind an antigen if it 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 -10 It can be M.

[0032] 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.

[0033] 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.

[0034] The structure and location of immunoglobulin variable domains 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).

[0035] 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), variants thereof, fusion proteins containing the antigen-binding portion 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 regard 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."

[0036] 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.

[0037] 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 made 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 LRP5 and / or LRP6 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.

[0038] 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.

[0039] In certain embodiments, the LRP5 / 6-binding antibodies described herein are in the form of diabodies. Diabodies are multimers of polypeptides, each of which contains 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).

[0040] A dAb fragment of an antibody consists of the VH domain (Ward, ES et al., Nature 341, 544-546 (1989)).

[0041] 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 ...

[0042] 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).

[0043] 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. For certain cancer cell surface antigens, monovalent binding may not stimulate cancer cell growth as effectively as bivalent antibodies with the same antigen specificity, making UniBody® technology a potential treatment option for some types of cancer that are resistant to traditional antibody therapy. The small size of UniBody® may be highly beneficial when treating some forms of cancer, potentially resulting in better molecular distribution in larger solid tumors and increased efficacy.

[0044] In certain embodiments, antibodies of the present disclosure can take the form of VHHs or sdAbs. VHHs or sdAbs are encoded by a single gene and are efficiently produced in nearly all prokaryotic and eukaryotic hosts, for example, 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 a long shelf life. The Nanoclone® method (see, e.g., WO 06 / 079372) is a unique method for generating VHHs or sdAbs against desired targets, based on automated high-throughput selection of B cells.

[0045] In certain embodiments, the anti-LRP5 / 6 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 remaining immunoglobulin structure of the molecule based on the structure and / or sequence of a human immunoglobulin. The antigen-binding site may comprise either a complete variable domain fused to a constant domain or only CDRs grafted onto appropriate framework regions within the variable domain. 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-LRP5 / 6 antibodies disclosed herein include those described in U.S. Patent No. 7,462,697.

[0046] 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.

[0047] 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-LRP5 / 6 antibody operably linked or fused to 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-LRP5 / 6 antigen-binding fragment of a chimeric antibody may include only one or more of the CDRs of an antibody described herein (e.g., 1, 2, 3, 4, 5, or 6 CDRs of an antibody described herein) or may include the entire variable domain (VL, VH, or both).

[0048] In certain embodiments, the Fc region of an antibody or fragment thereof can be derived from any of a variety of different Fc variants, 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.

[0049] 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.

[0050] In certain embodiments, the Wnt signaling pathway agonist fusion protein (or Wnt surrogate) comprises an anti-LRP5 / 6 antibody or antigen-binding fragment thereof fused to a polypeptide that specifically binds to one or more Frizzled (Fzd) receptors, as disclosed herein. In certain embodiments, the polypeptide that specifically binds to one or more Fzd receptors is an antibody or antigen-binding fragment thereof. In certain embodiments, it is an antibody or antigen-binding fragment thereof disclosed in U.S. Provisional Patent Application No. 62 / 607,877, filed December 19, 2017, attorney docket number SRZN-004 / 00US, entitled "Anti-Frizzled antibodies and Methods of Use," which application is incorporated herein by reference in its entirety.

[0051] In certain embodiments, the Fzd binding domain binds with high affinity, e.g., at least about 1×10 -7 M, at least 1 × 10 -8 M, at least 1 × 10 -9 M, at least 1 × 10 -10 The Fzd-binding domain can be selected from any domain that binds to Fzd with a KD of M. Suitable Fzd-binding domains include, but are not limited to, de novo designed Fzd-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; binding domains derived from VHHs or sdAbs; knottin-based engineered scaffolds; Norrin and Norrin-derived engineered binding fragments, natural Fzd-binding domains, etc. The Fzd-binding domain can be affinity-selective to enhance binding to one or more desired Fzd proteins, for example, to provide tissue selectivity.

[0052] In some embodiments, the Fzd binding domain binds to one, two, three, four, five, or more different Frizzled proteins, e.g., one or more of the human Frizzled proteins Fzdl, Fzd2, Fzd3, Fzd4, Fzd5, Fzd6, Fzd7, Fzd8, Fzd9, and FzdlO. In some embodiments, the Fzd binding domain binds to Fzdl, Fzd2, Fzd5, Fzd7, and Fzd8. In other embodiments, the Fzd binding domain is selective for one or more desired Frizzled proteins, 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.

[0053] In certain embodiments, the Fzd-binding domain comprises the six CDR regions of the pan-specific frizzled antibody OMP-18R5 (vantictumab). In certain embodiments, the Fzd-binding domain is an scFv comprising the six CDR regions of the pan-specific frizzled antibody OMP-18R5 (vantictumab). See, e.g., U.S. Patent No. 8,507,442 (specifically incorporated herein by reference). For example, the CDR sequences of OMP-18R5 comprise: (i) a heavy chain CDR1 comprising GFTFSHYTLS (SEQ ID NO: 25), a heavy chain CDR2 comprising VISGDGSYTYYADSVKG (SEQ ID NO: 26), and a heavy chain CDR3 comprising NFIKYVFAN (SEQ ID NO: 27); and (ii) a light chain CDR1 comprising SGDKLGKKYAS (SEQ ID NO: 28) or SGDNIGSFYVH (SEQ ID NO: 31), a light chain CDR2 comprising EKDNRPSG (SEQ ID NO: 29) or DKSNRPSG (SEQ ID NO: 32), and a light chain CDR3 comprising SSFAGNSLE (SEQ ID NO: 30) or QSYANTLSL (SEQ ID NO: 33). In certain embodiments, the frizzled binding domain is an antibody or derivative thereof, including, but not limited to, an scFv, a minibody, a VHH, or an sdAb, and various antibody mimetics comprising any of these CDR sequences. In certain embodiments, these CDR sequences comprise one or more amino acid modifications.

