WO2013126911A1 - Inhibitors and agonists of the low density lipoprotein receptor related domain of lrp6 and/or lrp5 and uses thereof - Google Patents

Abstract

Disclosed herein are inhibitors and agonists that specifically target the low density lipoprotein receptor related (LDLRR) domain of low density lipoprotein receptor-related protein (LRP) 6 and/or LRP5. More particularly, LDLRR binding antibodies and antigen-binding fragments of such antibodies are disclosed and methods of using such antibodies and fragments to treat diseases.

Description

Attorney Docket No.: 27527-0106WO1

INHIBITORS AND AGONISTS OF THE LOW DENSITY LIPOPROTEIN RECEPTOR RELATED DOMAIN OF LRP6 AND/OR LRP5 AND USES THEREOF

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority from U.S. Provisional Application Serial No. 61/602,853, filed on February 24, 2012, which is incorporated herein by reference in its entirety.

TECHNICAL FIELD

[0002] Disclosed herein are inhibitors that specifically target the low density lipoprotein receptor related (LDLRR) domain of low density lipoprotein receptor-related protein (LRP) 6 and/or LRP5. More particularly, LDLRR binding antibodies and antigen-binding fragments thereof are provided.

BACKGROUND

[0003] Wnt autocrine mechanisms are responsible for constitutive Wnt activation at high frequencies in several human solid tumors, including breast, ovarian and lung cancers, as well as in sarcomas and gliomas, which affect hundreds of thousands of new cancer patients yearly. Thus, autocrine Wnt signaling is a key target for therapies that could decrease morbidity and mortality from cancer.

[0004] Wnt signaling mediated through β-catenin stabilization acts in concert with TCF/LEF transcription factors to activate transcriptional target genes. Wnt canonical co- receptors, low density lipoprotein receptor-related proteins (LRP) LRP5 and LRP6 and Frizzled (Fzd), are coupled by canonical Wnts to transmit the canonical Wnt signal intracellularly. Fzd recruits the intracellular protein Dvl, which, in turn, brings to the membrane the axin-GSK3 complex, thereby promoting the initial phosphorylation of LRP6. Further phosphorylation of LRP6 by casein kinase 1 (CKl) is associated with clustering of different proteins including LRP 6, Dvl and Axin to form what has been defined as the LRP 6 signalosome. Phosphorylated LRP5/6 leads to inhibition of the so-called β-catenin destruction complex, which includes axin, GSK3, Dvl, CKl and the tumor suppressor adenomatous polyposis coli (APC), resulting in the stabilization and translocation of Attorney Docket No.: 27527-0106WO1 β-catenin in the nucleus, where it activates target genes through binding to TCF/LEF transcription factors.

[0005] Interfering with Wnt signaling by inhibiting the function of LRP5 or LRP6 has been demonstrated to be effective in inhibiting growth of cancer cells. For example, Vijayakumar et al. (Cancer Cell, 2011, 19:601-612) tested the effects of shRNA knockdown of either LRP5 or LRP6 in sarcoma cell lines, which exhibited high levels of endogenous LRP5 and LRP6 proteins, respectively. Downregulation of these receptors led to a concomitant decrease in Wnt reporter activity and inhibition of in vitro proliferation, implying the involvement of these overexpressed receptors in autocrine Wnt-mediated proliferation of these sarcoma lines.

[0006] Presently available inhibitors of canonical Wnt signaling are limited, because those inhibitors only inhibit signaling mediated by a small subgroup of Wnt ligands, and, in some cases, can actually augment rather than inhibit Wnt signaling in response to other Wnt ligands (see, Ettenberg, et al. (2010) Proc Natl Acad Sci U S A. 2010 Aug 31 ;107(35): 15473- 8). More specifically, known inhibitors of LRP6 and LRP5 are directed to those proteins' El- 2 or E3-4 domain (but not both). Since tumors with activated autocrine Wnt signaling produce a wide range of Wnt ligands that can bind to either El -2 or E3-4 domains, a given inhibitor targeting one of those domains cannot inhibit Wnt signaling mediated by Wnt ligand binding to the other domain. Moreover, signaling by Wnt ligands that bind El -2 can be enhanced by inhibitors (e.g., monoclonal antibodies) directed against E3-4, and signaling by Wnt ligands that bind E3-4 can be enhanced by inhibitors directed against El-2.

SUMMARY OF THE INVENTION

[0007] As follows from the Background section, above, there is a need in the art for novel and improved agents that specifically antagonize autocrine Wnt signaling for the treatment of cancers with activated autocrine Wnt signaling, as well as agents which specifically affect (both inhibit and increase) Wnt signaling mediated by a broader range of Wnt ligands. In a preferred embodiment, an inhibitor of the invention inhibits autocrine Wnt signaling mediated by all canonical Wnt ligands.

[0008] Described herein are isolated antibodies and antigen-binding fragments thereof which specifically bind to the low density lipoprotein receptor related (LDLRR) domain of Attorney Docket No.: 27527-0106WO1 low density lipoprotein receptor-related protein (LRP) 6 and/or LRP5. In one embodiment, the isolated antibody or antigen-binding fragment binds to an epitope found within or at least partially within the LDLRR domain consisting of amino acids 1247-1361 of SEQ ID NO: 2 (LDLRR domain of LRP6) and/or amino acid residues 1257-1370 of SEQ ID NO: 8 (LDLRR domain of LRP5).

[0009] In one embodiment, the antibody or antigen-binding fragment has the property of inhibiting autocrine Wnt signaling in a cell. In another embodiment, the antibody or antigen- binding fragment has the property of increasing Wnt ligand stimulation in a cell.

[0010] In one embodiment, the antibody or binding fragment is a monoclonal antibody. In another embodiment, the antibody or antigen-binding fragment comprises a member selected from the group consisting of a single chain antibody, an scFv fragment, a Fab fragment, a F(ab)2 fragment, a humanized antibody, a monovalent antibody, a multispecific antibody, and a chimeric antibody. In one specific embodiment, the antibody or antigen- binding fragment is a bispecific antibody which specifically binds to the LDLRR domain of LRP6 and the LDLRR domain of LRP5.

[001 1] Also described herein are pharmaceutical formulations comprising an effective amount for inhibiting autocrine Wnt signaling (or an effective amount for increasing Wnt ligand stimulation) of an antibody or antigen-binding fragment described herein, and a pharmaceutically acceptable carrier.

[0012] Also described herein is a method of inhibiting growth of a cancer cell, which comprises contacting the cell with an antibody or antigen-binding fragment described herein. In one embodiment, the cancer cell has activated autocrine Wnt signaling. In one embodiment, the cancer cell is selected from the group consisting of breast carcinoma cell, ovarian carcinoma cell, non-small cell lung cancer (NSCLC) cell, sarcoma cell, and glioma cell. In one embodiment, the cancer cell is in a patient. In one embodiment, the method further comprises treating the cancer cell with a chemotherapeutic agent or radiation.

[0013] Also described herein is a method for sensitizing a cancer cell to a treatment, which comprises contacting the cell with an antibody or antigen-binding fragment described herein. In one embodiment, the treatment is a chemotherapy or a radiation treatment. In one embodiment, the cancer cell has activated autocrine Wnt signaling. In one embodiment, the cancer cell is selected from the group consisting of breast carcinoma cell, ovarian carcinoma Attorney Docket No.: 27527-0106WO1 cell, non-small cell lung cancer (NSCLC) cell, sarcoma cell, and glioma cell. In one embodiment, the cancer cell is in a patient.

[0014] Also described herein is a method of inhibiting autocrine Wnt signaling mediated by canonical Wnt ligands in a cancer cell which comprises contacting the cell with an antibody or antigen binding fragment described herein.

[0015] Further described herein is a method of increasing Wnt ligand stimulation in a cell comprising contacting the cell with an antibody or antigen-binding fragment described herein. In one embodiment, the cell is in a patient (e.g., a patient afflicted with a disease treatable by increasing Wnt ligand stimulation, e.g., osteoporosis).

[0016] In a more general aspect, the invention provides a method of inhibiting growth of a cancer cell comprising contacting the cell with an LDLRR inhibitor of LRP6 and/or LRP5. In one embodiment, the cancer cell has activated autocrine Wnt signaling. In one embodiment, the cancer cell is selected from the group consisting of breast carcinoma cell, ovarian carcinoma cell, non-small cell lung cancer (NSCLC) cell, sarcoma cell, and glioma cell. In one embodiment, the cancer cell is in a patient.

[0017] In another aspect, the invention provides a method of sensitizing a cancer cell to a treatment comprising contacting the cell with an LDLRR inhibitor of LRP6 and/or LRP5. In one embodiment, the treatment is a chemotherapy or a radiation treatment. In one embodiment, the cancer cell has activated autocrine Wnt signaling. In one embodiment, the cancer cell is selected from the group consisting of breast carcinoma cell, ovarian carcinoma cell, non-small cell lung cancer (NSCLC) cell, sarcoma cell, and glioma cell. In one embodiment, the cancer cell is in a patient.

[0018] In yet another aspect, the invention provides a method of inhibiting an autocrine Wnt signaling in a cancer cell comprising contacting the cell with an LDLRR inhibitor of LRP6 and/or LRP5. In one embodiment, the cancer cell has activated autocrine Wnt signaling. In one embodiment, the cancer cell is selected from the group consisting of breast carcinoma cell, ovarian carcinoma cell, non-small cell lung cancer (NSCLC) cell, sarcoma cell, and glioma cell. In one embodiment, the cancer cell is in a patient.

[0019] In a further aspect, the invention provides a method of increasing Wnt ligand stimulation in a cell comprising contacting the cell with an agonist of the LDLRR domain of

LRP6 and/or LRP5. In one embodiment, the cell is in a patient (e.g., the patient afflicted with a disease treatable by increasing Wnt ligand stimulation, e.g., osteoporosis). Attorney Docket No.: 27527-0106WO1

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0021] Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is a diagram of the structural and functional domains of the human LRP6 amino acid sequence and their amino acid boundaries. The amino acid numbering corresponds to the numbering of SEQ ID NO: 2; "SP": signal peptide (corresponding to amino acids 1-20); "P I", "P2", "P3" and "P4": β-propellar extracellular domain 1, 2, 3 and 4, respectively (corresponding to amino acids 22-274, 330-581, 633-882, and 935-1194, respectively); "El", "E2", "E3" and "E4": EGF-like module extracellular domain 1, 2, 3 and 4, respectively (corresponding to amino acids 284-325, 590-628, 891-930, and 1205-1244, respectively); "LI", "L2" and "L3": extracellular subdomains within the low density lipoprotein receptor related (LDLRR) domain (corresponding to amino acids 1247-1286, 1287-1323, and 1325-1361, respectively); "TM": transmembrane domain (corresponding to amino acids 1371-1395); "CD": cytoplasmic domain (corresponding to amino acids 1396- 1613).

[0023] Figure 2 shows an amino acid sequence alignment of the relevant domain of human LDLR6 (amino acid residues 139-270 (SEQ ID NO: 12) of the sequence for LDLR6 having GenBank® Accession No. AAP36025.1 (SEQ ID NO: 10) and a fragment containing the LDLRR domain of LRP6 (SEQ ID NO: 1 1) (corresponding to residues 1239-1360 of SEQ ID NO: 2). The one-letter code for each amino acid is shown. Residues in the LDLR domain known to be associated with markedly diminished LDLR function when mutated are Attorney Docket No.: 27527-0106WO1 highlighted as follows: D (aspartic acid) (at position 72); C (cysteine) (at position 73); C (at position 197); S (serine) (at position 226).

[0024] Figures 3A and 3B are bar graphs quantifying TCF luciferase activity normalized to the expression of LRP6 receptor levels determined by the Bradford method in the absence (Fig. 3A) or presence (Fig. 3B) of Wnt3a in 293T cells overexpressing wild-type (WT) LRP6, the LRP6 mutant C1288Y, or the LRP6 mutant S1317P, or in control cells.

[0025] Figure 4 is a bar graph quantifying TCF (TOP) luciferase activity normalized to the expression of LRP6 receptor/tubulin in cells overexpressing wild-type (WT) LRP6 or various LRP6 mutants (W1268L, F1293Y, E1319D, D1315N, S 1317P, E1319K). Also shown are total LRP6 protein expression levels for each of the receptors analyzed.

[0026] Figure 5 shows the effect of Wnt3a on subcellular localization of wild-type (WT) and LDLRR mutant LRP6 (S1317P) in HeLa cells. HeLa cells were transfected with wild- type (WT) or LDLRR mutant LRP6 (S1317P) and 24 hours later treated with Wnt3a for various times. Cells were fixed and stained for LRP6 using flag antibody. Nuclei were stained with DAPI.

[0027] Figure 6 shows the effect of Wnt3a on subcellular localization of wild-type (WT) and various LDLRR mutant LRP6 (W1268L, D1315N, E1319K) in HeLa cells. HeLa cells were transfected with wild-type (WT) or LDLRR mutant LRP6 and 24 hours later treated with Wnt3a for various times. Cells were fixed and stained for LRP6 using flag antibody. Nuclei are stained with DAPI.

[0028] Figure 7 shows the effect of Wnt3a on internalization of LRP6 in 293T cells. Shown is PAGE of immunoprecipitated proteins isolated from 293T cells transfected with various LRP6 constructs (WT, C1288F, D1315N, S1317P, E1319K) and treated with Wnt3a. 50μg of WCL was run on a separate gel and used for input control. E-cadherin and tubulin are used as controls.

DESCRIPTION OF CERTAIN EMBODIMENTS

Overview

[0029] Provided herein are inhibitors that specifically target the low density lipoprotein receptor related (LDLRR) domain of low density lipoprotein receptor-related protein (LRP) 6 Attorney Docket No.: 27527-0106WO1 and/or LRP5 (referred to herein generally as "LDLRR inhibitors"). In a preferred embodiment, an inhibitor of the invention is an antibody or antigen-binding fragment thereof that binds to an epitope comprising an amino acid sequence that is at least partially or that is completely overlapping with an amino acid sequence comprised in the LDLRR domain of LRP6 and/or LRP5. In a preferred embodiment, the inhibitor is specific for a human LDLRR polypeptide.

[0030] In conjunction with the LDLRR inhibitors of the invention (e.g., LDLRR binding antibodies and antigen-binding fragments thereof), the invention also provides methods for inhibiting growth of cancer cells that have activated autocrine Wnt signaling and for sensitizing cancer cells that have activated autocrine Wnt signaling to a treatment (e.g., a chemotherapy and/or radiotherapy) comprising contacting the cell(s) with an LDLRR inhibitor (e.g., antibody or antigen-binding fragment thereof) of the invention. In a preferred embodiment, the cell is a cancer cell such as a breast carcinoma cell, ovarian carcinoma cell, non-small cell lung cancer ( SCLC) cell, sarcoma cell, or a glioma cell.

[0031] In other embodiments, the invention provides agonists of the LDLRR domain in Wnt receptors such as LRP6 and/or LRP5, which increase Wnt ligand stimulation. Such agonists are useful, e.g., for treating diseases such as osteoporosis, where there is evidence that increased Wnt canonical signaling (e.g., in mesenchymal stem cells) would be beneficial.

[0032] The present invention is based in part on the discovery of a new functional domain within the LRP6 receptor, its LDLRR domain, which is conserved among a large family of divergent cell surface receptors, and is presently discovered to be critical to LRP6 function. While not intending to be limited by one particular theory or mechanism of action, it is believed that the present inhibitors inhibit autocrine Wnt signaling through an entirely different mechanism of action that is independent of Wnt ligand interaction with LRP6 at the cell membrane.

[0033] As demonstrated in Example 1, below, single point mutations introduced into the LDLRR domain of LRP6 interfered with signaling in the absence and presence of canonical Wnt3a (an LRP6 ligand), indicating that this domain is critical for Wnt-activated signaling. Further, as discussed above, Wnt inhibitors presently available in the art are directed to other regions of LRP6 (e.g., EGF-like module (E) 1-2 or E3-4 domains), and are only capable of inhibiting signaling mediated by select Wnt ligands that bind to the same E domain, and often undesirably augment Wnt signaling mediated by Wnt ligands that bind to the other E domain. Attorney Docket No.: 27527-0106WO1

The discovery of the functional importance of the LDLRR domain of LRP6, which is not believed to be directly involved in Wnt ligand interactions with LRP6, thus provides a novel and needed target for inhibiting autocrine Wnt signaling mediated by a broader range of Wnt ligands in a broader range of cell types such as different types of cancer cells.

Definitions

[0034] As used herein, the terms "anti-LDLRR antibody", "LDLRR binding antibody" and "antibody which binds to an epitope within the LDLRR domain" are used interchangeably, and refer to an antibody or antigen-binding fragment thereof which binds to an epitope that is at least partially or completely comprised within the LDLRR domain (e.g., of LRP6 and/or LRP5).

[0035] An antibody "epitope" as the term is used herein refers to the specific site on an antigen (e.g., LRP6 or LRP5 LDLRR domain) to which an antibody or antigen-binding fragment thereof binds. Typically, antibody epitopes range from about 5 to about 8 amino acids in length.