[0054] In other embodiments, the Fzd-binding domain comprises a variable region sequence or CDRs from any of several Frizzled-specific antibodies, which are known in the art, commercially available, or can be generated de novo. Any Frizzled polypeptide can be used as an immunogen or in screening assays to develop antibodies. Non-limiting examples of Frizzled-binding domains include antibodies available from Biolegend, such as clone CH3A4A7 specific for human Frizzled 4 (CD344); clone W3C4E11 specific for human Fz9 (CD349); antibodies available from Abcam, such as ab64636 specific for Fz7; ab83042 specific for human Fz4; ab77379 specific for human Fz7; ab75235 specific for human Fz8; and ab102956 specific for human Fz9. Other examples of suitable antibodies are described in, among others, U.S. Patent Application No. 20140105917, U.S. Patent Application No. 20130230521, U.S. Patent Application No. 20080267955, U.S. Patent Application No. 20080038272, U.S. Patent Application No. 20030044409, each of which is specifically incorporated herein by reference.

[0055] The surrogate Fzd-binding moiety can be an engineered protein selected for structural homology to the Frizzled-binding region of a Wnt protein. Such proteins can be identified by screening structural databases for homology. In this manner, an initial protein (e.g., a microbial Bh1478 protein) is identified. The native protein can then be engineered to introduce amino acid substitutions that increase affinity, and further selected by affinity maturation to increase affinity and selectivity in binding to the desired Frizzled protein. Non-limiting examples of Frizzled-binding moieties include the Fz27 and Fz27-B12 proteins.

[0056] The anti-LRP5 / 6 antibody or antigen-binding fragment thereof and the Fzd-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 region of the Wnt surrogate 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 intensity 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.

[0057] 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.

[0058] In certain embodiments, a Wnt surrogate directly activates canonical Wnt signaling by binding to one or more Fzd proteins and LRP5 and / or LRP6, particularly by binding to these proteins on the cell surface, e.g., on the cell surface of human cells. 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.

[0059] 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.

[0060] In certain embodiments, the antibodies or antigen-binding fragments thereof disclosed herein inhibit Wnt pathway signaling. In certain embodiments, the anti-LRP5 / 6 antibodies or antigen-binding fragments thereof block or inhibit endogenous Wnt from binding to one or more LRP5 / 6 receptors on the cell surface, thereby reducing or inhibiting Wnt signaling.

[0061] 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, axin translocation from the cytoplasm to the plasma membrane, and binding to LRP. The canonical Wnt / β-catenin signaling pathway ultimately leads to changes in gene expression via the transcription factors TCF7, TCF7L1, TCF7L2, and LEF. The transcriptional response to Wnt activation has been characterized in 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.

[0062] 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.

[0063] 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.

[0064] 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.

[0065] 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.

[0066] 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.

[0067] 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 substance or negative control, as measured in an assay described above, e.g., a TOPFlash assay. Such assays may include a positive control.

[0068] "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.

[0069] 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.

[0070] "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+ ) Route (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.

[0071] 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.

[0072] In certain embodiments, the functional properties of anti-LRP5 / 6 antibodies and antigen-binding fragments thereof can be assessed using a variety of methods known to those skilled in the art, including, for example, affinity / binding assays (e.g., surface plasmon resonance, competitive inhibition assays), cytotoxicity assays, cell viability assays, in vitro or in vivo assays. These assays include cell proliferation or differentiation assays in response to Wnt, cancer cell, and / or tumor growth inhibition using 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 / LRP5 / 6-mediated responses. The antibodies and antigen-binding fragments thereof described herein may also be tested for their effect on LRP5 / 6 receptor internalization, in vitro and in vivo efficacy, etc. Such assays may be performed using well-established protocols known to those skilled in the art (see, 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 commercially available kits.

[0073] In certain embodiments, the LRP5 / 6 binding antibody comprises one or more of the CDRs 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). Also, McLane et al., PNAS (1995) 92: 5214-5218, Barbas et al. See also, al., J. Am. Chem. Soc. (1994) 116:2161-2162.

[0074] 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 domain to produce an antibody or antigen-binding fragment thereof that binds to LRP5 and / or LRP6. The repertoire can then be displayed in a suitable host system, such as the phage display system of WO 92 / 01047, to allow selection of a suitable antibody or antigen-binding fragment 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.

[0075] 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).

[0076] 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 LRP5 and / or LRP6). Such methods are described, for example, in Portolano et al., J. Immunol. (1993) 150:880-887; Clarkson et al., Nature (1991) 352:624-628.

[0077] Other methods can also be used to mix and match CDRs to identify antibodies with the desired binding activity (e.g., binding to LRP5 and / or LRP6). For example, Klimka et al., British Journal of Cancer (2000) 83:252-260, describe a screening process using a library of mouse VL and human VH in which CDR3 and FR4 were retained from the mouse VH. After obtaining antibodies, the VH was 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.

[0078] 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.

[0079] Also disclosed herein is a method for obtaining an antibody or antigen-binding domain specific for LRP5 and / or LRP6 antigens, 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 of the VH domain; 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 specific binding members of the antibody antigen-binding domain that are specific for LRP5 and / or LRP6 and optionally have 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.

[0080] In certain embodiments, anti-LRP5 / 6 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 of at least about 5% of the starting material from which it is purified, typically at least about 10%, 20%, or 30%, more typically about 40%, 50%, or 60%, and sometimes about 50%, about 90%, or more. The anti-LRP5 / 6 antibodies and antigen-binding fragments thereof (including Wnt surrogates) of the present invention typically form a substantially homogeneous aqueous solution at a concentration of at least 25 μM or greater, e.g., at least 25 μM, 40 μM, or 50 μM, typically 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.

[0081] The epitope that an antibody or polypeptide "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 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 LRP5 is one that binds to LRP5 with greater affinity, avidity, more readily, and / or with a longer duration than to LRP6 or non-LRP5 / 6 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.

[0082] 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.

[0083] In certain embodiments, the anti-LRP5 / 6 antibody is administered to LRP5 and / or LRP6 at a concentration of about 1×10 -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 at least about 1 × 10 -10 K of M D In certain embodiments, the anti-LRP5 / 6 antibodies described herein bind to LRP5 and / or LRP6 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-LRP5 / 6 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. Dand in some embodiments, the antibodies may have even higher affinity for LRP5 and / or LRP6.

[0084] The term "immunologically active" refers to an epitope that is immunologically active or "remains active," and refers to the ability of an antibody (e.g., an anti-LRP5 / 6 antibody) to bind to an epitope under various conditions, for example, after the epitope has been subjected to reducing and denaturing conditions.

[0085] Antibodies or antigen-binding fragments thereof according to certain preferred embodiments of the present application may compete for binding to LRP5 and / or LRP6 with any of the antibodies described herein, or any variant thereof, that (i) specifically bind to the antigen and (ii) comprise the VH and / or VL domains disclosed herein or comprise the VH CDR3s 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 enabling identification of 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 for binding to LRP5 and / or LRP6 described herein.

[0086] In this regard, as used herein, the terms "compete with," "inhibit binding," and "block binding" (e.g., referring to inhibition / blocking of Wnt binding to LRP5 and / or LRP6 or to inhibition / blocking of anti-LRP5 / 6 antibody binding to LRP5 and / or LRP6) are used interchangeably and encompass partial and complete inhibition / blocking. Inhibition / blocking of Wnt binding to LRP5 and / or LRP6 preferably reduces or alters the normal level or type of cell signaling that occurs when Wnt binds to LRP5 and / or LRP6 without inhibition or blockage. Inhibition and blocking are also intended to include any measurable decrease in binding of Wnt to LRP5 and / or LRP6 when contacted with an anti-LRP5 / 6 antibody disclosed herein, compared to a ligand that has not been contacted with an anti-LRP5 / 6 antibody, e.g., 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% blocking of Wnt to LRP5 and / or LRP6.

[0087] 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.

[0088] 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.

[0089] 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 FcIIIb 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).

[0090] 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 -5The 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 the efficacy of the anti-CD20 antibody rituximab (Rituxan®, a registered trademark of IDEC Pharmaceuticals Corporation). Patients with the V158 allotype respond favorably to rituximab treatment, whereas patients 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.

[0091] 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 ***-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)).

[0092] Thus, in certain embodiments, the invention provides anti-LRP5 / 6 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.

[0093] 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 fusions are with an Ig heavy-chain constant domain containing at least part of the Ig light-chain binding region. 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.

[0094] 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.

[0095] In another contemplated embodiment, the LRP5 / 6-specific antibodies 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 above therapeutic agents can be used as antibody conjugates.

[0096] 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.

[0097] In certain embodiments, the anti-LRP5 / 6 antibodies and antigen-binding fragments thereof are monoclonal antibodies. In certain embodiments, they are humanized.

[0098] 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 the 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 LRP5 and / or LRP6 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.

[0099] 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.

[0100] 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.

[0101] 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.

[0102] The term "natural nucleotides" includes deoxyribonucleotides and ribonucleotides. The term "modified nucleotides" includes nucleotides with modified or substituted sugar groups, etc.

[0103] The term "oligonucleotide linkage" includes oligonucleotide linkages such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoraniladate, phosphoramidate, and the like. 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 et al., U.S. Pat. No. 5,151,510; Uhlmann and Peyman, 1990, Chemical Reviews, 90:543 (the disclosures of which are incorporated herein by reference for all purposes). The oligonucleotide can include a detectable label to allow for detection of the oligonucleotide or its hybridization.

[0104] 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.

[0105] 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.

[0106] 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.

[0107] Thus, in accordance with these and related embodiments, the present disclosure also provides polynucleotides encoding the anti-LRP5 / 6 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.

[0108] 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 such polynucleotides will have minimal sequence identity to the nucleotide sequence of a native or original polynucleotide sequence encoding an antibody that binds to LRP5 and / or LRP6. Nevertheless, polynucleotides that vary 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.

[0109] 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.

[0110] 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.

[0111] 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.

[0112] 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.

[0113] 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.

[0114] 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.

[0115] 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.

[0116] 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.

[0117] Expression of antibodies and antigen-binding fragments 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.

[0118] 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.

[0119] 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.

[0120] 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 an LRP5- or LRP6-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 has been "transfected" when the foreign DNA has been introduced inside the cell membrane. Several transfection techniques are known in the art and are disclosed herein. See, for example, Graham et al. et al.,1973,Virology 52:456;Sambrook et al. al.,2001,MOLECULAR CLONING,A LABORATORY See, "Manual," Cold Spring Harbor Laboratories; Davis et al., 1986, "Basic Methods in Molecular Biology," Elsevier; and Chu et al., 1981, Gene 13:197. Such techniques can be used to introduce one or more foreign DNA moieties into a suitable host cell.

[0121] 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.

[0122] 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, and the polypeptide or protein may include multiple chains non-covalently and / or covalently linked together by peptide bonds that have the sequence of a native protein, i.e., a protein produced by a cell that is naturally occurring and not specifically recombinant, and may include molecules having the amino acid sequence of a native protein or molecules with deletions from, additions to, and / or substitutions of one or more amino acids of the native sequence. The terms "polypeptide" and "protein" specifically encompass antibodies that bind to LRP5 and / or LRP6 of the present disclosure, or sequences that have deletions from, additions to, and / or substitutions of one or more amino acids from anti-Fzd antibodies of the present disclosure. 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").

[0123] The term "isolated protein," as used herein, means that the subject protein (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, preventative, research, or other applications).

[0124] Amino acid sequence modifications 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 its chain, 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 LRP5 and / or LRP6). Amino acid changes can 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.

[0125] The present disclosure provides variants of the antibodies disclosed herein. In certain embodiments, such variant antibodies, or antigen-binding fragments or CDRs thereof, bind to LRP5 and / or LRP6 with at least about 50%, at least about 70%, and in certain embodiments, at least about 90% of the binding of the antibody sequences specifically described herein. In further embodiments, such variant antibodies, or antigen-binding fragments or CDRs thereof, bind to LRP5 and / or LRP6 with higher affinity than the antibodies described herein, e.g., binding quantitatively at least about 105%, 106%, 107%, 108%, 109%, or 110% of the binding of the antibody sequences specifically described herein.

[0126] 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 whose amino acid sequence is identical to the heavy chain CDR1 region of a selected antibody described herein; ii. a CDR2 region whose amino acid sequence is identical to the heavy chain CDR2 region of the selected antibody; and iii. a CDR3 region whose amino acid sequence is identical to the heavy chain CDR3 region of the selected antibody; and / or b) a light chain variable region comprising: i. a CDR1 region whose amino acid sequence is identical to the light chain CDR1 region of the selected antibody; ii. a CDR2 region whose amino acid sequence is identical to the light chain CDR2 region of the selected antibody; and iii. a CDR3 region whose amino acid sequence is identical to the light chain CDR3 region of the selected antibody, wherein the antibody specifically binds to a selected target (e.g., LRP5 and / or LRP6). In further embodiments, the antibody or antigen-binding fragment thereof is a variant antibody, 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.) 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, an antibody or antigen-binding fragment thereof comprises a CDRH1 comprising or consisting of any of SEQ ID NOs: 34-172, a CDRH2 comprising or consisting of any of SEQ ID NOs: 173-312, a CDRH3 comprising or consisting of any of SEQ ID NOs: 313-485, a CDRL1 comprising or consisting of any of SEQ ID NOs: 486-524, a CDRL2 comprising or consisting of any of SEQ ID NOs: 525-556, and / or a CDRL3 comprising or consisting of any of SEQ ID NOs: 557-607.