[0036] "Fragment", "antigen-binding fragment", "binding fragment" or "antibody fragment" as the terms are used herein in reference to an antibody refer to a polypeptide derived from an antibody polypeptide molecule (e.g., an antibody heavy or light chain polypeptide) that does not comprise a full length antibody polypeptide, but which still comprises at least a portion of a full length antibody polypeptide. Antibody fragments often comprise polypeptides that comprise a cleaved portion of a full length antibody polypeptide, although the term is not limited to such cleaved fragments. Since a fragment, as the term is used herein in reference to an antibody, encompasses fragments that comprise single polypeptide chains derived from antibody polypeptides (e.g., a heavy or light chain antibody polypeptides), it will be understood that an antibody fragment may or may not, on its own, bind an antigen. For example, an antibody fragment may comprise that portion of a heavy chain antibody polypeptide that would be contained in a Fab fragment; such an antibody fragment typically will not typically bind an antigen unless it associates with another antibody fragment derived from a light chain antibody polypeptide (e.g., that portion of a light chain antibody polypeptide that would be contained in a Fab fragment), such that the antigen-binding site is reconstituted. Antibody fragments can include, for example, polypeptides that would be contained in Fab fragments, F(ab')₂ fragments, scFv (single chain Attorney Docket No.: 27527-0106WO1

Fv) fragments, diabodies, linear antibodies, multispecific antibody fragments such as bispecific, trispecific, and multispecific antibodies (e.g., diabodies (e.g., a bispecific antibody which specifically binds to the LDLRR domain of LRP6 and LDLRR domain of LRP5), triabodies, tetrabodies), minibodies, chelating recombinant antibodies, tribodies or bibodies, intrabodies, nanobodies, small modular immunopharmaceuticals (SMIP), binding-domain immunoglobulin fusion proteins, camelized antibodies, and VHH containing antibodies.

[0037] As used herein, the term "isolated" means that the referenced material is removed from the environment in which it is normally found. Thus, an isolated biological material (e.g., antibody, binding fragment or other polypeptide of the invention) can be free of cellular components, i.e., components of the cells in which the material is found or produced. An isolated material may or may not be "purified," as defined herein. The term "purified" as used herein refers to a material (e.g., an antibody or binding fragment) that has been isolated under conditions that detectably reduce or eliminate the presence of other contaminating materials. Contaminants may or may not include native materials from which the purified material has been obtained. A purified material preferably contains less than about 90%, less than about 75%, less than about 50%, less than about 25%, less than about 10%, less than about 5%, or less than about 2% by weight of other components with which it was originally associated.

[0038] "Chimeric antibody" as the term is used herein refers to an antibody that has been engineered to comprise at least one human constant region. For example, one or all the variable regions of the light chain(s) and/or one or all the variable regions the heavy chain(s) of a mouse antibody (e.g., a mouse monoclonal antibody) may each be joined to a human constant region, such as, without limitation an IgGl human constant region. Chimeric antibodies are typically less immunogenic to humans, relative to non-chimeric antibodies, and thus offer therapeutic benefits in certain situations. Those skilled in the art will be aware of chimeric antibodies, and will also be aware of suitable techniques for their generation. See, for example, Cabilly et al, U.S. Pat. No. 4,816,567; Shoemaker et al, U.S. Pat. No. 4,978,775; Beavers et al, U.S. Pat. No. 4,975,369; and Boss et al, U.S. Pat. No. 4,816,397, each of which is incorporated herein by reference in its entirety.

[0039] "Complementarity determining region" or "CDR" as the terms are used herein refer to short polypeptide sequences within the variable region of both heavy and light chain polypeptides that are primarily responsible for mediating specific antigen recognition. CDRs Attorney Docket No.: 27527-0106WO1 have been described by Kabat, et al, J. Biol. Chem. 252, 6609-6616 1977; by Chothia, et al, J. Mol. Biol. 196:901-917, 1987; and by MacCallum, et al., J. Mol. Biol. 262:732-745, 1996, each of which is incorporated herein by reference in its entirety. There are three CDRs (termed CDR1, CDR2, and CDR3) within each V_L and each V_H.

[0040] "Humanized antibody" as the term is used herein refers to an antibody that has been engineered to comprise one or more human framework regions in the variable region together with non-human (e.g., mouse, rat, or hamster) complementarity-determining regions (CDRs) of the heavy and/or light chain. In certain embodiments, a humanized antibody comprises sequences that are entirely human except for the CDR regions. Humanized antibodies are typically less immunogenic to humans, relative to non-humanized antibodies, and thus offer therapeutic benefits in certain situations. Those skilled in the art will be aware of humanized antibodies, and will also be aware of suitable techniques for their generation. See for example, Hwang, W. Y. K., et al, Methods 36:35, 2005; Queen et al, Proc. Natl. Acad. Sci. USA, 86: 10029-10033, 1989; Jones et al, Nature, 321 :522-25, 1986; Riechmann et al, Nature, 332:323-27, 1988; Verhoeyen et al, Science, 239: 1534-36, 1988; Orlandi et al, Proc. Natl. Acad. Sci. USA, 86:3833-37, 1989; U.S. Pat. Nos. 5,225,539; 5,530, 101 ; 5,585,089; 5,693,761 ; 5,693,762; 6, 180,370; and Selick et al, WO 90/07861.

[0041] Within the meaning of the present invention, the term "inhibit" and its grammatical variations are used to refer to any level of reduction in a function or amount. As used herein, the phrase "inhibiting growth of a cancer cell" with respect to an inhibitor (e.g., antibody or antigen-binding fragment thereof) of the invention means the inhibitor causes reduced cancer cell growth. The decrease in cell growth can also be associated with apoptosis of the cell. An increase in apoptosis in a cell population is relative to the incidence of cell death or apoptosis observed in a population of the cell in the absence of an inhibitor of the invention (e.g., more of the cells are induced into the death process as compared to non- exposure to (contact with) the inhibitor. Thus, an effective amount of the inhibitor is introduced into the cancer cell to result in a decrease in cell growth and/or preventing any additional growth. Apoptosis is generally considered to be a form of programmed cell death in which a controlled sequence of events (or program) leads to the elimination of the cell.

[0042] Preferably, cancer cell growth is inhibited (decreased) by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, more preferably by at least about 50%, at least about 60%, at least about 70%, and most preferably by at least Attorney Docket No.: 27527-0106WO1 about 80%, at least about 90%, at least about 95%, at least about 99%, or at least about 100%. Inhibition of cancer cell growth can be determined by any suitable method, known in the art, such as, e.g., ³H-Thymidine incorporation, cell counting, apoptosis assays, e.g., (annexin V staining), or, e.g., for in vivo animal models of cancer, tumor size can be measured before and after treatment with an inhibitor of the invention and/or size or number of metastases can be determined.

[0043] As used herein, the term "activated autocrine Wnt signaling" refers to a situation when Wnt canonical ligands (e.g., Wnt 1, 2, 3, 3A, and 10B) are produced by a cell that contains functional receptors for the same ligands. The term encompasses tumors that contain mutations in intracellular components of Wnt signaling as long as there is a Wnt autocrine component driving tumor proliferation.

[0044] The term "canonical Wnt signaling" refers to a Wnt signaling pathway mediated by β-catenin activation as a transcription factor. As used herein, the term "all canonical Wnt ligands" includes at least Wntl, Wnt2, Wnt3, Wnt3a, Wnt6, Wnt7a, Wnt7b, Wnt9a, WntlOa, and WntlOb (see, Ettenberg, et al, supra), but also includes any additional ligand or ligands determined to bind to a Wnt receptor and activate a β-catenin-dependent Wnt signaling pathway.

[0045] The term "inhibit autocrine Wnt signaling" refers to any decrease in autocrine

Wnt signaling activation as measured, for example, by a decrease in TCF transcriptional reporter activity in a tumor cell line, a decrease in the expression level of a Wnt target gene (e.g., Axin2) in a tumor or tumor cell line, or by a decrease in levels of uncomplexed β- catenin in a tumor or tumor cell line.

[0046] As used herein, the term "uncomplexed β-catenin" refers to β-catenin within a cell that is not bound to a cadherin but is instead free in the cytosol and able to be transported to the nucleus to act in concert with TFC/LEF transcription factors to activate TCF target genes.

[0047] The term "increasing Wnt ligand stimulation" refers to increasing canonical Wnt signaling pathway. An increase in Wnt ligand stimulation can be assessed, for example, by detecting an increase in TCF transcriptional reporter activity or by detecting an increase in levels of uncomplexed β-catenin. Attorney Docket No.: 27527-0106WO1

[0048] Preferred inhibitors of the invention are those that decrease autocrine Wnt signaling in a cell by at least 10%, and more preferably at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or even at least 99%.

[0049] In certain embodiments, agonists (e.g., activating antibodies or antigen-binding fragments thereof) of autocrine Wnt signaling which target the LDLRR domain of LRP6 are provided, as well as methods for increasing autocrine Wnt signaling. The phrase "increasing autocrine Wnt signaling" refers to any measurable increase in autocrine Wnt signaling activation. Preferred agonists of the invention are those that increase autocrine Wnt signaling in a cell by at least 2-fold, at least 3-fold, at least 5-fold, at least 10-fold, or more.

[0050] In certain embodiments, an autocrine Wnt inhibitor of the invention inhibits metastasis of a cancer cell. As used herein, the term "inhibiting growth of a cancer cell" is used to refer to any decrease in the rate of cancer cell growth and/or in the size of a tumor and/or in the rate of local or distant cancer cell metastasis in the presence of an inhibitor of the Wnt signaling pathway as compared to the rate of cancer cell growth and/or in the size of the a tumor and/or in the rate of local or distant metastasis in the absence of such inhibitor. Preferably the inhibition of cancer cell growth is at least about 5%, at least about 10%, at least about 20%, at least about 30%, preferably at least about 40%, at least about 50%, at least about 60%, more preferably at least about 70%, at least about 80%, and most preferably at least about 90%, at least about 95%, and at least about 99%.

[0051] "About" as the term is used herein refers to a range around a given value. Generally, when used in reference to a given value, the term "about" refers to a range of values within +/- 25% of that value, e.g., +/- 20% of that value, +/- 15% of that value, +/- 10% of that value, +/- +/- 9% of that value, +/- 8% of that value, +/- 7% of that value, +/- 6% of that value, 5% of that value, +/- 4% of that value, +/- 3% of that value, +/- 2% of that value, +/- 1% of that value, or less. When used in reference to a given value, the term "about" encompasses the exact value, e.g., as determined within experimental error.

[0052] The terms "polypeptide" and "protein" may be used herein interchangeably to refer to the product (or corresponding synthetic product) encoded by a particular gene, such as LRP6. The term "protein" may also refer specifically to the polypeptide as expressed in cells. A "peptide" refers to a polypeptide often amino acids or less. Attorney Docket No.: 27527-0106WO1

[0053] The term "gene" is used herein to refer to a portion of an RNA or DNA molecule that includes a polypeptide coding sequence operatively associated with expression control sequences. Thus, a gene includes both transcribed and untranscribed regions. The transcribed region may include introns, which are spliced out of the mRNA, and 5'- and 3 '-untranslated (UTR) sequences along with protein coding sequences.

[0054] The term "homologous" as used in the art commonly refers to the relationship between nucleic acid molecules or proteins that possess a "common evolutionary origin," including nucleic acid molecules or proteins within superfamilies and nucleic acid molecules or proteins from different species (Reeck et al, Cell 1987;50:667). Such nucleic acid molecules or proteins have sequence homology, as reflected by their sequence similarity, whether in terms of substantial percent similarity or the presence of specific residues or motifs at conserved positions. As disclosed herein, the LDLRR domain of LRP5 and LRP6 are homologous to each other, and are both homologous to a region of LDLR.

[0055] The terms "percent (%) sequence similarity", "percent (%) sequence identity", and the like, generally refer to the degree of identity or correspondence between different nucleotide sequences of nucleic acid molecules or amino acid sequences of proteins that may or may not share a common evolutionary origin (see Reeck et al, supra). Sequence identity can be determined using any of a number of publicly available sequence comparison algorithms, such as BLAST, FASTA, DNA Strider, GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wisconsin), etc.

[0056] To determine the percent identity between two amino acid sequences or two nucleic acid molecules, the sequences are aligned for optimal comparison purposes. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences. In one embodiment, the two sequences are, or are about, of the same length. The percent identity between two sequences can be determined using techniques similar to those described below, with or without allowing gaps. In calculating percent sequence identity, typically exact matches are counted.

[0057] The determination of percent identity between two sequences can be accomplished using a mathematical algorithm. A non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 1990; 87:2264, modified as in Karlin and Altschul, Proc. Natl. Acad. Sci. USA 1993; 90:5873-5877. Such an algorithm is incorporated into the Attorney Docket No.: 27527-0106WO1

NBLAST and XBLAST programs of Altschul et al, J. Mol. Biol. 1990; 215:403. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, word length = 12, to obtain nucleotide sequences homologous to sequences of the invention. BLAST protein searches can be performed with the XBLAST program, score = 50, word length = 3, to obtain amino acid sequences homologous to protein sequences of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al, Nucleic Acids Res. 1997;25:3389. Alternatively, PSI-Blast can be used to perform an iterated search that detects distant relationship between molecules. See Altschul et al. (1997), supra. When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See ncbi.nlm.nih.gov/BLAST/ on the WorldWideWeb. Another non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, CABIOS 1988; 4: 11-17. Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

[0058] The term "host cell" means any cell of any organism that is selected, modified, transformed, grown, used or manipulated in any way, for the production of a substance by the cell, for example, the expression by the cell of a gene, a DNA or RNA sequence, a protein or an enzyme.

[0059] As used herein, the term "oligonucleotide" refers to a nucleic acid, generally of at least 8, preferably no more than 100 nucleotides, that is hybridizable to a genomic DNA molecule, a cDNA molecule, or an RNA molecule. Oligonucleotides can be labeled, e.g., with 32P-nucleotides or nucleotides to which a label, such as biotin, has been covalently conjugated. In one embodiment, an oligonucleotide of the invention can be used as antisense oligonucleotides to inhibit the expression level or activity of a target nucleic acid molecule (e.g., the LDLRR domain of LRP 5 and/or LPR 6, as described below). Generally, oligonucleotides are prepared synthetically, preferably on a nucleic acid synthesizer. Accordingly, oligonucleotides can be prepared with non-naturally occurring phosphoester analog bonds, such as thioester bonds, etc.

[0060] Generally, a nucleic acid is "antisense" to a nucleic acid molecule when, written in the 5' to 3' direction, it comprises the reverse complement of the corresponding region of the Attorney Docket No.: 27527-0106WO1 target nucleic acid. "Antisense compounds" are also often defined in the art to comprise the further limitation of, once hybridized to a target, being able to modulate expression levels, or function of the target compound. The term "antisense" nucleic acid molecule or oligonucleotide is used in the present disclosure to refer to a single stranded (ss) or double stranded (ds) nucleic acid molecule, which may be DNA, RNA, a DNA-RNA chimera, or a derivative thereof, which, upon hybridizing under physiological conditions with complementary bases in an RNA or DNA molecule of interest, inhibits the expression level or activity of the target molecule. As presently used, "antisense" broadly includes RNA-RNA interactions, RNA-DNA interactions, and RNase-H mediated arrest.

[0061] The term "RNA interference" or "RNAi" refers to the ability of double stranded RNA (dsRNA) to suppress the expression level or activity of a specific target nucleic acid molecule (e.g., the nucleic acid encoding the LDLRR domain of LRP5 and/or LRP6) of interest in a homology-dependent manner. While not intended to be bound by a particular theory or mechanism, it is currently believed that RNA interference acts post- transcriptionally by targeting RNA molecules for degradation. RNA interference commonly involves the use of dsRNAs that are greater than 500 bp; however, it can also be mediated through small interfering RNAs (siRNAs) or small hairpin RNAs (shRNAs), which can be 10 or more nucleotides in length and are typically 18 or more nucleotides in length. For reviews, see Bosner and Labouesse, Nature Cell Biol. 2000; 2:E31-E36 and Sharp and Zamore, Science 2000; 287:2431-2433.

[0062] As used herein, the term "triplex-forming oligonucleotide" or "triple helix forming oligonucleotide" or "TFO" refers to molecules that bind in the major groove of duplex DNA and by so doing produce triplex structures. TFOs bind to the purine-rich strand of the duplex through Hoogsteen or reverse Hoogsteen hydrogen bonding. They exist in two sequence motifs, either pyrimidine or purine. According to the present invention, TFOs can be employed as an alternative to antisense oligonucleotides to inhibit nucleic acid expression level. TFOs have also been shown to produce mutagenic events, even in the absence of tethered mutagens. TFOs can increase rates of recombination between homologous sequences in close proximity. TFOs of the present invention may be conjugated to active molecules (for a review, see Casey and Glazer, Prog. Nucleic Acid. Res. Mol. Biol. 2001 ; 67: 163-92).

[0063] The term "ribozyme" is used herein to refer to a catalytic RNA molecule capable of mediating catalytic reactions on (e.g., cleaving) RNA substrates (e.g., RNA encoding the Attorney Docket No.: 27527-0106WO1

LDLRR domain of LRP5 and/or LRP6) ). Ribozyme specificity is dependent on complementary RNA-RNA interactions (for a review, see Cech and Bass, Annu. Rev. Biochem. 1986; 55:599-629). Two types of ribozymes, hammerhead and hairpin, have been described. Each has a structurally distinct catalytic center. The present invention contemplates the use of ribozymes designed on the basis of the LRP 5 and LRP 6 LDLRR domain-encoding nucleic acid molecules of the invention to induce catalytic reaction (e.g., cleavage) of the target nucleic acid, thereby modulating (e.g., inhibiting) the expression of the LRP5 and/or LRP6 molecule. Ribozyme technology is described further in Intracellular Ribozyme Applications: Principals and Protocols, Rossi and Couture ed., Horizon Scientific Press, 1999.

[0064] The term "nucleic acid hybridization" refers to the pairing of complementary strands of nucleic acids, including triple-stranded nucleic acid hybridization. The mechanism of pairing involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases (nucleobases) of the strands of nucleic acids. For example, adenine and thymine are complementary nucleobases that pair through the formation of hydrogen bonds. Hybridization can occur under varying circumstances. Nucleic acid molecules are "hybridizable" to each other when at least one strand of one nucleic acid molecule can form hydrogen bonds with the complementary bases of another nucleic acid molecule under defined stringency conditions. Stringency of hybridization is determined, e.g., by (i) the temperature at which hybridization and/or washing is performed, and (ii) the ionic strength and (iii) concentration of denaturants such as formamide of the hybridization and washing solutions, as well as other parameters. Hybridization requires that the two strands contain substantially complementary sequences. Depending on the stringency of hybridization, however, some degree of mismatches may be tolerated. Under "low stringency" conditions, a greater percentage of mismatches are tolerable (i.e., will not prevent formation of an anti- parallel hybrid). See Molecular Biology of the Cell, Alberts et al, 3rd ed., New York and London: Garland Publ, 1994, Ch. 7.

[0065] Typically, hybridization of two strands at high stringency requires that the sequences exhibit a high degree of complementarity over an extended portion of their length. Examples of high stringency conditions include: hybridization to filter-bound DNA in 0.5 M NaHP04, 7% SDS, 1 mM EDTA at 65°C, followed by washing in O. lx SSC/0.1% SDS (where lx SSC is 0.15 M NaCl, 0.15 M Na citrate) at 68°C or for oligonucleotide inhibitors Attorney Docket No.: 27527-0106WO1 washing in 6xSSC/0.5% sodium pyrophosphate at about 37°C (for 14 nucleotide-long oligos), at about 48°C (for about 17 nucleotide-long oligos), at about 55°C (for 20 nucleotide-long oligos), and at about 60°C (for 23 nucleotide-long oligos).

[0066] Conditions of intermediate or moderate stringency (such as, for example, an aqueous solution of 2xSSC at 65°C; alternatively, for example, hybridization to filter-bound DNA in 0.5 M NaHP04, 7% SDS, 1 mM EDTA at 65°C followed by washing in 0.2 x SSC/0.1% SDS at 42°C) and low stringency (such as, for example, an aqueous solution of 2xSSC at 55°C), require correspondingly less overall complementarity for hybridization to occur between two sequences. Specific temperature and salt conditions for any given stringency hybridization reaction depend on the concentration of the target DNA or RNA molecule and length and base composition of the probe, and are normally determined empirically in preliminary experiments, which are routine (see Southern, J. Mol. Biol. 1975;98:503; Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 2, ch. 9.50, CSH Laboratory Press, 1989; Ausubel et al. (eds.), 1989, Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc., and John Wiley & Sons, Inc., New York, at p. 2.10.3).

[0067] As used herein, the term "standard hybridization conditions" refers to hybridization conditions that allow hybridization of two nucleotide molecules having at least 50% sequence identity. According to a specific embodiment, hybridization conditions of higher stringency may be used to allow hybridization of only sequences having at least 75% sequence identity, at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 99% sequence identity.

[0068] The phrases "specifically hybridizable" or "hybridizes specifically to" and other similar phrases refer to the association of a nucleic acid with a target nucleic acid molecule (e.g., the nucleic acid encoding the LDLRR domain of LRP5 and/or LRP6), resulting in interference with the expression and/or function of the target molecule (e.g. by altering the activity, disrupting the function, or modulating the expression level of the LRP5 and/or LRP6). Where a nucleic acid is "specifically hybridizable," to a target nucleic acid molecule (e.g., the nucleic acid encoding the LDLRR domain of LRP5 and/or LRP6) , there is a sufficient degree of complementarity to avoid non-specific binding of the nucleic acid to nucleic acid sequences other than the intended target under conditions in which specific hybridization is desired (e.g. under physiological conditions in the case of in vivo assays or Attorney Docket No.: 27527-0106WO1 therapeutic treatment, and under standard assay conditions in the case of in vitro assays). The sequence of the nucleic acid need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable. Moreover, the nucleic acid may hybridize over one or more segments of the target nucleic acid such that intervening or adjacent segments are not involved in the hybridization (e.g., a bulge, a loop structure or a hairpin structure).

[0069] Nucleic acid molecules that "hybridize" to a target nucleic acid molecule (e.g., the nucleic acid encoding the LDLRR domain of LRP5 and/or LRP6) of the present invention may be of any length. In one embodiment, such nucleic acid molecules are at least 10, at least 15, at least 20, or at least 25 nucleotides in length. In another embodiment, nucleic acid molecules that hybridize are of about the same length as the particular nucleic acid molecule (e.g., the nucleic acid encoding the LDLRR domain of LRP5 and/or LRP6) targeted by the nucleic acid molecule. In another embodiment, more than one inhibitor can be assembled into one vector of about more than 25 nucleotides.

[0070] The terms "individual", "subject", "patient" and "animal" are used interchangeably to refer to any animal (including humans) that can develop a tumor having an activated autocrine Wnt signaling pathway.

[0071] As used herein, the term "small molecules" refers to organic compounds, whether naturally-occurring or artificially created (e.g., via chemical synthesis) that have relatively low molecular weight and are typically non-peptide molecules. Typically, small molecules have a molecular weight of less than about 1500 g/mol.

[0072] As used herein, the terms "treat", "treatment", and the like mean to relieve or alleviate at least one symptom associated with such condition, or to slow or reverse the progression of such condition. For example, in relation to cancer, the term "treat" may mean to relieve or alleviate at least one symptom selected from the group consisting of cancer cell growth, metastasis, sensitivity of cancer cells to treatments such as chemotherapy, radiation therapy, thermotherapy, etc. The term "treat" also denotes to arrest, delay the onset (i.e., the period prior to clinical manifestation of a disease) and/or reduce the risk of developing or worsening a disease. In a specific embodiment, treating cancer comprises inhibiting metastasis.

[0073] As used herein the term "therapeutically effective" applied to dose or amount refers to that quantity of a compound or composition (e.g., pharmaceutical composition) that is sufficient to result in a desired activity upon administration to an animal in need thereof. Attorney Docket No.: 27527-0106WO1

Thus, within the context of the present invention, the term "therapeutically effective amount" refers to that quantity of a compound or composition that is sufficient to treat at least one symptom of a cancer, such as but not limited to cancer cell growth, proliferation, tumor growth, resistance to apoptosis, and angiogenesis, and/or to inhibit metastasis of a cancer cell. When a combination of active ingredients is administered, an effective amount of the combination may or may not include amounts of each ingredient that would have been effective if administered individually. A "prophylactically effective amount" is an amount of a pharmaceutical composition that, when administered to a subject, will have the intended prophylactic effect, e.g., preventing or delaying the onset (or recurrence) of cancer, or reducing the likelihood of the onset (or recurrence) of cancer or cancer symptoms. The full prophylactic effect does not necessarily occur by administration of one dose, and may occur only after administration of a series of doses. Thus, a prophylactically effective amount may be administered in one or more administrations.

[0074] As used herein, the term "cancer" includes all cancers in which the cancer cells have activated autocrine Wnt signaling, including tumors that contain mutations in intracellular components of Wnt signaling as long as there is a Wnt autocrine component driving tumor proliferation. Non-limiting examples of preferred cancer cells having activated autocrine Wnt signaling include, e.g., ovarian and breast carcinoma cells, non-small cell lung cancer (NSCLC) cells, sarcoma cells, and glioma cells. Methods for determining whether a cancer cell has activated autocrine Wnt signaling are described in detail in PCT Publication No. WO 2010/121269.

[0075] The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable when administered to a human. Preferably, as used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

[0076] The term "carrier" applied to pharmaceutical compositions of the invention refers to a diluent, excipient, or vehicle with which a compound (e.g., an anti-LDLRR antibody or antigen-binding fragment thereof) is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water or aqueous solution, saline solutions, and aqueous dextrose and glycerol solutions are preferably Attorney Docket No.: 27527-0106WO1 employed as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin, 18th Edition.

[0077] The terms "polynucleotide" or "nucleotide sequence" mean a series of nucleotide bases (also called "nucleotides") in DNA and RNA, and mean any chain of two or more nucleotides. A nucleotide sequence can carry genetic information, including the information used by cellular machinery to make proteins and enzymes. These terms include double or single stranded genomic and cDNA, RNA, any synthetic and genetically manipulated polynucleotide, and both sense and anti-sense polynucleotide. This includes single- and double-stranded molecules, i.e., DNA -DNA, DNA-RNA and RNA-RNA hybrids.

[0078] The polynucleotides herein may be flanked by natural regulatory (expression control) sequences, or may be associated with heterologous sequences, including promoters, internal ribosome entry sites (IRES) and other ribosome binding site sequences, enhancers, response elements, suppressors, signal sequences, polyadenylation sequences, introns, 5'- and 3'- non-coding regions, and the like. The nucleic acids may also be modified by many means known in the art. Non-limiting examples of such modifications include methylation, "caps", substitution of one or more of the naturally occurring nucleotides with an analog, and internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoroamidates, carbamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.). Polynucleotides may contain one or more additional covalently linked moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), intercalators (e.g., acridine, psoralen, etc.), chelators (e.g., metals, radioactive metals, iron, oxidative metals, etc.), and alkylators. The polynucleotides may be derivatized by formation of a methyl or ethyl phosphotriester or an alkyl phosphoramidate linkage. Furthermore, the polynucleotides herein may also be modified with a label capable of providing a detectable signal, either directly or indirectly. Exemplary labels include radioisotopes, fluorescent molecules, biotin, and the like.

[0079] The terms "express" and "expression" mean allowing or causing the information in a gene or DNA sequence to become manifest, for example, producing an non-coding (untranslated) RNA or a protein by activating the cellular functions involved in transcription and translation of a corresponding gene or DNA sequence. A DNA sequence is expressed in or by a cell to form an "expression product" such as RNA or a protein. The expression Attorney Docket No.: 27527-0106WO1 product itself, e.g. the resulting RNA or protein, may also be said to be "expressed" by the cell.

[0080] The terms "vector", "cloning vector" and "expression vector" mean the vehicle by which a DNA or RNA sequence can be introduced into a host cell, so as to transform the host and clone the vector or promote expression of the introduced sequence. Vectors include plasmids, cosmids, phages, viruses, etc. Vectors may further comprise selectable markers.

[0081] A sequence "encoding" an expression product, such as a polypeptide or protein (e.g., antibody), is a minimum nucleotide sequence that, when expressed, results in the production of that the polypeptide or protein.

[0082] In accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 1989 (herein "Sambrook et al, 1989"); DNA Cloning: A Practical Approach, Volumes I and II (D.N. Glover ed. 1985); Oligonucleotide Synthesis (M.J. Gait ed. 1984); Nucleic Acid Hybridization [B.D. Hames & S.J. Higgins eds.

(1985) ]; Transcription And Translation [B.D. Hames & S.J. Higgins, eds. (1984)]; Animal Cell Culture [R.I. Freshney, ed. (1986)]; Immobilized Cells And Enzymes [IRL Press,

(1986) ]; B. Perbal, A Practical Guide To Molecular Cloning (1984); Ausubel, F.M. et al. (eds.). Current Protocols in Molecular Biology. John Wiley & Sons, Inc., 1994. These techniques include site directed mutagenesis as described in Kunkel, Proc. Natl. Acad. Sci. USA 82: 488- 492 (1985), U. S. Patent No. 5,071, 743, Fukuoka et al. , Biochem. Biophys. Res. Commun. 263 : 357-360 (1999); Kim and Maas, BioTech. 28: 196-198 (2000); Parikh and Guengerich, BioTech. 24: 4 28-431 (1998); Ray and Nickoloff, BioTech. 13 : 342-346 (1992); Wang et al, BioTech. 19: 556-559 (1995); Wang and Malcolm, BioTech. 26: 680- 682 (1999); Xu and Gong, BioTech. 26: 639-641 (1999), U.S. Patents Nos. 5,789, 166 and 5,932, 419, Hogrefe, Strategies 14. 3 : 74-75 (2001), U. S. Patents Nos. 5,702,931, 5,780,270, and 6,242,222, Angag and Schutz, Biotech. 30: 486-488 (2001), Wang and Wilkinson, Biotech. 29: 976-978 (2000), Kang et al, Biotech. 20: 44-46 (1996), Ogel and McPherson, Protein Engineer. 5: 467-468 (1992), Kirsch and Joly, Nuc. Acids. Res. 26: 1848-1850 (1998), Rhem and Hancock, J. Bacteriol. 178: 3346-3349 (1996), Boles and Miogsa, Curr. Genet. 28: 197-198 (1995), Barrenttino et al, Nuc. Acids. Res. 22: 541-542 (1993), Tessier Attorney Docket No.: 27527-0106WO1 and Thomas, Meths. Molec. Biol. 57: 229-237, and Pons et al, Meth. Molec. Biol. 67: 209- 218.

Structure of LRP6 and LRP5

[0083] LRP6 is a single-pass transmembrane protein. Human LRP6 shares 71% sequence identity with human LRP5. The extracellular domain of LRP6 and LRP5 is comprised of four alternating epidermal growth factor (EGF) YWTD propeller domains, and three cysteine-rich LDL domains (LDLRR).

[0084] The human gene and amino acid sequences of LRP6 are known and have been described in the literature. The cDNA sequence of human LRP6 has GenBank® Accession Number BC 126405 and the following nucleic acid sequence:

1 tcgccggtga gagaagagaa cgcgagaagg gaagatgggg gccgtcctga ggagcctcct 61 ggcctgcagc ttctgtgtgc tcctgagagc ggcccctttg ttgctttatg caaacagacg 121 ggacttgcga ttggttgatg ctacaaatgg caaagagaat gctacgattg tagttggagg 181 cttggaggat gcagctgcgg tggactttgt gtttagtcat ggcttgatat actggagtga

241 tgtcagcgaa gaagccatta aacgaacaga atttaacaaa actgagagtg tgcagaatgt 301 tgttgtttct ggattattgt cccccgatgg gctggcatgt gattggcttg gagaaaaatt

361 gtactggaca gattctgaaa ctaatcggat tgaagtttct aatttagatg gatctttacg

421 aaaagtttta ttttggcaag agttggatca acccagagct attgccttag atccttcaag

481 tgggttcatg tactggacag actggggaga agtgccaaag atagaacgtg ctggaatgga 541 tggttcaagt cgcttcatta taataaacag tgaaatttac tggccaaatg gactgacttt

601 ggattatgaa gaacaaaagc tttattgggc agatgcaaaa cttaatttca tccacaaatc

661 aaatctggat ggaacaaatc ggcaggcagt ggttaaaggt tcccttccac atccttttgc 721 cttgacgtta tttgaggaca tattgtactg gactgactgg agcacacact ccattttggc

781 ttgcaacaag tatactggtg agggtctgcg tgaaatccat tctgacatct tctctcccat

841 ggatatacat gccttcagcc aacagaggca gccaaatgcc acaaatccat gtggaattga 901 caatgggggt tgttcccatt tgtgtttgat gtctccagtc aagccttttt atcagtgtgc

961 ttgccccact ggggtcaaac tcctggagaa tggaaaaacc tgcaaagatg gtgccacaga 1021 attattgctt ttagctcgaa ggacagactt gagacgcatt tctttggata caccagattt

1081 tacagacatt gttctgcagt tagaagacat ccgtcatgcc attgccatag attacgatcc

1141 tgtggaaggc tacatctact ggactgatga tgaagtgagg gccatacgcc gttcatttat 1201 agatggatct ggcagtcagt ttgtggtcac tgctcaaatt gcccatcctg atggtattgc

1261 tgtggactgg gttgcacgaa atctttattg gacagacact ggcactgatc gaatagaagt Attorney Docket No.: 27527-0106WO1