[0127] In certain embodiments, a subject antibody (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 the heavy chain variable region of an anti-LRP5 / 6 antibody 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 the light chain variable region of an anti-LRP5 / 6 antibody described herein. Exemplary amino acid sequences of such heavy and / or light chain regions are set forth in SEQ ID NOS: 1-24.

[0128] Determination of the three-dimensional structure of a representative polypeptide (e.g., a variant LRP% / 6-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 to determine whether the resulting structural variant retains 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 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 LRP5 / 6-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 ... al.(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 (e.g., Schrodinger (Munich, Germany)).

[0129] In certain embodiments, the present disclosure provides antibodies or antigen-binding fragments thereof that bind to the E3 E4 region of LRP6. In certain embodiments, they bind to the E3 β-propeller region of LRP6. 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 described 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.

[0130] In another embodiment of the present invention, anti-LRP5 / 6 antibodies and humanized versions thereof are derived from rabbit monoclonal antibodies, specifically, 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 technology (see, e.g., U.S. Pat. No. 7,462,697). Accordingly, exemplary methods for producing anti-*** 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-LRP5 / 6 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.

[0131] composition Also disclosed are pharmaceutical compositions comprising an anti-LRP5 / 6 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.

[0132] In further embodiments, pharmaceutical compositions are also disclosed comprising a polynucleotide comprising a nucleic acid encoding an anti-LRP5 / 6 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-LRP5 / 6 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.

[0133] 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-LRP5 / 6 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 includes 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-LRP5 / 6 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).

[0134] 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-LRP5 / 6 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-LRP5 / 6 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).

[0135] The present disclosure contemplates a pharmaceutical composition comprising a first molecule for delivering an anti-LRP5 / 6 antibody or an 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) containing nucleic acid sequences encoding the first or second active agent, and cells (optionally expression cassettes) containing nucleic acid sequences encoding the first or second active agent.

[0136] 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.

[0137] 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 to the extent that easy syringability exists. 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.

[0138] Sterile solutions can be prepared by incorporating the required amount of anti-LRP5 / 6 antibody or antigen-binding fragment thereof (or 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 powders containing the active ingredient and any additional desired ingredient from a previously sterile-filtered solution.

[0139] 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.

[0140] 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.

[0141] 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.

[0142] 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.

[0143] The present invention includes pharmaceutically acceptable salts of the anti-LRP5 / 6 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 the 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 later 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.

[0144] 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.

[0145] In some embodiments, pharmaceutical compositions provided herein comprise a therapeutically effective amount of an anti-LRP5 / 6 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 glycols. Preferably, the formulation is stable at 4°C for at least 6 months.

[0146] 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.

[0147] 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, for example, to modulate the Wnt signaling pathway, e.g., to increase or decrease Wnt signaling, as well as methods for administering LRP5 / 6-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, the antibodies or antigen-binding fragments thereof of the present invention are provided to subjects with diseases associated with inappropriate or deregulated Wnt signaling, e.g., increased or decreased Wnt signaling.

[0148] Increasing the Wnt signaling pathway and related therapeutic methods In certain embodiments, anti-LRP5 / 6 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-LRP5 / 6 antibody or antigen-binding fragment thereof (e.g., a Wnt surrogate) disclosed herein, wherein the anti-LRP5 / 6 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.

[0149] 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-LRP5 / 6 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-LRP5 / 6 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 polynucleotide.

[0150] 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-LRP5 / 6 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-LRP5 / 6 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.

[0151] 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-LRP5 / 6 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-LRP5 / 6 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-LRP5 / 6 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).

[0152] Anti-LRP5 / 6 antibodies or antigen-binding fragments thereof (e.g., Wnt surrogates) can be used to treat diseases, disorders, or conditions, for example, by increasing Wnt signaling in targeted cells, tissues, or organs. Thus, in some embodiments, the present invention provides a method for treating a disease or condition in a subject in need thereof, for example, 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-LRP5 / 6 antibody or its antigen-binding fragment (e.g., a Wnt surrogate); a polynucleotide, e.g., DNA or mRNA, optionally modified mRNA, comprising a nucleic acid sequence encoding an anti-LRP5 / 6 antibody or its antigen-binding fragment (e.g., a Wnt surrogate); a vector, e.g., an expression vector or viral vector, comprising a nucleic acid sequence encoding an anti-LRP5 / 6 antibody or its antigen-binding fragment (e.g., a Wnt surrogate); or a cell comprising a nucleic acid sequence encoding an anti-LRP5 / 6 antibody or its antigen-binding fragment (e.g., a Wnt surrogate), e.g., a cell transduced with an expression vector or viral vector encoding an anti-LRP5 / 6 antibody or its antigen-binding fragment (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, hi some embodiments, the contacting occurs in vivo, i.e., a subject composition is administered to a subject.

[0153] 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-LRP5 / 6 antibody or an 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.

[0154] 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-LRP5 / 6 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-LRP5 / 6 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.

[0155] 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-LRP5 / 6 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-LRP5 / 6 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., the same expression cassette.

[0156] 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-LRP5 / 6 antibody or an 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-LRP5 / 6 antibody or an 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-LRP5 / 6 antibody or an 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).

[0157] Wnt signaling plays an important 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-LRP5 / 6 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.

[0158] 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 injuries including vertebral compression fractures, preoperative spinal surgery optimization, orthopedic device osseointegration, tendon-osseous integration, tooth growth and regeneration, dental implants, periodontal disease, maxillofacial reconstruction, and treatment of osteonecrosis of the jaw.The compositions of the present invention can also be used to treat alopecia; enhance sensory organ regeneration, for example, treatment of hearing loss (including inner and outer hair cell regeneration and treatment of vestibular dysfunction), treatment of macular degeneration, treatment of retinopathies (including vitreoretinopathy, diabetic retinopathy, and other retinal degenerative diseases), Fuchs' dystrophy, other corneal diseases, etc.; 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.