1321 gacaaggctc aatgggacca tgaggaagat cttgatttca gaggacttag aggaaccccg

1381 ggctattgtg ttagatccca tggttgggta catgtattgg actgactggg gagaaattcc

1441 gaaaattgag cgagcagctc tggatggttc tgaccgtgta gtattggtta acacttctct

1501 tggttggcca aatggtttag ccttggatta tgatgaaggc aaaatatact ggggagatgc

1561 caaaacagac aagattgagg ttatgaatac tgatggcact gggagacgag tactagtgga

1621 agacaaaatt cctcacatat ttggatttac tttgttgggt gactatgttt actggactga

1681 ctggcagagg cgtagcattg aaagagttca taaacgaagt gcagagaggg aagtgatcat

1741 agatcagctg cctgacctca tgggcctaaa ggctacaaat gttcatcgag tgattggttc

1801 caacccctgt gctgaggaaa acgggggatg tagccatctc tgcctctata gacctcaggg

1861 ccttcgctgt gcttgcccta ttggctttga actcatcagt gacatgaaga cctgcattgt

1921 cccagaggct ttccttttgt tttcacggag agcagatatc agacgaattt ctctggaaac

1981 aaacaataat aatgtggcta ttccactcac tggtgtcaaa gaagcttctg ctttggattt

2041 tgatgtgaca gacaaccgaa tttattggac tgatatatca ctcaagacca tcagcagagc

2101 ctttatgaat ggcagtgcac tggaacatgt ggtagaattc ggcttagatt atccagaagg

2161 catggcagta gactggcttg ggaagaactt gtactgggca gacacaggaa cgaatcgaat

2221 tgaggtgtca aagttggatg ggcagcaccg acaagttttg gtgtggaaag acctagatag

2281 tcccagagct ctcgcgttgg accctgccga aggatttatg tattggactg aatggggtgg

2341 aaaacctaag atagacagag ctgcaatgga tggaagtgaa cgtactacct tagttccaaa

2401 tgtggggcgg gcaaacggcc taactattga ttatgctaaa aggaggcttt attggacaga

2461 cctggacacc aacttaatag aatcttcaaa tatgcttggg ctcaaccgtg aagttatagc

2521 agatgacttg cctcatcctt ttggcttaac tcagtaccaa gattatatct actggacgga

2581 ctggagccga cgcagcattg agcgtgccaa caaaaccagt ggccaaaacc gcaccatcat 2641 tcagggccat ttggattatg tgatggacat cctcgtcttt cactcatctc gacagtcagg

2701 gtggaatgaa tgtgcttcca gcaatgggca ctgctcccac ctctgcttgg ctgtgccagt

2761 tgggggtttt gtttgtggat gccctgccca ctactctctt aatgctgaca acaggacttg

2821 tagtgctcct acgactttcc tgctcttcag tcaaaagagt gccatcaacc gcatggtgat

2881 tgatgaacaa cagagccccg acatcatcct tcccatccac agccttcgga atgtccgggc

2941 cattgactat gacccactgg acaagcaact ctattggatt gactcacgac aaaacatgat

3001 ccgaaaggca caagaagatg gcagccaggg ctttactgtg gttgtgagct cagttccgag

3061 tcagaacctg gaaatacaac cctatgacct cagcattgat atttacagcc gctacatcta

3121 ctggacttgt gaggctacca atgtcattaa tgtgacaaga ttagatggga gatcagttgg

3181 agtggtgctg aaaggcgagc aggacagacc tcgagccgtt gtggtaaacc cagagaaagg 3241 gtatatgtat tttaccaatc ttcaggaaag gtctcctaaa attgaacggg ctgctttgga Attorney Docket No.: 27527-0106WO1

3301 tgggacagaa cgggaggtcc tctttttcag tggcttaagt aaaccaattg ctttagccct 3361 tgatagcagg ctgggcaagc tcttttgggc tgattcagat ctccggcgaa ttgaaagcag 3421 tgatctctca ggtgctaacc ggatagtatt agaagactcc aatatcttgc agcctgtggg

3481 acttactgtg tttgaaaact ggctctattg gattgataaa cagcagcaaa tgattgaaaa

3541 aattgacatg acaggtcgag agggtagaac caaagtccaa gctcgaattg cccagcttag 3601 tgacattcat gcagtaaagg agctgaacct tcaagaatac agacagcacc cttgtgctca 3661 ggataatggt ggctgttcac atatttgtct tgtaaagggg gatggtacta caaggtgttc

3721 ttgccccatg cacctggttc tacttcaaga tgagctatca tgtggagaac ctccaacatg

3781 ttctcctcag cagtttactt gtttcacggg ggaaattgac tgtatccctg tggcttggcg

3841 gtgcgatggg tttactgaat gtgaagacca cagtgatgaa ctcaattgtc ctgtatgctc

3901 agagtcccag ttccagtgtg ccagtgggca gtgtattgat ggtgccctcc gatgcaatgg 3961 agatgcaaac tgccaggaca aatcagatga gaagaactgt gaagtgcttt gtttaattga 4021 tcagttccgc tgtgccaatg gtcagtgcat tggaaagcac aagaagtgtg atcataatgt 4081 ggattgcagt gacaagtcag atgaactgga ttgttatccg actgaagaac cagcaccaca 4141 ggccaccaat acagttggtt ctgttattgg cgtaattgtc accatttttg tgtctggaac

4201 tgtatacttt atctgccaga ggatgttgtg tccacgtatg aagggagatg gggaaactat 4261 gactaatgac tatgtagttc atggaccagc ttctgtgcct cttggttatg tgccacaccc

4321 aagttctttg tcaggatctc ttccaggaat gtctcgaggt aaatcaatga tcagctccct

4381 cagtatcatg gggggaagca gtggaccccc ctatgaccga gcccatgtta caggagcatc 4441 atcaagtagt tcttcaagca ccaaaggcac ttacttccct gcaattttga accctccacc

4501 atccccagcc acagagcgat cacattacac tatggaattt ggatattctt caaacagtcc 4561 ttccactcat aggtcataca gctacaggcc atatagctac cggcactttg caccccccac 4621 cacaccctgc agcacagatg tttgtgacag tgactatgct cctagtcgga gaatgacctc 4681 agtggcaaca gccaagggct ataccagtga cttgaactat gattcagaac ctgtgccccc 4741 acctcccaca ccccgaagcc aatacttgtc agcagaggag aactatgaaa gctgcccacc 4801 ttctccatac acagagagga gctattctca tcacctctac ccaccgccac cctctccctg 4861 tacagactcc tcctgaggag gggccctcct cctctgactg cctccaacg (SEQ ID NO: 1).

[0085] The amino acid sequence of human LRP6 has GenBank® Accession Number AAI26406 and the following amino acid sequence:

1 mgavlrslla csfcvllraa plllyanrrd lrlvdatngk enatiwggl edaaavdfvf 61 shgliywsdv seeaikrtef nktesvqnvv vsgllspdgl acdwlgekly wtdsetnrie 121 vsnldgslrk vlfwqeldqp raialdpssg fmywtdwgev pkieragmdg ssrfiiinse Attorney Docket No.: 27527-0106WO1

181 iywpngltld yeeqklywad aklnfihksn ldgtnrqavv kgslphpfal tlfedilywt 241 dwsthsilac nkytgeglre ihsdifspmd ihafsqqrqp natnpcgidn ggcshlclms 301 pvkpfyqcac ptgvklleng ktckdgatel lllarrtdlr risldtpdft divlqledir

361 haiaidydpv egyiywtdde vrairrsfid gsgsqfvvta qiahpdgiav dwvarnlywt 421 dtgtdrievt rlngtmrkil isedleepra ivldpmvgym ywtdwgeipk ieraaldgsd 481 rvvlvntslg wpnglaldyd egkiywgdak tdkievmntd gtgrrvlved kiphifgftl 541 lgdyvywtdw qrrsiervhk rsaereviid qlpdlmglka tnvhrvigsn pcaeenggcs 601 hlclyrpqgl rcacpigfel isdmktcivp eafllfsrra dirrisletn nnnvaipltg

661 vkeasaldfd vtdnriywtd islktisraf mngsalehw efgldypegm avdwlgknly 721 wadtgtnrie vskldgqhrq vlvwkdldsp ralaldpaeg fmywtewggk pkidraamdg 781 serttlvpnv grangltidy akrrlywtdl dtnliessnm lglnreviad dlphpfgltq

841 yqdyiywtdw srrsierank tsgqnrtiiq ghldyvmdil vfhssrqsgw necassnghc 901 shlclavpvg gfvcgcpahy slnadnrtcs apttfllfsq ksainrmvid eqqspdiilp 961 ihslrnvrai dydpldkqly widsrqnmir kaqedgsqgftvwssvpsq nleiqpydls 1021 idiysryiyw tceatnvinv trldgrsvgv vlkgeqdrpr awvnpekgy myftnlqers 1081 pkieraaldg terevlffsg lskpialald srlgklfwad sdlrriessd lsganrivle

1141 dsnilqpvgl tvfenwlywi dkqqqmieki dmtgregrtk vqariaqlsd ihavkelnlq 1201 eyrqhpcaqd nggcshiclv kgdgttrcsc pmhlvllqde lscgepptcs pqqftcftge 1261 idcipvawrc dgftecedhs delncpvcse sqfqcasgqc idgalrcngd ancqdksdek 1321 ncevlclidq frcangqcig khkkcdhnvd csdksdeldc ypteepapqa tntvgsvigv 1381 ivtifvsgtv yficqrmlcp rmkgdgetmt ndywhgpas vplgyvphps slsgslpgms 1441 rgksmissls imggssgppy drahvtgass ssssstkgty fpailnppps patershytm 1501 efgyssnsps thrsysyrpy syrhfapptt pcstdvcdsd yapsrrmtsv atakgytsdl 1561 nydsepvppp ptprsqylsa eenyescpps pytersyshh lyppppspct dss (SEQ ID NO

[0086] The structural and functional domains of human LRP6 are depicted in the schematic diagram shown in Figure 1. Residues 1-21 of the amino acid sequence of human LRP6 (numbers corresponding to the numbering of the sequence given above in SEQ ID NO: 2) correspond to the signal sequence, residues 1371-1395 correspond to the transmembrane domain, and residues 1396-1613 correspond to the cytoplasmic domain.

[0087] The LRP6 extracellular domain comprises four tandem β-propeller-EGF-like domain (PE) pairs that harbor binding sites for Wnt morphogens and their antagonists (Chen, Attorney Docket No.: 27527-0106WO1

S. et al. (2011) Dev. Cell; 21: 848-861), having the following amino acid boundaries, the numbering of which corresponds to the numbering of the amino acid sequence given above (SEQ ID NO: 2):

[0088] The LDLRR domain consists of domains L1-L3 (spanning residues 1247-1361 of SEQ ID NO: 2), which have the following amino acid boundaries (numbering corresponds to the numbering of the sequence given in SEQ ID NO: 2, above): LI: residues 1247-1286; L2: residues 1287-1323; and L3: residues 1325-1361. Thus, the entire LDLRR domain has the following amino acid sequence: PTCSPQQFTCFTGEIDCIPVAWRCDGFTECEDHSDELN CPVCSESQFQCASGQCIDGALRCNGDANCQDKSDEKNCEVLCLIDQFRCANGQCIGK HKKCDHNVDCSDKSDELDCY (SEQ ID NO: 3), the LI domain has the amino acid sequence: PTCSPQQFTCFTGEIDCIPVAWRCDGFTECEDHSDELNCP (SEQ ID NO: 4); the L2 domain has the amino acid sequence:

VCSESQFQCASGQCIDGALRCNGDANCQDKSDEKNCE (SEQ ID NO: 5); and the L3 domain has the amino acid sequence:

LCLIDQFRCANGQCIGKHKKCDHNVDCSDKSDELDCY (SEQ ID NO: 6).

[0089] The human LRP5 nucleic acid sequence has GenBank® Accession No. NM_002335 and the following sequence:

1 cgcgcgagga gccgccgccg ccgcgccatg gagcccgagt gagcgcggcg cgggcccgtc 61 cggccgccgg acaacatgga ggcagcgccg cccgggccgc cgtggccgct gctgctgctg 121 ctgctgctgc tgctggcgct gtgcggctgc ccggcccccg ccgcggcctc gccgctcctg 181 ctatttgcca accgccggga cgtacggctg gtggacgccg gcggagtcaa gctggagtcc 241 accatcgtgg tcagcggcct ggaggatgcg gccgcagtgg acttccagtt ttccaaggga 301 gccgtgtact ggacagacgt gagcgaggag gccatcaagc agacctacct gaaccagacg

361 ggggccgccg tgcagaacgt ggtcatctcc ggcctggtct ctcccgacgg cctcgcctgc 421 gactgggtgg gcaagaagct gtactggacg gactcagaga ccaaccgcat cgaggtggcc Attorney Docket No.: 27527-0106WO1

481 aacctcaatg gcacatcccg gaaggtgctc ttctggcagg accttgacca gccgagggcc

541 atcgccttgg accccgctca cgggtacatg tactggacag actggggtga gacgccccgg

601 attgagcggg cagggatgga tggcagcacc cggaagatca ttgtggactc ggacatttac

661 tggcccaatg gactgaccat cgacctggag gagcagaagc tctactgggc tgacgccaag

721 ctcagcttca tccaccgtgc caacctggac ggctcgttcc ggcagaaggt ggtggagggc

781 agcctgacgc accccttcgc cctgacgctc tccggggaca ctctgtactg gacagactgg

841 cagacccgct ccatccatgc ctgcaacaag cgcactgggg ggaagaggaa ggagatcctg 901 agtgccctct actcacccat ggacatccag gtgctgagcc aggagcggca gcctttcttc

961 cacactcgct gtgaggagga caatggcggc tgctcccacc tgtgcctgct gtccccaagc

1021 gagcctttct acacatgcgc ctgccccacg ggtgtgcagc tgcaggacaa cggcaggacg

1081 tgtaaggcag gagccgagga ggtgctgctg ctggcccggc ggacggacct acggaggatc 1141 tcgctggaca cgccggactt caccgacatc gtgctgcagg tggacgacat ccggcacgcc

1201 attgccatcg actacgaccc gctagagggc tatgtctact ggacagatga cgaggtgcgg

1261 gccatccgca gggcgtacct ggacgggtct ggggcgcaga cgctggtcaa caccgagatc 1321 aacgaccccg atggcatcgc ggtcgactgg gtggcccgaa acctctactg gaccgacacg 1381 ggcacggacc gcatcgaggt gacgcgcctc aacggcacct cccgcaagat cctggtgtcg 1441 gaggacctgg acgagccccg agccatcgca ctgcaccccg tgatgggcct catgtactgg

1501 acagactggg gagagaaccc taaaatcgag tgtgccaact tggatgggca ggagcggcgt 1561 gtgctggtca atgcctccct cgggtggccc aacggcctgg ccctggacct gcaggagggg 1621 aagctctact ggggagacgc caagacagac aagatcgagg tgatcaatgt tgatgggacg

1681 aagaggcgga ccctcctgga ggacaagctc ccgcacattt ttgggttcac gctgctgggg

1741 gacttcatct actggactga ctggcagcgc cgcagcatcg agcgggtgca caaggtcaag

1801 gccagccggg acgtcatcat tgaccagctg cccgacctga tggggctcaa agctgtgaat

1861 gtggccaagg tcgtcggaac caacccgtgt gcggacagga acggggggtg cagccacctg 1921 tgcttcttca caccccacgc aacccggtgt ggctgcccca tcggcctgga gctgctgagt

1981 gacatgaaga cctgcatcgt gcctgaggcc ttcttggtct tcaccagcag agccgccatc

2041 cacaggatct ccctcgagac caataacaac gacgtggcca tcccgctcac gggcgtcaag

2101 gaggcctcag ccctggactt tgatgtgtcc aacaaccaca tctactggac agacgtcagc

2161 ctgaagacca tcagccgcgc cttcatgaac gggagctcgg tggagcacgt ggtggagttt

2221 ggccttgact accccgaggg catggccgtt gactggatgg gcaagaacct ctactgggcc

2281 gacactggga ccaacagaat cgaagtggcg cggctggacg ggcagttccg gcaagtcctc 2341 gtgtggaggg acttggacaa cccgaggtcg ctggccctgg atcccaccaa gggctacatc

2401 tactggaccg agtggggcgg caagccgagg atcgtgcggg ccttcatgga cgggaccaac Attorney Docket No.: 27527-0106WO1

2461 tgcatgacgc tggtggacaa ggtgggccgg gccaacgacc tcaccattga ctacgctgac

2521 cagcgcctct actggaccga cctggacacc aacatgatcg agtcgtccaa catgctgggt

2581 caggagcggg tcgtgattgc cgacgatctc ccgcacccgt tcggtctgac gcagtacagc

2641 gattatatct actggacaga ctggaatctg cacagcattg agcgggccga caagactagc

2701 ggccggaacc gcaccctcat ccagggccac ctggacttcg tgatggacat cctggtgttc

2761 cactcctccc gccaggatgg cctcaatgac tgtatgcaca acaacgggca gtgtgggcag

2821 ctgtgccttg ccatccccgg cggccaccgc tgcggctgcg cctcacacta caccctggac

2881 cccagcagcc gcaactgcag cccgcccacc accttcttgc tgttcagcca gaaatctgcc

2941 atcagtcgga tgatcccgga cgaccagcac agcccggatc tcatcctgcc cctgcatgga

3001 ctgaggaacg tcaaagccat cgactatgac ccactggaca agttcatcta ctgggtggat

3061 gggcgccaga acatcaagcg agccaaggac gacgggaccc agccctttgt tttgacctct

3121 ctgagccaag gccaaaaccc agacaggcag ccccacgacc tcagcatcga catctacagc 3181 cggacactgt tctggacgtg cgaggccacc aataccatca acgtccacag gctgagcggg

3241 gaagccatgg gggtggtgct gcgtggggac cgcgacaagc ccagggccat cgtcgtcaac 3301 gcggagcgag ggtacctgta cttcaccaac atgcaggacc gggcagccaa gatcgaacgc 3361 gcagccctgg acggcaccga gcgcgaggtc ctcttcacca ccggcctcat ccgccctgtg

3421 gccctggtgg tggacaacac actgggcaag ctgttctggg tggacgcgga cctgaagcgc 3481 attgagagct gtgacctgtc aggggccaac cgcctgaccc tggaggacgc caacatcgtg

3541 cagcctctgg gcctgaccat ccttggcaag catctctact ggatcgaccg ccagcagcag

3601 atgatcgagc gtgtggagaa gaccaccggg gacaagcgga ctcgcatcca gggccgtgtc 3661 gcccacctca ctggcatcca tgcagtggag gaagtcagcc tggaggagtt ctcagcccac

3721 ccatgtgccc gtgacaatgg tggctgctcc cacatctgta ttgccaaggg tgatgggaca

3781 ccacggtgct catgcccagt ccacctcgtg ctcctgcaga acctgctgac ctgtggagag

3841 ccgcccacct gctccccgga ccagtttgca tgtgccacag gggagatcga ctgtatcccc

3901 ggggcctggc gctgtgacgg ctttcccgag tgcgatgacc agagcgacga ggagggctgc 3961 cccgtgtgct ccgccgccca gttcccctgc gcgcggggtc agtgtgtgga cctgcgcctg

4021 cgctgcgacg gcgaggcaga ctgtcaggac cgctcagacg aggcggactg tgacgccatc 4081 tgcctgccca accagttccg gtgtgcgagc ggccagtgtg tcctcatcaa acagcagtgc

4141 gactccttcc ccgactgtat cgacggctcc gacgagctca tgtgtgaaat caccaagccg

4201 ccctcagacg acagcccggc ccacagcagt gccatcgggc ccgtcattgg catcatcctc

4261 tctctcttcg tcatgggtgg tgtctatttt gtgtgccagc gcgtggtgtg ccagcgctat

4321 gcgggggcca acgggccctt cccgcacgag tatgtcagcg ggaccccgca cgtgcccctc 4381 aatttcatag ccccgggcgg ttcccagcat ggccccttca caggcatcgc atgcggaaag Attorney Docket No.: 27527-0106WO1

4441 tccatgatga gctccgtgag cctgatgggg ggccggggcg gggtgcccct ctacgaccgg 4501 aaccacgtca caggggcctc gtccagcagc tcgtccagca cgaaggccac gctgtacccg 4561 ccgatcctga acccgccgcc ctccccggcc acggacccct ccctgtacaa catggacatg 4621 ttctactctt caaacattcc ggccactgcg agaccgtaca ggccctacat cattcgagga 4681 atggcgcccc cgacgacgcc ctgcagcacc gacgtgtgtg acagcgacta cagcgccagc 4741 cgctggaagg ccagcaagta ctacctggat ttgaactcgg actcagaccc ctatccaccc 4801 ccacccacgc cccacagcca gtacctgtcg gcggaggaca gctgcccgcc ctcgcccgcc 4861 accgagagga gctacttcca tctcttcccg ccccctccgt ccccctgcac ggactcatcc 4921 tgacctcggc cgggccactc tggcttctct gtgcccctgt aaatagtttt aaatatgaac

4981 aaagaaaaaa atatatttta tgatttaaaa aataaatata attgggattt taaaaacatg

5041 agaaatgtga actgtgatgg ggtgggcagg gctgggagaa ctttgtacag tggagaaata 5101 tttataaact taattttgta aaacagaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 5161 a (SEQ ID NO: 7).

[0090] The human LRP5 amino acid sequence has GenBank® Accession No. NP_002326 and the following sequence:

1 meaappgppw alcgcpapaa asplllfanr rdvrlvdagg vklestiws 61 gledaaavdf qfskgavywt dvseeaikqt ylnqtgaavq nwisglvsp dglacdwvgk 121 klywtdsetn rievanlngt srkvlfwqdl dqpraialdp ahgymywtdw getprierag 181 mdgstrkiiv dsdiywpngl tidleeqkly wadaklsfih ranldgsfrq kwegslthp 241 faltlsgdtl ywtdwqtrsi hacnkrtggk rkeilsalys pmdiqvlsqe rqpffhtrce 301 ednggcshlc llspsepfyt cacptgvqlq dngrtckaga eevlllarrt dlrrisldtp

361 dftdivlqvd dirhaiaidy dplegyvywt ddevrairra yldgsgaqtl vnteindpdg 421 iavdwvarnl ywtdtgtdri evtrlngtsr kilvsedlde praialhpvm glmywtdwge 481 npkiecanld gqerrvlvna slgwpnglal dlqegklywg daktdkievi nvdgtkrrtl 541 ledklphifg ftllgdfiyw tdwqrrsier vhkvkasrdv iidqlpdlmg lkavnvakw 601 gtnpcadrng gcshlcfftp hatrcgcpig lellsdmktc ivpeaflvft sraaihrisl

661 etnnndvaip ltgvkeasal dfdvsnnhiy wtdvslktis rafmngssve hwefgldyp 721 egmavdwmgk nlywadtgtn rievarldgq frqvlvwrdl dnprslaldp tkgyiywtew 781 ggkprivraf mdgtncmtlv dkvgrandlt idyadqrlyw tdldtnmies snmlgqerw 841 iaddlphpfg ltqysdyiyw tdwnlhsier adktsgrnrt liqghldfvm dilvfhssrq 901 dglndcmhnn gqcgqlclai pgghrcgcas hytldpssrn csppttfllf sqksaisrmi 961 pddqhspdli lplhglrnvk aidydpldkf iywvdgrqni krakddgtqp fvltslsqgq Attorney Docket No.: 27527-0106WO1

1021 npdrqphdls idiysrtlfw tceatntinv hrlsgeamgv vlrgdrdkpr aivvnaergy

1081 lyftnmqdra akieraaldg terevlfttg lirpvalwd ntlgklfwvd adlkriescd

1141 lsganrltle danivqplgl tilgkhlywi drqqqmierv ekttgdkrtr iqgrvahltg

1201 ihaveevsle efsahpcard nggcshicia kgdgtprcsc pvhlvllqnl ltcgepptcs

1261 pdqfacatge idcipgawrc dgfpecddqs deegcpvcsa aqfpcargqc vdlrlrcdge

1321 adcqdrsdea dcdaiclpnq frcasgqcvl ikqqcdsfpd cidgsdelmc eitkppsdds

1381 pahssaigpv igiilslfvm ggvyfvcqrv vcqryagang pfpheyvsgt phvplnfiap

1441 ggsqhgpftg iacgksmmss vslmggrggv plydrnhvtg assssssstk atlyppilnp

1501 ppspatdpsl ynmdmfyssn ipatarpyrp yiirgmappt tpcstdvcds dysasrwkas

1561 kyyldlnsds dpypppptph sqylsaedsc ppspatersy fhlfppppsp ctdss (SEQ ID NO:

8).

[0091] The LDLRR domain of LRP5 has the following sequence (corresponding to amino acid residues 1257-1370 of SEQ ID NO: 8): PPTCSPDQFACATGEIDCIPGAWRCD GFPECDDQSDEEGCPVCSAAQFPCARGQCVDLRLRDGEADCQDRSDEVDCDAICLP NQFRCASGQCVLIKQQCDSFPDCIDGSDELMCEI (SEQ ID NO: 9). The LI domain of LRP5 (also known in the art as "repeat 1", has the following amino acid sequence (corresponding to amino acid residues 1257-1294 of SEQ ID NO: 8): PPTCSPDQFACATGEIDCIPGAWRCDG FPECDDQSDEEG (SEQ ID NO: 13); the L2 domain ("repeat 2") of LRP5 has the following amino acid sequence (corresponding to amino acid residues 1298-1333 of SEQ ID NO: 8):

CSAAQFPCARGQCVDLRLRDGEADCQDRSDEVDCD (SEQ ID NO: 14); and the L3 domain ("repeat 3") has the following amino acid sequence (corresponding to amino acid residues 1336-1370 of SEQ ID NO: 8): CLPNQFRCASGQCVLIKQQCDSFPDCIDGSDE LMCE (SEQ ID NO: 15).

[0092] It is to be understood that the sequences given above for human LRP6 and LRP5 LDLRR domains are exemplary, and the present invention also encompasses inhibitors of homologous mammalian LDLRR domains as well as variants and/or mutants (e.g., having amino acid substitutions, deletions, translocations, and/or other modifications known in the art) of such LDLRR domains. In a preferred embodiment, an LDLRR binding antibody or fragment thereof that binds to a human LDLRR domain cross reacts with the homologous domain in cynomolgous monkey and/or rat and/or mouse. Attorney Docket No.: 27527-0106WO1

[0093] In certain embodiments, an LDLRR binding antibody or antigen-binding fragment thereof may cross react with one or more LDLRR domains of other polypeptides (e.g., other Wnt receptors). Thus, for example, an LDLRR binding antibody may bind to both LRP5 and LRP6. In other embodiments, an LDLRR binding antibody or antigen-binding fragment thereof is specific for a single LDLRR domain and does not cross react with homologous LDLRR domains in other polypeptides.

LDLRR Antagonists and Agonists

Antibodies and Binding Fragments

[0094] In a preferred embodiment, an LDLRR inhibitor of the invention is an LDLRR binding antibody or antigen-binding fragment thereof. Preferably, an LDLRR binding antibody or antigen-binding fragment thereof binds to an epitope having an amino acid sequence that is at least partially or that is completely overlapping (identical) with an amino acid sequence located within the LDLRR domain (e.g., of LRP6 and/or LRP5).

[0095] In another preferred embodiment, the LDLRR binding antibody or antigen- binding fragment thereof binds to the human LDLRR domain (e.g., of LRP6 and/or LRP5). Preferably an LDLRR binding antibody of the invention cross-reacts with the homologous cynomolgus monkey ("cyno") LDLRR polypeptide and binds to the cyno polypeptide within 5-fold affinity (Kd) of the human polypeptide. It is also preferred that an LDLRR binding antibody or binding fragment of the invention cross-reacts with the homologous mouse and/or rat LDLRR domain.

[0096] In another preferred embodiment, an LDLRR binding antibody or binding fragment thereof has a binding affinity (Kd) of less than 50 nM, preferably less than 30 nM, less than 20 nM, less than 10 nM, more preferably, less than 5 nM, and most preferably, less than 1 nM, less than 500 pM, less than 250 pM, less than 100 pM, less than 50 pM, less than 25 pM, less than 10 pM, less than 5 pM, less than 3 pM, less than 1 pM, less than 0.75 pM, less than 0.5 pM, or less than 0.3 pM.

[0097] As discussed above, like LRP6 and LRP5, other polypeptides (e.g., other Wnt receptors) comprise homologous LDLRR domains, and thus, antibodies and antigen-binding fragments thereof that target homologous LDLRR domains in other Wnt receptors that activate autocrine Wnt signaling are also encompassed by the present invention.

[0098] In a specific embodiment, an LDLRR binding antibody or antigen-binding fragment thereof binds to an epitope having an amino acid sequence found at least partially or Attorney Docket No.: 27527-0106WO1 completely within the amino acid sequence given in SEQ ID NO: 3 (the amino acid sequence for the complete LDLRR domain of LRP6), SEQ ID NO: 4 (the LI domain of LRP6 LDLRR), SEQ ID NO: 5 (the L2 of LRP6 LDLRR) or SEQ ID NO: 6 (the L3 domain of LRP6 LDLRR).

[0099] In another embodiment, an LDLRR binding antibody or antigen-binding fragment thereof binds to an epitope having an amino acid sequence found at least partially or completely within the amino acid sequence given in SEQ ID NO: 9 (the amino acid sequence for the LDLRR domain of LRP5); SEQ ID NO: 13 (the LI domain of LRP5 LDLRR), SEQ ID NO: 14 (the L2 of LRP5 LDLRR) or SEQ ID NO: 15 (the L3 domain of LRP5 LDLRR).

[00100] Typically, although not necessarily, an epitope will range from about 5 to about 8 amino acids in length. As an example of an epitope that has an amino acid sequence that is partially within the LDLRR domain sequence, e.g., SEQ ID NO: 3, if the last 5 amino acid sequences of an 8 amino acid epitope are the first 5 amino acids of SEQ ID NO: 3 (residues 1247-1251 of SEQ ID NO: 2), then the first 3 amino acid residues of the epitope are the 3 amino acids occurring just before the LDLRR domain (i.e. adjacent and upstream) in the full length sequence of LRP6 (i.e., residues 1244-1246 of SEQ ID NO: 2).

[00101] The LDLRR (e.g., LRP6 and/or LRP5 LDLRR domain) binding antibodies of the present invention may be provided as polyclonal antibodies, monoclonal antibodies (mAbs), recombinant antibodies, chimeric antibodies, CDR-grafted antibodies, fully human antibodies, single chain antibodies, and/or bispecific antibodies, as well as fragments, including variants and derivatives thereof, provided by known techniques, including, but not limited to enzymatic cleavage, peptide synthesis or recombinant techniques. In a preferred embodiment, an LDLRR binding antibody or binding fragment thereof is a non-depleting human IgGl .

[00102] Antibodies generally comprise two heavy chain polypeptides and two light chain polypeptides, though single domain antibodies having one heavy chain and one light chain, and heavy chain antibodies devoid of light chains are also contemplated. There are five types of heavy chains, called alpha, delta, epsilon, gamma and mu, based on the amino acid sequence of the heavy chain constant domain. These different types of heavy chains give rise to five classes of antibodies, IgA (including IgAi and IgA₂), IgD, IgE, IgG and IgM, respectively, including four subclasses of IgG, namely IgGl, IgG2, IgG3 and IgG4. There are also two types of light chains, called kappa (κ) or lambda (λ) based on the amino acid Attorney Docket No.: 27527-0106WO1 sequence of the constant domains. A full-length antibody includes a constant domain and a variable domain. The constant region need not be present in an antigen binding fragment of an antibody. Antigen binding fragments of an antibody disclosed herein can include Fab, Fab', F(ab')2, and F(v) antibody fragments. As discussed in more detail below, LDLRR binding fragments encompass antibody fragments and antigen-binding polypeptides that will bind the LDLRR domain (e.g., of LRP6 and/or LRP5).

[00103] Each of the heavy chain and light chain sequences of an antibody, or antigen binding fragment thereof, includes a variable region with three complementarity determining regions (CDRs) as well as non-CDR framework regions (FRs). The terms "heavy chain" and "light chain", as used herein, mean the heavy chain variable region and the light chain variable region, respectively, unless otherwise noted. Heavy chain CDRs are referred to herein as CDR-H1, CDR-H2, and CDR-H3. Light chain CDRs are referred to herein as CDR- Ll, CDR-L2, and CDR-L3. Variable regions and CDRs in an antibody sequence can be identified (i) according to general rules that have been developed in the art or (ii) by aligning the sequences against a database of known variable regions. Methods for identifying these regions are described in Kontermann and Dubel, eds., Antibody Engineering, Springer, New York, N.Y., 2001, and Dinarello et al, Current Protocols in Immunology, John Wiley and Sons Inc., Hoboken, N.J., 2000. Databases of antibody sequences are described in and can be accessed through "The Kabatman" database at www.bioinf.org.uk/abs (maintained by A. C. Martin in the Department of Biochemistry & Molecular Biology University College London, London, England) and VBASE2 at www.vbase2.org, as described in Retter et al., Nucl. Acids Res., 33(Database issue): D671-D674 (2005). The "Kabatman" database web site also includes general rules of thumb for identifying CDRs. The term "CDR", as used herein, is as defined in Kabat et al., Sequences of Immunological Interest, 5.sup.th ed., U.S. Department of Health and Human Services, 1991, unless otherwise indicated.

[00104] Polyclonal antibodies are preferably raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and an adjuvant. An improved antibody response may be obtained by conjugating the relevant antigen to a protein that is immunogenic in the species to be immunized, e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor using a bifunctional or derivatizing agent, for example, maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride or other agents known in the art. Attorney Docket No.: 27527-0106WO1

[00105] Animals are immunized against the antigen, immunogenic conjugates, or derivatives by combining, e.g., 100 μg or 5 μg of the protein or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites. One month later, the animals are boosted with 1/5 to 1/10 the original amount of peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites. At 7-14 days post-booster injection, the animals are bled and the serum is assayed for antibody titer. Animals are boosted until the titer plateaus. Preferably, the animal is boosted with the conjugate of the same antigen, but conjugated to a different protein and/or through a different cross-linking reagent. Conjugates also can be made in recombinant cell culture as protein fusions. Also, aggregating agents such as alum are suitably used to enhance the immune response.

[00106] Monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies. Monoclonal antibodies are generally highly specific, and may be directed against a single antigenic site, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants (epitopes). In addition to their specificity, the monoclonal antibodies are advantageous in that they are synthesized by the homogeneous culture, uncontaminated by other immunoglobulins with different specificities and characteristics.

[00107] Monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al, (Nature, 256:495-7, 1975), or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The monoclonal antibodies may also be isolated from phage antibody libraries using the techniques described in, for example, Clackson et al, (Nature 352:624-628, 1991) and Marks et al, (J. Mol. Biol. 222:581-597, 1991).