[0159] 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-LRP5 / 6 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.

[0160] 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-LRP5 / 6 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-LRP5 / 6 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.

[0161] Compositions comprising one or more anti-LRP5 / 6 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, such as 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.

[0162] Anti-LRP5 / 6 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.

[0163] 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.

[0164] Anti-LRP5 / 6 antibodies or antigen-binding fragments thereof (e.g., Wnt surrogates) can also be used to regenerate retinal tissue. In the retina of adult mammals, 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.

[0165] 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.

[0166] 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.

[0167] 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-LRP5 / 6 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-LRP5 / 6 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), pulmonary fibrosis, particularly idiopathic pulmonary fibrosis (IPF), 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.

[0168] 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.

[0169] Stroke, traumatic brain injury, Alzheimer's disease, multiple sclerosis, and other conditions affecting the blood-brain barrier (BBB) can be treated using anti-LRP5 / 6 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. Additionally, 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.

[0170] Wnt signaling also plays a role in angiogenesis. Anti-LRP5 / 6 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 present invention, which may be administered systemically or locally.

[0171] 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.

[0172] Reduction of the Wnt signaling pathway and related therapeutic methods In certain embodiments, anti-LRP5 / 6 antibodies or antigen-binding fragments thereof can be used to reduce or inhibit Wnt signaling in tissues or cells. Thus, in some aspects, the present invention provides a method for reducing or inhibiting Wnt signaling in tissues or cells, comprising contacting the tissue or cells with an effective amount of an anti-LRP5 / 6 antibody or antigen-binding fragment thereof disclosed herein, wherein the anti-LRP5 / 6 antibody or antigen-binding fragment thereof is a Wnt signaling pathway antagonist or inhibitor. In some embodiments, the contacting is carried out in vitro, ex vivo, or in vivo. In certain embodiments, the cells are cultured cells, and the contacting is carried out in vitro. Performed in vitro.

[0173] 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-LRP5 / 6 antibody or antigen-binding fragment thereof of the present invention, wherein the anti-LRP5 / 6 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.

[0174] In a related aspect, the present invention provides a method for reducing or inhibiting Wnt signaling in tissues or cells, comprising contacting the tissues or cells with an effective amount of a vector comprising a nucleic acid sequence encoding an anti-LRP5 / 6 antibody or its antigen-binding fragment, wherein the anti-LRP5 / 6 antibody or its antigen-binding fragment is a Wnt signaling pathway antagonist or inhibitor. In certain embodiments, the vector is an expression vector and can comprise a promoter operably linked to the nucleic acid sequence. In certain embodiments, the vector is a viral vector.

[0175] 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-LRP5 / 6 antibody or its antigen-binding fragment, wherein the anti-LRP5 / 6 antibody or its antigen-binding fragment 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-LRP5 / 6 antibody or its antigen-binding fragment, wherein the anti-LRP5 / 6 antibody or its antigen-binding fragment 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).

[0176] Anti-LRP5 / 6 antibodies and antigen-binding fragments thereof, wherein the anti-LRP5 / 6 antibodies or antigen-binding fragments thereof are Wnt signaling pathway antagonists or inhibitors, can be used to treat diseases, disorders, or conditions, for example, by reducing or inhibiting Wnt signaling in cells, tissues, or organs. Thus, in some embodiments, the present invention provides a method for treating a disease or condition in a subject in need thereof, for example, a disease or disorder associated with increased or deregulated Wnt signaling, or a disease or disorder in which reduced Wnt signaling is believed to provide therapeutic benefit, comprising contacting the subject with an effective amount of a composition comprising an anti-LRP5 / 6 antibody or antigen-binding fragment thereof, wherein the anti-LRP5 / 6 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-LRP5 / 6 antibody or antigen-binding fragment thereof; a polynucleotide, e.g., DNA or mRNA, optionally modified mRNA, comprising a nucleic acid sequence encoding an anti-LRP5 / 6 antibody or antigen-binding fragment thereof; a vector, e.g., an expression vector or viral vector, comprising a nucleic acid sequence encoding an anti-LRP5 / 6 antibody or antigen-binding fragment thereof; or a cell comprising a nucleic acid sequence encoding an anti-LRP5 / 6 antibody or antigen-binding fragment thereof, e.g., a cell transduced with an expression vector or viral vector encoding an anti-LRP5 / 6 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 is performed in vivo, i.e., the subject composition is administered to a subject.

[0177] 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-LRP5 / 6 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 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.

[0178] 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 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 vector comprising a nucleic acid sequence encoding an anti-LRP5 / 6 antibody or its antigen-binding fragment, wherein the antibody or its antigen-binding fragment 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.

[0179] 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 cells comprising a nucleic acid sequence encoding an anti-LRP5 / 6 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-LRP5 / 6 antibody or antigen-binding fragment thereof. In certain embodiments, the cells are stem cells (e.g., adipose-derived stem cells or hematopoietic stem cells).

[0180] 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-LRP5 / 6 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.

[0181] In certain embodiments, the method of treating a disease or disorder in a subject in need thereof by providing the subject with an effective amount of an anti-LRP5 / 6 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, such as vasculitis and conditions associated with abnormal angiogenesis.

[0182] In certain embodiments, the method for treating a disease or disorder in a subject in need thereof by providing the subject with an effective amount of an anti-LRP5 / 6 antibody or its antigen-binding fragment 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, idiopathic pulmonary fibrosis), renal fibrosis (e.g., end-stage renal disease), hepatic fibrosis, congenital liver storage disease, and cardiac fibrosis.

[0183] 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-LRP5 / 6 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.

[0184] 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-LRP5 / 6 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.

[0185] 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.

[0186] 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.

[0187] 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 anti-LRP5 / 6 antibody or antigen-binding fragment thereof (e.g., 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.

[0188] 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-LRP5 / 6 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 preferably 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.

[0189] 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.

[0190] 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.

[0191] 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.

[0192] 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.

[0193] 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.

[0194] 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).

[0195] 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.