[00108] In the hybridoma method, a mouse or other appropriate host animal, such as a hamster or macaque monkey, is immunized as herein described to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization. Alternatively, lymphocytes may be immunized in vitro. Lymphocytes then are fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). Attorney Docket No.: 27527-0106WO1

[00109] The hybridoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.

[001 10] Preferred myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a medium. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133 : 3001 (1984); Brodeur et al, Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)). Exemplary murine myeloma lines include those derived from MOP-21 and M.C.-l 1 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, Calif. USA, and SP-2 or X63-Ag8-653 cells available from the American Type Culture Collection, Rockville, Md. USA.

[001 11] Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). The binding affinity of the monoclonal antibody can, for example, be determined by Scatchard analysis (Munson et al, Anal. Biochem., 107:220 (1980)).

[001 12] After hybridoma cells are identified that produce antibodies of the desired specificity, affinity, and/or activity, the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, DMEM or RPMI-1640 medium. In addition, the hybridoma cells may be grown in vivo as ascites tumors in an animal. The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A- Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography. Attorney Docket No.: 27527-0106WO1

[001 13] It is further contemplated that antibodies of the invention may be used as smaller antigen binding fragments of the antibody well-known in the art and described herein.

[001 14] The present invention encompasses LDLRR (e.g., LRP6 and/or LRP5 LDLRR domain) binding antibodies that include two full length heavy chains and two full length light chains. Alternatively, the LDLRR binding antibodies can be constructs such as single chain antibodies or "mini" antibodies that retain binding activity to LDLRR. Such constructs can be prepared by methods known in the art such as, for example, the PCR mediated cloning and assembly of single chain antibodies for expression in E. coli (as described in Antibody Engineering, The practical approach series, J. McCafferty, H. R. Hoogenboom, and D. J. Chiswell, editors, Oxford University Press, 1996). In this type of construct, the variable portions of the heavy and light chains of an antibody molecule are PCR amplified from cDNA. The resulting amplicons are then assembled, for example, in a second PCR step, through a linker DNA that encodes a flexible protein linker composed of the amino acids Gly and Ser. This linker allows the variable heavy and light chain portions to fold in such a way that the antigen binding pocket is regenerated and antigen is bound with affinities often comparable to the parent full-length dimeric immunoglobulin molecule.

[001 15] The LDLRR (e.g. LRP6 and/or LRP5 LDLRR domain) binding antibodies and fragments of the present invention encompass variants of the exemplary antibodies, fragments and sequences disclosed herein. Variants include peptides and polypeptides comprising one or more amino acid sequence substitutions, deletions, and/or additions that have the same or substantially the same affinity and specificity of epitope binding as one or more of the exemplary antibodies, fragments and sequences disclosed herein. Thus, variants include peptides and polypeptides comprising one or more amino acid sequence substitutions, deletions, and/or additions to the exemplary antibodies, fragments and sequences disclosed herein where such substitutions, deletions and/or additions do not cause substantial changes in affinity and specificity of epitope binding. For example, a variant of an antibody or fragment may result from one or more changes to an antibody or fragment, where the changed antibody or fragment has the same or substantially the same affinity and specificity of epitope binding as the starting sequence. Variants may be naturally occurring, such as allelic or splice variants, or may be artificially constructed. Variants may be prepared from the corresponding nucleic acid molecules encoding the variants. Variants of the present antibodies and LDLRR binding fragments may have changes in light and/or heavy chain amino acid sequences that are naturally occurring or are introduced by in vitro engineering of Attorney Docket No.: 27527-0106WO1 native sequences using recombinant DNA techniques. Naturally occurring variants include "somatic" variants which are generated in vivo in the corresponding germ line nucleotide sequences during the generation of an antibody response to a foreign antigen.

[001 16] Variants of LDLRR (e.g., LRP6 and/or LRP5 LDLRR domain) binding antibodies and binding fragments may also be prepared by mutagenesis techniques. For example, amino acid changes may be introduced at random throughout an antibody coding region and the resulting variants may be screened for binding affinity for LDLRR or for another property. Alternatively, amino acid changes may be introduced in selected regions of an LDLRR antibody, such as in the light and/or heavy chain CDRs, and/or in the framework regions, and the resulting antibodies may be screened for binding to LDLRR or some other activity. Amino acid changes encompass one or more amino acid substitutions in a CDR, ranging from a single amino acid difference to the introduction of multiple permutations of amino acids within a given CDR, such as CDR3. In another method, the contribution of each residue within a CDR to LDLRR binding may be assessed by substituting at least one residue within the CDR with alanine. Lewis et al. (1995), Mol. Immunol. 32: 1065-72. Residues which are not optimal for binding to LDLRR may then be changed in order to determine a more optimum sequence. Also encompassed are variants generated by insertion of amino acids to increase the size of a CDR, such as CDR3. For example, most light chain CDR3 sequences are nine amino acids in length. Light chain sequences in an antibody which are shorter than nine residues may be optimized for binding to LDLRR by insertion of appropriate amino acids to increase the length of the CDR.

[001 17] "Framework region" as the term is used herein refers to amino acid sequences within the variable region of both heavy and light chain polypeptides that are not CDR sequences, and are primarily responsible for maintaining correct positioning of the CDR sequences to permit antigen binding. Although the framework regions themselves typically do not directly participate in antigen binding, as is known in the art, certain residues within the framework regions of certain antibodies can directly participate in antigen binding or can affect the ability of one or more amino acids in CDRs to interact with antigen. Framework regions are sometimes referred to as "FR."

[001 18] Variants may also be prepared by "chain shuffling" of light or heavy chains. Marks et al. (1992), Biotechnology 10: 779-83. A single light (or heavy) chain can be combined with a library having a repertoire of heavy (or light) chains and the resulting Attorney Docket No.: 27527-0106WO1 population is screened for a desired activity, such as binding to LDLRR. This permits screening of a greater sample of different heavy (or light) chains in combination with a single light (or heavy) chain than is possible with libraries comprising repertoires of both heavy and light chains.

[001 19] The LDLRR (e.g., LRP6 and/or LRP5 LDLRR domain) binding antibodies and fragments of the present invention encompass derivatives of the exemplary antibodies, fragments and sequences disclosed herein. Derivatives include polypeptides or peptides, or variants, fragments or derivatives thereof, which have been chemically modified. Examples include covalent attachment of one or more polymers, such as water soluble polymers, N- linked, or O-linked carbohydrates, sugars, phosphates, and/or other such molecules. The derivatives are modified in a manner that is different from naturally occurring or starting peptide or polypeptides, either in the type or location of the molecules attached. Derivatives further include deletion of one or more chemical groups which are naturally present on the peptide or polypeptide.

[00120] The LDLRR (e.g., LRP6 and/or LRP5 LDLRR domain) binding antibodies and fragments of the present invention can be bispecific. Bispecific antibodies or fragments can be of several configurations. For example, bispecific antibodies may resemble single antibodies (or antibody fragments) but have two different antigen binding sites (variable regions). Bispecific antibodies can be produced by chemical techniques (Kranz et al. (1981), Proc. Natl. Acad. Sci. USA, 78: 5807), by "polydoma" techniques (U.S. Pat. No. 4,474,893) or by recombinant DNA techniques. Bispecific antibodies of the present invention can have binding specificities for at least two different epitopes, at least one of which is an epitope of LDLRR. The LDLRR binding antibodies and fragments can also be heteroantibodies. Heteroantibodies are two or more antibodies, or antibody binding fragments (Fab) linked together, each antibody or fragment having a different specificity.

[00121] Techniques for creating recombinant DNA versions of the antigen-binding regions of antibody molecules which bypass the generation of monoclonal antibodies are contemplated for the present LDLRR (e.g., LRP6 or LRP5 LDLRR domain) binding antibodies and fragments. DNA is cloned into a bacterial expression system. One example of such a technique suitable for the practice of this invention uses a bacteriophage lambda vector system having a leader sequence that causes the expressed Fab protein to migrate to the periplasmic space (between the bacterial cell membrane and the cell wall) or to be secreted. Attorney Docket No.: 27527-0106WO1

One can rapidly generate and screen great numbers of functional Fab fragments for those which bind LDLRR. Such LDLRR binding agents (Fab fragments with specificity for an LDLRR polypeptide) are specifically encompassed within the LDLRR binding antibodies and fragments of the present invention.

[00122] The present LDLRR (e.g., LRP6 and/or LRP5 LDLRR domain) binding antibodies and fragments can be humanized or human engineered antibodies. As used herein, a humanized antibody, or antigen binding fragment thereof, is a recombinant polypeptide that comprises a portion of an antigen binding site from a non-human antibody and a portion of the framework and/or constant regions of a human antibody. A human engineered antibody or antibody fragment is a non-human (e.g., mouse) antibody that has been engineered by modifying (e.g., deleting, inserting, or substituting) amino acids at specific positions so as to reduce or eliminate any detectable immunogenicity of the modified antibody in a human.

[00123] Humanized antibodies include chimeric antibodies and CDR-grafted antibodies. Chimeric antibodies are antibodies that include a non-human antibody variable region linked to a human constant region. Thus, in chimeric antibodies, the variable region is mostly non- human, and the constant region is human. Chimeric antibodies and methods for making them are described in Morrison, et al, Proc. Natl. Acad. Sci. USA, 81 : 6841-6855 (1984), Boulianne, et al, Nature, 312: 643-646 (1984), and PCT Application Publication WO 86/01533. Although, they can be less immunogenic than a mouse monoclonal antibody, administrations of chimeric antibodies have been associated with human anti-mouse antibody responses (HAMA) to the non-human portion of the antibodies. Chimeric antibodies can also be produced by splicing the genes from a mouse antibody molecule of appropriate antigen- binding specificity together with genes from a human antibody molecule of appropriate biological activity, such as the ability to activate human complement and mediate ADCC. Morrison et al. (1984), Proc. Natl. Acad. Sci., 81 : 6851 ; Neuberger et al. (1984), Nature, 312: 604. One example is the replacement of an Fc region with that of a different isotype.

[00124] CDR-grafted antibodies are antibodies that include the CDRs from a non-human "donor" antibody linked to the framework region from a human "recipient" antibody. Generally, CDR-grafted antibodies include more human antibody sequences than chimeric antibodies because they include both constant region sequences and variable region (framework) sequences from human antibodies. Thus, for example, a CDR-grafted humanized antibody of the invention can comprise a heavy chain that comprises a contiguous Attorney Docket No.: 27527-0106WO1 amino acid sequence (e.g., about 5 or more, 10 or more, or even 15 or more contiguous amino acid residues) from the framework region of a human antibody (e.g., FR-1, FR-2, or FR-3 of a human antibody) or, optionally, most or all of the entire framework region of a human antibody. CDR-grafted antibodies and methods for making them are described in, Jones et al, Nature, 321 : 522-525 (1986), Riechmann et al, Nature, 332: 323-327 (1988), and Verhoeyen et al, Science, 239: 1534-1536 (1988)). Methods that can be used to produce humanized antibodies also are described in U.S. Pat. Nos. 4,816,567, 5,721,367, 5,837,243, and 6, 180,377. CDR-grafted antibodies are considered less likely than chimeric antibodies to induce an immune reaction against non-human antibody portions. However, it has been reported that framework sequences from the donor antibodies are required for the binding affinity and/or specificity of the donor antibody, presumably because these framework sequences affect the folding of the antigen-binding portion of the donor antibody. Therefore, when donor, non-human CDR sequences are grafted onto unaltered human framework sequences, the resulting CDR-grafted antibody can exhibit, in some cases, loss of binding avidity relative to the original non-human donor antibody. See, e.g., Riechmann et al, Nature, 332: 323-327 (1988), and Verhoeyen et al, Science, 239: 1534-1536 (1988).

[00125] Human engineered antibodies include for example "veneered" antibodies and antibodies prepared using HUMAN ENGINEERING™ technology (U.S. Pat. No. 5,869,619). HUMAN ENGINEERING™ technology is commercially available, and involves altering a non-human antibody or antibody fragment, such as a mouse or chimeric antibody or antibody fragment, by making specific changes to the amino acid sequence of the antibody so as to produce a modified antibody with reduced immunogenicity in a human that nonetheless retains the desirable binding properties of the original non-human antibodies. Generally, the technique involves classifying amino acid residues of a non-human (e.g., mouse) antibody as "low risk", "moderate risk", or "high risk" residues. The classification is performed using a global risk/reward calculation that evaluates the predicted benefits of making particular substitution (e.g., for immunogenicity in humans) against the risk that the substitution will affect the resulting antibody's folding and/or antigen-binding properties. Thus, a low risk position is one for which a substitution is predicted to be beneficial because it is predicted to reduce immunogenicity without significantly affecting antigen binding properties. A moderate risk position is one for which a substitution is predicted to reduce immunogenicity, but is more likely to affect protein folding and/or antigen binding. High risk positions contain residues most likely to be involved in proper folding or antigen binding. Attorney Docket No.: 27527-0106WO1

Generally, low risk positions in a non-human antibody are substituted with human residues, high risk positions are rarely substituted, and humanizing substitutions at moderate risk positions are sometimes made, although not indiscriminately. Positions with prolines in the non-human antibody variable region sequence are usually classified as at least moderate risk positions.

[00126] The particular human amino acid residue to be substituted at a given low or moderate risk position of a non-human (e.g., mouse) antibody sequence can be selected by aligning an amino acid sequence from the non-human antibody's variable regions with the corresponding region of a specific or consensus human antibody sequence. The amino acid residues at low or moderate risk positions in the non-human sequence can be substituted for the corresponding residues in the human antibody sequence according to the alignment. Techniques for making human engineered proteins are described in greater detail in Studnicka et al, Protein Engineering, 7: 805-814 (1994), U.S. Pat. Nos. 5,766,886, 5,770, 196, 5,821, 123, and 5,869,619, and PCT Application Publication WO 93/1 1794.

[00127] "Veneered" antibodies are non-human or humanized (e.g., chimeric or CDR- grafted antibodies) antibodies that have been engineered to replace certain solvent-exposed amino acid residues so as to further reduce their immunogenicity or enhance their function. As surface residues of a chimeric antibody are presumed to be less likely to affect proper antibody folding and more likely to elicit an immune reaction, veneering of a chimeric antibody can include, for instance, identifying solvent-exposed residues in the non-human framework region of a chimeric antibody and replacing at least one of them with the corresponding surface residues from a human framework region. Veneering can be accomplished by any suitable engineering technique, including the use of the above-described HUMAN ENGINEERING™ technology.

[00128] In a different approach, a recovery of binding avidity can be achieved by "dehumanizing" a CDR-grafted antibody. De-humanizing can include restoring residues from the donor antibody's framework regions to the CDR grafted antibody, thereby restoring proper folding. Similar "de-humanization" can be achieved by (i) including portions of the "donor" framework region in the "recipient" antibody or (ii) grafting portions of the "donor" antibody framework region into the recipient antibody (along with the grafted donor CDRs). Attorney Docket No.: 27527-0106WO1

[00129] For a further discussion of antibodies, humanized antibodies, human engineered, and methods for their preparation, see Kontermann and Dubel, eds., Antibody Engineering, Springer, New York, N.Y., 2001.

[00130] Exemplary humanized or human engineered antibodies include IgG, IgM, IgE, IgA, and IgD antibodies. The present antibodies can be of any class (IgG, IgA, IgM, IgE, IgD, etc.) or isotype and can comprise a kappa or lambda light chain. For example, a human antibody can comprise an IgG heavy chain or defined fragment, such as at least one of isotypes, IgGl, IgG2, IgG3 or IgG4. As a further example, the present antibodies or fragments can comprise an IgGl heavy chain and an IgGl light chain.

[00131] The present antibodies and fragments can be human antibodies, such as antibodies which bind LDLRR (e.g., LRP6 and/or LRP5 LDLRR domain) polypeptides and are encoded by nucleic acid sequences which are naturally occurring somatic variants of human germline immunoglobulin nucleic acid sequence, and fragments, synthetic variants, derivatives and fusions thereof. Such antibodies may be produced by any method known in the art, such as through the use of transgenic mammals (such as transgenic mice) in which the native immunoglobulin repertoire has been replaced with human V-genes in the mammal chromosome. Such mammals appear to carry out VDJ recombination and somatic hypermutation of the human germline antibody genes in a normal fashion, thus producing high affinity antibodies with completely human sequences.

[00132] Human antibodies to target protein can also be produced using transgenic animals that have no endogenous immunoglobulin production and are engineered to contain human immunoglobulin loci. For example, WO 98/24893 discloses transgenic animals having a human Ig locus wherein the animals do not produce functional endogenous immunoglobulins due to the inactivation of endogenous heavy and light chain loci. WO 91/00906 also discloses transgenic non-primate mammalian hosts capable of mounting an immune response to an immunogen, wherein the antibodies have primate constant and/or variable regions, and wherein the endogenous immunoglobulin encoding loci are substituted or inactivated. WO 96/30498 and U.S. Pat. No. 6,091,001 disclose the use of the Cre/Lox system to modify the immunoglobulin locus in a mammal, such as to replace all or a portion of the constant or variable region to form a modified antibody molecule. WO 94/02602 discloses non-human mammalian hosts having inactivated endogenous Ig loci and functional human Ig loci. U.S. Pat. No. 5,939,598 discloses methods of making transgenic mice in which the mice lack Attorney Docket No.: 27527-0106WO1 endogenous heavy chains, and express an exogenous immunoglobulin locus comprising one or more xenogeneic constant regions. See also, U.S. Pat. Nos. 6, 114,598 6,657, 103 and 6,833,268.

[00133] Using a transgenic animal described above, an immune response can be produced to a selected antigenic molecule, and antibody producing cells can be removed from the animal and used to produce hybridomas that secrete human monoclonal antibodies. Immunization protocols, adjuvants, and the like are known in the art, and are used in immunization of, for example, a transgenic mouse as described in WO 96/33735, which discloses monoclonal antibodies against a variety of antigenic molecules including IL-6, IL- 8, TNFa, human CD4, L selectin, gp39, and tetanus toxin. The monoclonal antibodies can be tested for the ability to inhibit or neutralize the biological activity or physiological effect of the corresponding protein. WO 96/33735 discloses that monoclonal antibodies against IL-8, derived from immune cells of transgenic mice immunized with IL-8, blocked IL-8 induced functions of neutrophils. Human monoclonal antibodies with specificity for the antigen used to immunize transgenic animals are also disclosed in WO 96/34096 and U.S. Patent Application Publication Nos. 2003/0194404 and 2003/0031667.

[00134] Additional transgenic animals useful to make monoclonal antibodies include the Medarex HuMAb-MOUSE® described in U.S. Pat. No. 5,770,429 and Fishwild, et al. (Nat. Biotechnol. 14:845-851, 1996), which contains gene sequences from unrearranged human antibody genes that code for the heavy and light chains of human antibodies. Immunization of a HuMAb-MOUSE® enables the production of fully human monoclonal antibodies to the target protein.

[00135] Also, Ishida et al. (Cloning Stem Cells. 4:91-102, 2002) describes the TransChromo Mouse (TCMOUSE™) which comprises megabase-sized segments of human DNA and which incorporates the entire human immunoglobulin (hlg) loci. The TCMOUSE™ has a fully diverse repertoire of hlgs, including all the subclasses of IgGs (IgGl-G4). Immunization of the TC MOUSE™ with various human antigens produces antibody responses comprising human antibodies.

[00136] See also Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al, Nature, 362:255-258 (1993); Bruggermann et al, Year in Immunol, 7:33 (1993); and U.S. Pat. Nos. 5,591,669, 5,589,369, 5,545,807; and U.S. Patent Publication No. 20020199213. U.S. Patent Publication No. 20030092125 describes methods for biasing the Attorney Docket No.: 27527-0106WO1 immune response of an animal to the desired epitope. Human antibodies may also be generated by in vitro activated B cells (see U.S. Pat. Nos. 5,567,610 and 5,229,275).

[00137] Human antibodies can also be generated through the in vitro screening of antibody display libraries. See Hoogenboom et al. (1991), J. Mol. Biol. 227: 381; and Marks et al. (1991), J. Mol. Biol. 222: 581. Various antibody-containing phage display libraries have been described and may be readily prepared. Libraries may contain a diversity of human antibody sequences, such as human Fab, Fv, and scFv fragments, which may be screened against an appropriate target. Phage display libraries may comprise peptides or proteins other than antibodies which may be screened to identify selective binding agents of LDLRR.

[00138] The development of technologies for making repertoires of recombinant human antibody genes, and the display of the encoded antibody fragments on the surface of filamentous bacteriophage, has provided a means for making human antibodies directly. The antibodies produced by phage technology are produced as antigen binding fragments-usually Fv or Fab fragments-in bacteria and thus lack effector functions. Effector functions can be introduced by one of two strategies: The fragments can be engineered either into complete antibodies for expression in mammalian cells, or into bispecific antibody fragments with a second binding site capable of triggering an effector function.

[00139] The invention contemplates a method for producing target-specific antibody or antigen-binding portion thereof comprising the steps of synthesizing a library of human antibodies on phage, screening the library with target protein or a portion thereof, isolating phage that bind target, and obtaining the antibody from the phage. By way of example, one method for preparing the library of antibodies for use in phage display techniques comprises the steps of immunizing a non-human animal comprising human immunoglobulin loci (see, Lonberg N. Nat Biotechnol. 2005 Sep;23(9): 1 117-25) with target antigen or an antigenic portion thereof to create an immune response, extracting antibody producing cells from the immunized animal; isolating RNA from the extracted cells, reverse transcribing the RNA to produce cDNA, amplifying the cDNA using a primer, and inserting the cDNA into a phage display vector such that antibodies are expressed on the phage. Recombinant target-specific antibodies of the invention may be obtained in this way.

[00140] Phage-display processes mimic immune selection through the display of antibody repertoires on the surface of filamentous bacteriophage, and subsequent selection of phage by their binding to an antigen of choice. One such technique is described in WO 99/10494, Attorney Docket No.: 27527-0106WO1 which describes the isolation of high affinity and functional agonistic antibodies for MPL and msk receptors using such an approach. Antibodies of the invention can be isolated by screening of a recombinant combinatorial antibody library, preferably a scFv phage display library, prepared using human VL and VH CDNAS prepared from mRNA derived from human lymphocytes. Methodologies for preparing and screening such libraries are known in the art. See e.g., U.S. Pat. No. 5,969, 108. There are commercially available kits for generating phage display libraries (e.g., the Pharmacia Recombinant Phage Antibody System, catalog no. 27- 9400-01; and the Stratagene SurfZAP™ phage display kit, catalog no. 240612). There are also other methods and reagents that can be used in generating and screening antibody display libraries (see, e.g., Ladner et al. U.S. Pat. No. 5,223,409; Kang et al. PCT Publication No. WO 92/18619; Dower et al. PCT Publication No. WO 91/17271 ; Winter et al. PCT Publication No. WO 92/20791; Markland et al. PCT Publication No. WO 92/15679; Breitling et al. PCT Publication No. WO 93/01288; McCafferty et al. PCT Publication No. WO 92/01047; Garrard et al. PCT Publication No. WO 92/09690; Fuchs et al. (1991) Bio/Technology 9: 1370-1372; Hay et al. (1992) Hum. Antibod. Hybridomas 3 :81-85; Huse et al. (1989) Science 246: 1275-1281; McCafferty et al, Nature (1990) 348:552-554; Griffiths et al. (1993) EMBO J 12:725-734; Hawkins et al. (1992) J. Mol. Biol. 226:889-896; Clackson et al. (1991) Nature 352:624-628; Gram et al. (1992) Proc. Natl. Acad. Sci. USA 89:3576-3580; Garrad et al. (1991) Bio/Technology 9: 1373-1377; Hoogenboom et al. (1991) Nuc Acid Res 19:4133-4137; and Barbas et al. (1991) Proc. Natl. Acad. Sci. USA 88:7978-7982.

[00141] In one embodiment, to isolate human antibodies specific for the target antigen with the desired characteristics, a human VH and VL library are screened to select for antibody fragments having the desired specificity. The antibody libraries used in this method are preferably scFv libraries prepared and screened as described herein and in the art (McCafferty et al., PCT Publication No. WO 92/01047, McCafferty et al, (Nature 348:552- 554, 1990); and Griffiths et al, (EMBO J 12:725-734, 1993). The scFv antibody libraries preferably are screened using target protein as the antigen.

[00142] Alternatively, the Fd fragment (VH-CHI) and light chain (VL-CL) of antibodies are separately cloned by PCR and recombined randomly in combinatorial phage display libraries, which can then be selected for binding to a particular antigen. The Fab fragments are expressed on the phage surface, i.e., physically linked to the genes that encode them. Thus, selection of Fab by antigen binding co-selects for the Fab encoding sequences, which can be Attorney Docket No.: 27527-0106WO1 amplified subsequently. Through several rounds of antigen binding and re-amplification, a procedure termed panning, Fab specific for the antigen are enriched and finally isolated.

[00143] In 1994, an approach for the humanization of antibodies, called "guided selection", was described. Guided selection utilizes the power of the phage display technique for the humanization of mouse monoclonal antibody (See Jespers, L. S., et al, Bio/Technology 12, 899-903 (1994)). For this, the Fd fragment of the mouse monoclonal antibody can be displayed in combination with a human light chain library, and the resulting hybrid Fab library may then be selected with antigen. The mouse Fd fragment thereby provides a template to guide the selection. Subsequently, the selected human light chains are combined with a human Fd fragment library. Selection of the resulting library yields entirely human Fab.

[00144] A variety of procedures have been described for deriving human antibodies from phage-display libraries (See, for example, Hoogenboom et al, J. Mol. Biol, 227:381 (1991); Marks et al, J. Mol. Biol, 222:581-597 (1991); U.S. Pat. Nos. 5,565,332 and 5,573,905; Clackson, T., and Wells, J. A., TIBTECH 12, 173-184 (1994)). In particular, in vitro selection and evolution of antibodies derived from phage display libraries has become a powerful tool (See Burton, D. R., and Barbas III, C. F., Adv. Immunol. 57, 191-280 (1994); Winter, G., et al, Annu. Rev. Immunol. 12, 433-455 (1994); U.S. patent publication no. 20020004215 and WO 92/01047; U.S. patent publication no. 20030190317; and U.S. Pat. Nos. 6,054,287 and 5,877,293.

[00145] Watkins, "Screening of Phage-Expressed Antibody Libraries by Capture Lift", Methods in Molecular Biology, Antibody Phage Display: Methods and Protocols 178: 187- 193 (2002), and U.S. patent publication no. 20030044772, published Mar. 6, 2003, describe methods for screening phage-expressed antibody libraries or other binding molecules by capture lift, a method involving immobilization of the candidate binding molecules on a solid support.

[00146] Fv fragments are displayed on the surface of phage, by the association of one chain expressed as a phage protein fusion (e.g., with M13 gene III) with the complementary chain expressed as a soluble fragment. It is contemplated that the phage may be a filamentous phage such as one of the class I phages: fd, M13, fl, Ifl, Ike, ZJ/Z, Ff and one of the class II phages Xf, Pfl and Pf3. The phage may be Ml 3, or fd or a derivative thereof. Attorney Docket No.: 27527-0106WO1

[00147] Once initial human VL and VH segments are selected, "mix and match" experiments, in which different pairs of the initially selected VL and VH segments are screened for target binding, are performed to select preferred VL/VH pair combinations. Additionally, to further improve the quality of the antibody, the VL and VH segments of the preferred VL VH pair(s) can be randomly mutated, preferably within the any of the CDR1, CDR2 or CDR3 region of VH and/or VL, in a process analogous to the in vivo somatic mutation process responsible for affinity maturation of antibodies during a natural immune response. This in vitro affinity maturation can be accomplished by amplifying VL and VH regions using PCR primers complimentary to the VH CDRl, CDR2, and CDR3, or VL CDRl, CDR2, and CDR3, respectively, which primers have been "spiked" with a random mixture of the four nucleotide bases at certain positions such that the resultant PCR products encode VL and VH segments into which random mutations have been introduced into the VH and/or VL CDR3 regions. These randomly mutated VL and VH segments can be rescreened for binding to target antigen.

[00148] Following screening and isolation of a target specific antibody from a recombinant immunoglobulin display library, nucleic acid encoding the selected antibody can be recovered from the display package (e.g., from the phage genome) and subcloned into other expression vectors by standard recombinant DNA techniques. If desired, the nucleic acid can be further manipulated to create other antibody forms of the invention, as described below. To express a recombinant human antibody isolated by screening of a combinatorial library, the DNA encoding the antibody is cloned into a recombinant expression vector and introduced into a mammalian host cell, as described herein.

[00149] It is contemplated that the phage display method may be carried out in a mutator strain of bacteria or host cell. A mutator strain is a host cell which has a genetic defect which causes DNA replicated within it to be mutated with respect to its parent DNA. Example mutator strains are NR9046mutD5 and NR9046 mut Tl .

[00150] It is also contemplated that the phage display method may be carried out using a helper phage. This is a phage which is used to infect cells containing a defective phage genome and which functions to complement the defect. The defective phage genome can be a phagemid or a phage with some function encoding gene sequences removed. Examples of helper phages are M13K07, M13K07 gene III no. 3; and phage displaying or encoding a binding molecule fused to a capsid protein. Attorney Docket No.: 27527-0106WO1

[00151 ] Antibodies are also generated via phage display screening methods using the hierarchical dual combinatorial approach as disclosed in WO 92/01047 in which an individual colony containing either an H or L chain clone is used to infect a complete library of clones encoding the other chain (L or H) and the resulting two-chain specific binding member is selected in accordance with phage display techniques such as those described therein. This technique is also disclosed in Marks et al, (Bio/Technology, 10:779-783, 1992).

[00152] Methods for display of peptides on the surface of yeast and microbial cells have also been used to identify antigen specific antibodies. See, for example, U.S. Pat. No. 6,699,658. Antibody libraries may be attached to yeast proteins, such as agglutinin, effectively mimicking the cell surface display of antibodies by B cells in the immune system.

[00153] In addition to phage display methods, antibodies may be isolated using ribosome mRNA display methods and microbial cell display methods. Selection of polypeptide using ribosome display is described in Hanes et al, (Proc Natl Acad Sci USA, 94:4937-4942, 1997) and U.S. Pat. Nos. 5,643,768 and 5,658,754 issued to Kawasaki. Ribosome display is also useful for rapid large scale mutational analysis of antibodies. The selective mutagenesis approach also provides a method of producing antibodies with improved activities that can be selected using ribosomal display techniques.

[00154] The LDLRR (e.g., LRP6 and/or LRP5 LDLRR domain) binding antibodies and fragments may comprise one or more portions that do not bind LDLRR but instead are responsible for other functions, such as circulating half-life, direct cytotoxic effect, detectable labeling, or activation of the recipient's endogenous complement cascade or endogenous cellular cytotoxicity. The antibodies or fragments may comprise all or a portion of the constant region and may be of any isotype, including IgA (e.g., IgAl or IgA2), IgD, IgE, IgG (e.g. IgGl , IgG2, IgG3 or IgG4), or IgM. In addition to, or instead of, comprising a constant region, antigen-binding compounds of the invention may include an epitope tag, a salvage receptor epitope, a label moiety for diagnostic or purification purposes, or a cytotoxic moiety such as a radionuclide or toxin.

[00155] The constant region (when present) of the present antibodies and fragments may be of the γΐ , y2, γ3, γ4, μ, β2, or δ or ε type, preferably of the γ type, whereas the constant part of a human light chain may be of the κ or λ type (which includes the λι, λ2 and λ3 subtypes) but is preferably of the κ type. Attorney Docket No.: 27527-0106WO1

[00156] Variants also include antibodies or fragments comprising a modified Fc region, wherein the modified Fc region comprises at least one amino acid modification relative to a wild-type Fc region. The variant Fc region may be designed, relative to a comparable molecule comprising the wild-type Fc region, so as to bind Fc receptors with a greater or lesser affinity.

[00157] For example, the present LDLRR binding antibodies and fragments may comprise a modified Fc region. Fc region refers to naturally occurring or synthetic polypeptides homologous to the IgG C-terminal domain that is produced upon papain digestion of IgG. IgG Fc has a molecular weight of approximately 50 kD. In the present antibodies and fragments, an entire Fc region can be used, or only a half-life enhancing portion. In addition, many modifications in amino acid sequence are acceptable, as native activity is not in all cases necessary or desired.

[00158] The Fc region can be mutated, if desired, to inhibit its ability to fix complement and bind the Fc receptor with high affinity. For murine IgG Fc, substitution of Ala residues for Glu 318, Lys 320, and Lys 322 renders the protein unable to direct ADCC. Substitution of Glu for Leu 235 inhibits the ability of the protein to bind the Fc receptor with high affinity. Various mutations for human IgG also are known (see, e.g., Morrison et al, 1994, The Immunologist 2: 1 19 124 and Brekke et al, 1994, The Immunologist 2: 125).

[00159] In some embodiments, the present an antibodies or fragments are provided with a modified Fc region where a naturally-occurring Fc region is modified to increase the half-life of the antibody or fragment in a biological environment, for example, the serum half-life or a half-life measured by an in vitro assay. Methods for altering the original form of an Fc region of an IgG also are described in U.S. Pat. No. 6,998,253.

[00160] In certain embodiments, it may be desirable to modify the antibody or fragment in order to increase its serum half-life, for example, adding molecules such as PEG or other water soluble polymers, including polysaccharide polymers, to antibody fragments to increase the half-life. This may also be achieved, for example, by incorporation of a salvage receptor binding epitope into the antibody fragment (e.g., by mutation of the appropriate region in the antibody fragment or by incorporating the epitope into a peptide tag that is then fused to the antibody fragment at either end or in the middle, e.g., by DNA or peptide synthesis) (see, International Publication No. WO 96/32478). Salvage receptor binding Attorney Docket No.: 27527-0106WO1 epitope refers to an epitope of the Fc region of an IgG molecule (e.g., IgGl, IgG2, IgG3, or IgG4) that is responsible for increasing the in vivo serum half-life of the IgG molecule.

[00161] A salvage receptor binding epitope can include a region wherein any one or more amino acid residues from one or two loops of an Fc domain are transferred to an analogous position of the antibody fragment. Even more preferably, three or more residues from one or two loops of the Fc domain are transferred. Still more preferred, the epitope is taken from the CH2 domain of the Fc region (e.g., of an IgG) and transferred to the CHI, CH3, or VH region, or more than one such region, of the antibody. Alternatively, the epitope is taken from the Cm domain of the Fc region and transferred to the CL region or VL region, or both, of the antibody fragment. See also International applications WO 97/34631 and WO 96/32478 which describe Fc variants and their interaction with the salvage receptor.