[0196] 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 LRP5 and / or LRP6. Accordingly, the present disclosure provides methods for detecting LRP5 and / or LRP6 in a sample, e.g., methods for detecting cells or tissues expressing LRP5 or LRP6. 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 a tissue or cell (e.g., obtained from a subject) with an anti-Fzd antibody or antigen-binding fragment thereof disclosed herein, then determining the amount of antibody or antigen-binding fragment thereof that binds to the tissue or cell, and then determining the presence or amount of LRP5 and / or LRP6 receptor(s) in the tissue or cell.

[0197] 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.

[0198] 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-LRP5 / 6 antibody or its antigen-binding fragment covalently linked to a detectable label. In the present invention, DNA probes, RNA probes, monoclonal antibodies, their antigen-binding fragments, and antibody derivatives thereof, such as single-chain variable fragment antibodies or epitope-tagged antibodies, can all be covalently linked to a detectable label. In "direct detection," only one detectable antibody, i.e., a detectable primary antibody, is used. Therefore, direct detection means that an antibody conjugated to a detectable label can be detected by itself without the need for the addition of a second antibody (secondary antibody).

[0199] 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.

[0200] 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.

[0201] 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).

[0202] 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.

[0203] 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.

[0204] 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.

[0205] 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).

[0206] 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).

[0207] 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.

[0208] 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).

[0209] The present invention further provides kits for detecting one or more LRP receptors or cells or tissues expressing one or more LRP 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.

[0210] 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.

[0211] 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]

[0212] Example 1 Characterization of anti-LRP5 / 6 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 LRP receptors.

[0213] 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.

[0214] 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.

[0215] Protein concentrations were determined 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.

[0216] The binding kinetics of antibody fragments to the LRP5 extracellular domain (LRP5 ECD) and / or LRP6 extracellular domain (LRP6 ECD) protein targets 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 LRP ECD 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 LRP5 or LRP6, the SA biosensor with captured biotinylated LRP5 or LRP6 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 a well with 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:

[0217] Binding measurements were also performed by surface plasmon resonance on a BIAcore T100 (GE Healthcare, Pittsburgh, PA). All proteins were purified by SEC before the experiment. Biotinylated Lrp6 E3E4 was bound to streptavidin on an SA sensor chip (GE Healthcare, Pittsburgh, PA) at low density. An unrelated biotinylated protein was captured at a binding density equivalent to that of a control flow cell. Increasing concentrations of scFv-Nab fusion molecules (e.g., 18R5 scFv-LRP6-binding Nab) were flowed over the chip in 1x HBS-P (GE Healthcare, Pittsburgh, PA) containing 40 μl / ml of 0.5% BSA. After each injection, the chip surface was regenerated for 60 seconds with 2 M MgCl2 in HBS-P. Curves were baseline subtracted and all data were analyzed using the Biacore T100 evaluation software version 2.0 with a 1:1 Langmuir binding model to determine KD values.

[0218] Table 1A lists the heavy chain CDRs (CDRH1, CDRH2, and CDRH3) and light chain CDRs (CDRL1, CDRL2, and CDRL3) for the indicated antibody clones, and indicates the first LRP5 or LRP6 to which the antibody fragments showed binding. 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)).

[0219] If the light chain CDRs are not shown, then the antibody fragment did not contain a light chain. [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

[0220] Table 1B provides the sequence identification numbers of antibody heavy chain fragments (HC) of exemplary clones having only the heavy chain region, and their binding characteristics. In certain embodiments, the LRP5 / 6 binding domain is a Fab or is derived from a Fab, such that Table 1B contains VH and CH1 sequences, but not CH2 or CH3 sequences. In certain embodiments, the LRP5 / 6 binding domain is a VHH or sdAb or is derived from a VHH or sdAb, such that Table 2B contains VHH domains. K at various Fzd receptors as determined by Octet BLI D Data are shown. The "Confirmed Binding" column indicates binding results from Octet BLI binding or BIAcore SPR. Blank cells indicate that binding to specific Fzd receptors has not yet been determined. As shown in Table 1B, Octet BLI or BIAcore sensorgrams for the anti-LRP5 / 6 antibody fragments demonstrated a range of affinities for LRP5 and / or LRP6. [Table 1B]

[0221] Table 1C provides further binding characteristics for certain clones, including relative binding affinities for the LRP6E1E2 and LRP6E3E4 domains. The designation "nb" indicates no binding. [Table 1C-1] [Table 1C-2]

[0222] Example 2 Alanine scanning mutagenesis of anti-LRP6 antibody fragments One antibody fragment, 009S-E04, was selected for alanine scanning mutagenesis of the CDRs, and the LRP6 binding affinities of various mutants were determined by Octet BLI as described in Example 1. As shown in Table 2, many mutants bound to LRP6 with affinities similar to the wild-type antibody fragment, demonstrating that LRP6 antibodies and their antigen-binding fragments can tolerate amino acid modifications within the CDRs. [Table 2]

[0223] Example 3 Characterization of 18R5scFv-Lrp VHH Wnt surrogate fusion proteins in 293 and A375 Wnt-dependent reporter assays We demonstrated the ability of Wnt surrogate fusion proteins containing scFv regions that bind one or more Fzds and VHH or sdAb (or single-domain antibody Nab) regions that bind LRP5 and / or LRP6. The Fzd-binding agent 18R5 in scFv format was fused to various Lrp5 and Lrp6-binding agents disclosed herein. The LRP-binding agents, designated Nabs, were fused to the C-terminus of the 18R5 scFv using a six-amino acid linker. Fusion proteins containing a C-terminal poly-His tag were expressed in expi293 cells according to the manufacturer's protocol and purified from conditioned medium using complete His-tag purification resin and Superdex 200 10 / 300 GL equilibrated in HBS (10 mM HEPES, pH 7.3, 150 mM NaCl). Fractions containing the monomeric fusions were pooled and concentrated.

[0224] The ability of the fusion proteins to induce Wnt pathway signaling was tested using a Wnt-dependent reporter assay in 293 and A375 cell lines under the following conditions: 10,000 A375 and HEK293 Wnt reporter cells were stably transfected with the STF Wnt reporter plasmid or its variants, seeded in triplicate in 96-well plates for each condition, and stimulated with the fusion proteins for 16–20 h in the presence or absence of 25 nM Fc-Rspo2. -2 nM~10 3 Various concentrations of each fusion protein were tested, ranging from 0.1 nM to 0.1 nM. After washing the cells with PBS, the cells in each well were lysed in 30 μl of passive lysis buffer (Promega). 10 μl of lysate per well was assayed using a firefly luciferase assay kit (Promega).

[0225] The results demonstrated that multiple Wnt surrogates tested activated the Wnt signaling pathway in a concentration-dependent manner, which was further enhanced by treatment with Fc-Rspo2 (data not shown).

[0226] Example 4 Crystal structure of the Lrp6E3E4-binding substance complex Lrp6 (low-density lipoprotein receptor-related protein 6) is a 1,613 amino acid, single-pass membrane protein that plays a crucial role in Wnt-mediated activation of β-catenin signaling. Lrp6 and the related Lrp5 share 70% sequence identity between them. The extracellular regions of Lrp6 and Lrp5 contain four β-propeller domains, referred to herein as the E1, E2, E3, and E4 domains. In this section, we describe the structure of the Lrp6 E3E4 domain (residues 631–1246 of Uniprot entry O75581 (https: / / www.uniprot.org / uniprot / O75581)). The sequence of Lrp6 E3E4, which contains a biotin acceptor peptide (BAP) and an eight-histidine motif at the C-terminus, used for structural studies, is as follows: LRP6E3E4_O75581_631-1246: (SEQ ID NO: 619)

[0227] Example 5 Expression and purification of Lrp6E3E4 for structural studies Stable cell lines expressing the Lrp6E3E4 domain were generated in Expi293 cells by G418 selection. For large-scale expression, a frozen vial of Expi293 cells expressing Lrp6E3E4 was thawed in 20 mL of Expi293 medium (Thermofisher). Cell viability was monitored, and cells were cultured over separate days at approximately 3.0–4.0 × 10 cells in the desired volume (typically 6–10 L). 6The cells were expanded to reach 1000 cells / mL. At this stage, cells were treated with 2 mM valproic acid and continued to grow to a high density. After approximately 48 hours, the medium was harvested by centrifugation. Fzd CRD_Xtal protein was purified from the medium by incubation with HisComplete resin (1 mL per mL of medium; Roche) pre-equilibrated in PBS (50 mM sodium dihydrogen phosphate pH 8.0, 300 mM NaCl) and eluted with 250 mM imidazole. The 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 (20 mM HEPES pH 7.4, and 150 mM sodium chloride). 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 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 LRP6E3E4 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 determined 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.

[0228] Example 6 Expression and purification of VHH or sdAb or Fab binders Plasmids expressing the light and heavy chains (containing a hexahistidine at their C-terminus) in the case of Fabs or such plasmids expressing VHH binders were transfected into Expi293 cells for 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 harvested by centrifugation, bound to Complete His resin (2.5 mL per liter of medium; Roche) pre-equilibrated in PBS, and eluted under gravity flow with 250 mM imidazole in PBS. The eluate containing the Fab binders 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 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 Fab or VHH binders were concentrated to approximately 3 mg / mL and frozen at -80°C in the presence of 10% glycerol for storage until further use. Protein concentrations were determined 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.

[0229] Example 7 Lrp6E3E4: VHH / Fab complex formation, crystallization, and structure determination Purified Lrp6E3E4 and VHH / Fab binders were mixed at 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 / 300 GL) column pre-equilibrated in HBS. Fractions containing the complex were further checked by SDS-PAGE and concentrated to 10–25 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, as well as grid screens or microseed matrix screens [MMS; Microseed matrix screening for optimization in protein Optimization by [crystallization: what have we learned? D'Arcy, A., Bergfors, T., Cowan-Jacob SW, and Marshd, M. Acta Cryst. F70, 1117-1126 (2014)] was performed using a Mosquito (TTP LabTech) pipettor and equilibrated at 18°C in an EchoTherm incubator (Torrey Pines Scientific). 96-well plate crystal screening experiments were periodically monitored manually 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, or 1.1–2.5 M sodium malonate, pH 7.0). X-ray diffraction data sets were collected at the Berkeley Center for Structural Biology at the Advanced Light Source (ALS) (Berkeley, CA) and processed with the programs 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]. The structure of the Lrp6E3E4:VHH / Fab complex was analyzed using Phaser [Phaser crystallographic software. AJ McCoy, RW Grosse-Kunstleve, PDA Dams, MD Winn, LC Storoni, and RJ Read.J Appl Crystallogr 40,658-674(2007)] and the Lrp6E3E4 fragment [PDB file:4A0P;Chen S,Bubeck 21. MacDonald BT, Liang WX, Mao JH, Malinauskas T, Llorca O, Aricescu AR, Siebold C, He X, Jones EY.Dev.Cell [ PubMed ] 848-61(2011)] and sdAb in VHH[PDB file:6B20,chain E;Gulati S,Jin H,Masuho I,Orban T,Cai Y,Pardon E,Martemyanov KA,Kiser PD,Stewart PL,Ford CP,SteyaertJ,Palczewski K.Nat Commun 9 1996(2018)] and is a comprehensive Python-based system for Phenix macromolecular structure solution.PDAdams,PVAfonine,G.Bunkoczi,VBChen,IWDavis,N.Echols,JJHeadd,LWHung,GJKapral,RWGrosse-Kunstleve,AJMcCoy,NWMoriarty,R.Oeffner,RJRead,DCRichardson,JSRichardson,TCTerwilliger,and PHZwart.Acta Cryst.D66,213-221(2010);MolProbity: all-atom structure validation for macromolecular crystallography.D66, 12-21 (2010)]. COOT [Features and development of Coot. P. Emsley, B. Lohkamp, W.G. Scott, and K. Cowtan. Acta. Crystallographic models were manually inspected and constructed using [Cryst. D66, 486-501 (2010)]. Analysis and imaging of the refined crystal structures was performed using MOE (CCG) and PyMol (Schrodinger).

[0230] Example 8 Structure of the Lrp6E3E4:VHH26 complex Sequence of VHH26(009S-E04): DVQLVESGGGLVQAGGSLRLACAGSGRIFAIYDIAWYRHPPGNQRELVAMIRPVVTEIDYADSVKGRFTISRNNAMKTVYLQMNNLKPEDTAVYYCNAKRPWGSRDEYWGQGTQVTVSSGSGSGHHHHHH (SEQ ID NO: 620) Diffraction-quality crystals of Lrp6E3E4:VHH26 (concentration = 12.4 mg / mL) were obtained by MMS in G8 conditions containing PACT screen, 0.2 M sodium sulfate, 0.1 M bis-tris-propane pH 7.5, and 20% (v / v) PEG3350. Crystals were cryoprotected using 27% glycerol in the well solution. The Lrp6E3E4:VHH26 complex crystallized with one complex molecule per asymmetric unit in the P3121 space group (a = b = 136.64 Å and c = 104.70 Å). The structure of the Lrp6E3E4:VHH26 complex was determined at 2.40 Å resolution, and R 結晶 Factors and R 遊離 The factors were refined to 18.5% and 23.0%, respectively.

[0231] The overall structure of the Lrp6E3E4:VHH26 complex shown in Figure 3.1(A) indicates that the epitope on Lrp6E3E4 for VHH26 (Figure 4.1(B)) is located on the E3 of Lrp6. The complex structure reveals that Lrp6E3E4 is localized within the β-propeller. The epitope of Lrp6E3E4 for VHH26 can be identified, with the following residues of Lrp6E3E4 defining the core interaction site (atomic distance between Lrp6E3E4 and VHH26 is 5.0 Å or less): Arg639, Ala640, Lys622, Glu663, Ile681, Ser682, Lys684, Asp705, Tyr706, Glu708, Thr724, Gly725, Arg751, Trp767, Gly768, Gly769, Arg792, Leu810, Asp811, His834, Phe836, Trp850, Ser851, Arg853, Asp874, Tyr875, and Met877.

[0232] In addition, the following residues on Lrp6E3E4 are considered to be identified as direct interaction sites (atomic distance between Lrp6E3E4 and VHH26 is greater than 5.0 Å and less than 8.0 Å): Arg638, Asp641, Val661, Ala664, Ser665, Asp680, Leu683, Thr685, Leu704, Pro707, Asp723, Thr726, Asn727, Asp748, Ser749, Pro750, Lys770, Pro771, Gly791, Asn794, Asp809, Thr812, Asn813, Pro833, Pro835, Asp849, and Arg852.

[0233] Furthermore, from the structure of the Lrp6E3E4:VHH26 complex, the following residues on VHH26 can also be identified as core interaction sites (atomic distance between Lrp6E3E4 and VHH26 is 5.0 Å or less): Gly26, Arg27, Phe29, Ala30, Ile31, Tyr32, Arg52, Pro53, Val54, Val55, Glu57, Asn74, Ala75, Lys77, Arg100, Pro101, Trp102, Gly103, Ser104, Arg105, Asp106, and Tyr108.

[0234] Furthermore, from the structure of the Lrp6E3E4:VHH26 complex, the following residues on VHH26 can also be identified as core interaction sites (atomic distance between Lrp6E3E4 and VHH26 is greater than 5.0 Å and less than 8.0 Å): Gly24, Ser25, Ile28, Asp33, Met50, Ile51, Thr56, Arg72, Asn73, Met76, Lys99, and Glu107.

[0235] Example 9 Structure of the Lrp6E3E4:VHH36 complex Sequence of VHH36 (013S-D05): QVKLEESGGGLVQAGGSLRLSCAASGRIFSIYDMGWFRQAPGKEREFVSGIRWSGGTSYADSVKGRFTISKDNAKNTIYLQMNNLKAEDTAVYYCGSRGYWGQGTLVTVSSGSGSGHHHHHH (SEQ ID NO: 621) Diffraction-quality crystals of Lrp6E3E4:VHH36 (concentration = 14.0 mg / mL) were obtained by grid screen optimization under conditions containing 1.6 M ammonium sulfate and 0.1 M Tris pH 8.2. Crystals were cryoprotected using 1.1 M sodium malonate pH 7.0 in 1.6 M ammonium sulfate and Tris pH 9.0. The Lrp6E3E4:VHH36 complex crystallized with one complex molecule per asymmetric unit in the P65 space group (a = b = 180.61 Å and c = 98.63 Å). The structure of the Lrp6E3E4:VHH36 complex was determined at 2.70 Å resolution and analyzed by R. 結晶 Factors and R 遊離 The factors were refined to 18.6% and 22.8%, respectively.

[0236] The overall structure of the Lrp6E3E4:VHH36 complex shown in Figure 3.1(A) indicates that the epitope on Lrp6E3E4 for VHH36 (Figure 4.1(B)) is located on the E3 of Lrp6. The complex structure reveals that Lrp6E3E4 is localized within the β-propeller. The epitope of Lrp6E3E4 for VHH36 can be identified, with the following residues of Lrp6E3E4 defining the core interaction site (atomic distance between Lrp6E3E4 and VHH36 is 5.0 Å or less): Glu663, Ser665, Ile681, Tyr706, Glu708, Thr724, Ser749, Arg751, Trp767, Gly768, Arg792, Leu810, Asn813, Pro833, His834, Phe836, Trp850, Ser851, Arg853, Asp874, Tyr875, and Met877.

[0237] In addition, the following residues on Lrp6E3E4 are considered to be identified as direct interaction sites (atomic distance between Lrp6E3E4 and VHH36 is greater than 5.0 Å and less than 8.0 Å): Ser637, Arg638, Arg639, Lys662, Ala664, Ala666, Thr679, Asp680, Ser682, Lys684, Pro707, Gly725, Asn727, Asp748, Pro750, Glu766, Gly769, Pro771, Asn794, Thr808, Asp809, Asp811, Thr812, Leu814, Leu832, Pro835, Asp849, Arg852, His872, Leu873, Val876, and Asp878.

[0238] Furthermore, from the structure of the Lrp6E3E4:VHH36 complex, the following residues on VHH36 can also be identified as core interaction sites (atomic distance between Lrp6E3E4 and VHH36 is 5.0 Å or less): Gln1 (modified as pyroglutamic acid), Val2, Lys3, Ala24, Ser25, Gly26, Arg27, Ile28, Ser30, Ile31, Tyr32, Trp53, Asn73, Asn76, Arg98, and Tyr100.

[0239] Furthermore, from the structure of the Lrp6E3E4:VHH36 complex, the following residues on VHH36 can also be identified as core interaction sites (atomic distance between Lrp6E3E4 and VHH36 is greater than 5.0 Å and less than 8.0 Å): Leu4, Ala23, Phe29, Asp33, Arg52, Ser54, Lys71, Asp72, Ala74, Lys75, Ser97, and Gly99. [Table 3-1] [Table 3-2]

[0240] 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.

[0241] 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.

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