[00162] Mutation of residues within Fc receptor binding sites can result in altered effector function, such as altered ADCC or CDC activity, or altered half-life. Potential mutations include insertion, deletion or substitution of one or more residues, including substitution with alanine, a conservative substitution, a non-conservative substitution, or replacement with a corresponding amino acid residue at the same position from a different IgG subclass (e.g. replacing an IgGl residue with a corresponding IgG2 residue at that position). For example it has been reported that mutating the serine at amino acid position 241 in IgG4 to proline (found at that position in IgGl and IgG2) led to the production of a homogeneous antibody, as well as extending serum half-life and improving tissue distribution compared to the original chimeric IgG4. (Angal et al, Mol Immunol. 30: 105-8, 1993).

[00163] Antibody fragments are portions of an intact full length antibody, such as an antigen binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); multispecific antibody fragments such as bispecific, trispecific, and multispecific antibodies (e.g., diabodies, triabodies, tetrabodies); minibodies; chelating recombinant antibodies; tribodies or bibodies; intrabodies; nanobodies; small modular immunopharmaceuticals (SMIP), adnectins, binding-domain immunoglobulin fusion proteins; camelized antibodies; VHH containing antibodies; and any other polypeptides formed from antibody fragments.

[00164] The present invention includes LDLRR (e.g., LRP6 and/or LRP5 LDLRR domain) binding antibody fragments comprising any of the foregoing heavy or light chain Attorney Docket No.: 27527-0106WO1 sequences and which bind LDLRR. The term fragments as used herein refers to any 3 or more contiguous amino acids (e.g., 4 or more, 5 or more 6 or more, 8 or more, or even 10 or more contiguous amino acids) of the antibody and encompasses Fab, Fab', F(ab')2, and F(v) fragments, or the individual light or heavy chain variable regions or portion thereof. LDLRR binding fragments include, for example, Fab, Fab', F(ab')2, Fv and scFv. These fragments lack the Fc fragment of an intact antibody, clear more rapidly from the circulation, and can have less non-specific tissue binding than an intact antibody. See Wahl et al. (1983), J. Nucl. Med., 24: 316-25. These fragments can be produced from intact antibodies using well known methods, for example by proteolytic cleavage with enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab¾ fragments).

[00165] Antibodies and antigen-binding fragments thereof that bind to an epitope comprised at least partially or completely within the LDLRR domain (e.g. the LRP6 LDLRR domain having amino acid sequence PTCSPQQFTCFTGEIDCIPVAWRCDGFTECEDHSD ELNCPVCSESQFQCASGQCIDGALRCNGDANCQDKSDEKNCEVLCLIDQFRCANGQ CIGKHKKCDHNVDCSDKSDELDCY (SEQ ID NO: 3), which corresponds to amino acid residues 1247-1361 of the mature human LDLRR protein (SEQ ID NO: 2), or the LRP5 LDLRR domain corresponding to SEQ ID NO: 9, or the LI, L2, or L3 subdomains corresponding to SEQ ID NO: 13-15, respectively) can be identified by assays known in the art. For example, the key amino acid residues (epitope) bound by an LDLRR binding antibody or fragment may be determined using a peptide array, such as for example, a PepSpot™ peptide array (JPT Peptide Technologies, Berlin, Germany), wherein a scan of twelve amino-acid peptides, spanning the entire LDLRR amino acid sequence, each peptide overlapping by 11 amino acid to the previous one, is synthesized directly on a membrane. The membrane carrying the peptides is then probed with the antibody for which epitope binding information is sought, for example at a concentration of 2 μg/ml, for 2 hr at room temperature. Binding of antibody to membrane bound peptides may be detected using a secondary HRP-conjugated goat anti-human (or mouse, when appropriate) antibody, followed by enhanced chemiluminescence (ECL). The peptides spot(s) corresponding to particular amino acid residues or sequences of the mature LDLRR protein, and which score positive for antibody binding, are indicative of the epitope bound by the particular antibody.

[00166] An antibody of unknown specificity may also be assayed in a binding competition assay with a known antibody to determine if it binds to an epitope contained within the

LDLRR domain sequence. Binding competition assays may be performed, for example, to Attorney Docket No.: 27527-0106WO1 compare binding of an antibody with unknown specificity to an antibody with a known binding specificity, using, e.g., a BIACORE® instrument for kinetic analysis of binding interactions or by ELISA. In such an assay, the antibody of unknown epitope specificity is evaluated for its ability to compete for binding against the known comparator antibody. Competition for binding to a particular epitope is determined by a reduction in binding to the LDLRR epitope of at least about 50%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 95%, or at least about 99% or about 100% for the known comparator antibody and is indicative of binding to substantially the same epitope.

[00167] Antigen-binding fragments of an antibody include fragments that retain the ability to specifically bind to an antigen, generally by retaining the antigen-binding portion of the antibody. It is well established that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of antigen-binding portions include (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment which is the VH and CHI domains; (iv) a Fv fragment which is the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al, (1989) Nature 341 :544-546), which is a V_H domain; and (vi) an isolated complementarity determining region (CDR). Single chain antibodies are also encompassed within the term antigen-binding portion of an antibody. The LDLRR binding antibodies and fragments of the present invention also encompass monovalent or multivalent, or monomeric or multimeric (e.g. tetrameric), CDR-derived binding domains with or without a scaffold (for example, protein or carbohydrate scaffolding).

[00168] The present LDLRR binding antibodies or fragments may be part of a larger immunoadhesion molecules, formed by covalent or non-covalent association of the antibody or antibody portion with one or more other proteins or peptides. Examples of such immunoadhesion molecules include use of the streptavidin core region to make a tetrameric scFv molecule (Kipriyanov, S. M., et al. (1995) Human Antibodies and Hybridomas 6:93-

101) and use of a cysteine residue, a marker peptide and a C-terminal polyhistidine tag to make bivalent and biotinylated scFv molecules (Kipriyanov, S. M., et al. (1994) Mol.

Immunol. 31 : 1047-1058). Antibodies and fragments comprising immunoadhesion molecules can be obtained using standard recombinant DNA techniques, as described herein. Preferred antigen binding portions are complete domains or pairs of complete domains. Attorney Docket No.: 27527-0106WO1

[00169] The LDLRR binding antibodies and fragments of the present invention also encompass domain antibody (dAb) fragments (Ward et al, Nature 341 :544-546, 1989) which consist of a VH domain. The LDLRR binding antibodies and fragments of the present invention also encompass diabodies, which are bivalent antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see e.g., EP 404,097; WO 93/11 161; Holliger et al, Proc. Natl. Acad. Sci. USA 90:6444- 6448, 1993, and Poljak et al, Structure 2: 1 121-1123, 1994). Diabodies can be bispecific or monospecific.

[00170] The LDLRR binding antibodies and fragments of the present invention also encompass single-chain antibody fragments (scFv) that bind to LDLRR. An scFv comprises an antibody heavy chain variable region (VH) operably linked to an antibody light chain variable region (VL) wherein the heavy chain variable region and the light chain variable region, together or individually, form a binding site that binds LDLRR. An scFv may comprise a VH region at the amino-terminal end and a VL region at the carboxy -terminal end. Alternatively, scFv may comprise a VL region at the amino-terminal end and a VH region at the carboxy -terminal end. Furthermore, although the two domains of the Fv fragment, VH and VL, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VH and VL regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883).

[00171] An scFv may optionally further comprise a polypeptide linker between the heavy chain variable region and the light chain variable region. Such polypeptide linkers generally comprise between 1 and 50 amino acids, alternatively between 3 and 12 amino acids, alternatively 2 amino acids. An example of a linker peptide for linking heavy and light chains in an scFv comprises the 5 amino acid sequence Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 16). Other examples comprise one or more tandem repeats of this sequence (for example, a polypeptide comprising two to four repeats of Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 16) to create linkers. Attorney Docket No.: 27527-0106WO1

[00172] The LDLRR binding antibodies and binding fragments of the present invention also encompass heavy chain antibodies (HCAb). Exceptions to the H2L2 structure of conventional antibodies occur in some isotypes of the immunoglobulins found in camelids (camels, dromedaries and llamas; Hamers-Casterman et al, 1993 Nature 363: 446; Nguyen et al, 1998 J. Mol. Biol. 275: 413), wobbegong sharks (Nuttall et al, Mol Immunol. 38:313-26, 2001), nurse sharks (Greenberg et al, Nature 374: 168-73, 1995; Roux et al, 1998 Proc. Nat. Acad. Sci. USA 95: 11804), and in the spotted ratfish (Nguyen, et al, "Heavy -chain antibodies in Camelidae; a case of evolutionary innovation", 2002 Immunogenetics 54(1): 39-47). These antibodies can apparently form antigen-binding regions using only heavy chain variable regions, in that these functional antibodies are dimers of heavy chains only (referred to as "heavy-chain antibodies" or "HCAbs"). Accordingly, some embodiments of the present LDLRR binding antibodies and binding fragments may be heavy chain antibodies that specifically bind to LDLRR. For example, heavy chain antibodies that are a class of IgG and devoid of light chains are produced by animals of the genus Camelidae which includes camels, dromedaries and llamas (Hamers-Casterman et al, Nature 363 :446-448 (1993)). HCAbs have a molecular weight of about 95 kDa instead of the about 160 kDa molecular weight of conventional IgG antibodies. Their binding domains consist only of the heavy- chain variable domains, often referred to as VHH to distinguish them from conventional VH Muyldermans et al, J. Mol. Recognit. 12: 131-140 (1999). The variable domain of the heavy- chain antibodies is sometimes referred to as a nanobody (Cortez-Retamozo et al, Cancer Research 64:2853-57, 2004). A nanobody library may be generated from an immunized dromedary as described in Conrath et al, (Antimicrob Agents Chemother 45: 2807-12, 2001) or using recombinant methods.

[00173] Since the first constant domain (CHI) is absent (spliced out during mRNA processing due to loss of a splice consensus signal), the variable domain (VHH) is immediately followed by the hinge region, the CH2 and the CH3 domains (Nguyen et al, Mol. Immunol. 36:515-524 (1999); Woolven et al, Immunogenetics 50:98-101 (1999)). Camelid VHH reportedly recombines with IgG2 and IgG3 constant regions that contain hinge, CH2, and CH3 domains and lack a CHI domain (Hamers-Casterman et al, supra). For example, llama IgGl is a conventional (H2L2) antibody isotype in which VH recombines with a constant region that contains hinge, CHI, CH2 and Cm domains, whereas the llama IgG2 and IgG3 are heavy chain-only isotypes that lack CHI domains and that contain no light chains. Attorney Docket No.: 27527-0106WO1

[00174] Although the HCAbs are devoid of light chains, they have an antigen-binding repertoire. The genetic generation mechanism of HCAbs is reviewed in Nguyen et al. Adv. Immunol 79:261-296 (2001) and Nguyen et al, Immunogenetics 54:39-47 (2002). Sharks, including the nurse shark, display similar antigen receptor-containing single monomeric V- domains. Irving et al, J. Immunol. Methods 248:31-45 (2001); Roux et al, Proc. Natl. Acad. Sci. USA 95: 1 1804 (1998).

[00175] VHH comprise small intact antigen-binding fragments (for example, fragments that are about 15 kDa, 118-136 residues). Camelid VHH domains have been found to bind to antigen with high affinity (Desmyter et al, J. Biol. Chem. 276:26285-90, 2001), with V_HH affinities typically in the nanomolar range and comparable with those of Fab and scFv fragments. VHHS are highly soluble and more stable than the corresponding derivatives of scFv and Fab fragments. VH fragments have been relatively difficult to produce in soluble form, but improvements in solubility and specific binding can be obtained when framework residues are altered to be more Vn_H-like. (See, for example, Reichman et al, J Immunol Methods 1999, 231 :25-38.) VHHS carry amino acid substitutions that make them more hydrophilic and prevent prolonged interaction with BiP (immunoglobulin heavy-chain binding protein), which normally binds to the H-chain in the Endoplasmic Reticulum (ER) during folding and assembly, until it is displaced by the L-chain. Because of the VHHS' increased hydrophilicity, secretion from the ER is improved.

[00176] Functional VHHS may be obtained by proteolytic cleavage of HCAb of an immunized camelid, by direct cloning of V.sub.HH genes from B-cells of an immunized camelid resulting in recombinant VHHS, or from naive or synthetic libraries. VHHS with desired antigen specificity may also be obtained through phage display methodology. Using VHHS in phage display is much simpler and more efficient compared to Fabs or scFvs, since only one domain needs to be cloned and expressed to obtain a functional antigen-binding fragment. Muyldermans, Biotechnol. 74:277-302 (2001); Ghahroudi et al, FEBS Lett. 414:521-526 (1997); and van der Linden et al, J. Biotechnol. 80:261-270 (2000). Methods for generating antibodies having camelid heavy chains are also described in U.S. Patent Publication Nos. 20050136049 and 20050037421.

[00177] Ribosome display methods may be used to identify and isolate scFv and/or VHH molecules having the desired binding activity and affinity. Irving et al, J. Immunol. Methods Attorney Docket No.: 27527-0106WO1

248:31-45 (2001). Ribosome display and selection has the potential to generate and display large libraries (10¹⁴).

[00178] Other embodiments provide Vn_H-like molecules generated through the process of camelization, by modifying non-Camelidae VHS, such as human VHHS, to improve their solubility and prevent non-specific binding. This is achieved by replacing residues on the VLS side of VHS with Vn_H-like residues, thereby mimicking the more soluble VHH fragments. Camelised VH fragments, particularly those based on the human framework, are expected to exhibit a greatly reduced immune response when administered in vivo to a patient and, accordingly, are expected to have significant advantages for therapeutic applications. Davies et al, FEBS Lett. 339:285-290 (1994); Davies et al, Protein Eng. 9:531-537 (1996); Tanha et al, J. Biol. Chem. 276:24774-24780 (2001); and Riechmann et al, Immunol. Methods 231 :25-38 (1999).

[00179] A wide variety of expression systems are available for the production of LDLRR fragments including Fab fragments, scFv, and VHHS. For example, expression systems of both prokaryotic and eukaryotic origin may be used for the large-scale production of antibody fragments and antibody fusion proteins. Particularly advantageous are expression systems that permit the secretion of large amounts of antibody fragments into the culture medium.

[00180] Production of bispecific Fab-scFv ("bibody") and trispecific Fab-(scFv)(2) ("tribody") are described in Schoonjans et al. (J Immunol. 165:7050-57, 2000) and Willems et al. (J Chromatogr B Analyt Technol Biomed Life Sci. 786: 161-76, 2003). For bibodies or tribodies, a scFv molecule is fused to one or both of the VL-CL (L) and VH-CHI (Fd) chains, e.g., to produce a tribody two scFvs are fused to C-term of Fab while in a bibody one scFv is fused to C-term of Fab. A "minibody" consisting of scFv fused to CH3 via a peptide linker (hingeless) or via an IgG hinge has been described in Olafsen, et al, Protein Eng Des Sel. 2004 April; 17(4):315-23.

[00181] Intrabodies are single chain antibodies which demonstrate intracellular expression and can manipulate intracellular protein function (Biocca, et al, EMBO J. 9: 101-108, 1990; Colby et al, Proc Natl Acad Sci U S A. 101 : 17616-21, 2004). Intrabodies, which comprise cell signal sequences which retain the antibody construct in intracellular regions, may be produced as described in Mhashilkar et al (EMBO J14: 1542-51, 1995) and Wheeler et al. (FASEB J. 17: 1733-5. 2003). Transbodies are cell-permeable antibodies in which a protein Attorney Docket No.: 27527-0106WO1 transduction domains (PTD) is fused with single chain variable fragment (scFv) antibodies Heng et al., (Med Hypotheses. 64: 1105-8, 2005).

[00182] The LDLRR binding antibodies and fragments of the present invention also encompass antibodies that are SMIPs or binding domain immunoglobulin fusion proteins specific for target protein. These constructs are single-chain polypeptides comprising antigen binding domains fused to immunoglobulin domains necessary to carry out antibody effector functions. See e.g., WO03/041600, U.S. Patent publication 20030133939 and US Patent Publication 200301 18592.

[00183] The LDLRR binding antibodies and fragments of the present invention also encompass immunoadhesins. One or more CDRs may be incorporated into a molecule either covalently or noncovalently to make it an immunoadhesin. An immunoadhesin may incorporate the CDR(s) as part of a larger polypeptide chain, may covalently link the CDR(s) to another polypeptide chain, or may incorporate the CDR(s) noncovalently. The CDRs disclosed herein permit the immunoadhesin to specifically bind to LDLRR.

[00184] The LDLRR binding antibodies and fragments of the present invention also encompass antibody mimics comprising one or more LDLRR binding portions built on an organic or molecular scaffold (such as a protein or carbohydrate scaffold). Proteins having relatively defined three-dimensional structures, commonly referred to as protein scaffolds, may be used as reagents for the design of antibody mimics. These scaffolds typically contain one or more regions which are amenable to specific or random sequence variation, and such sequence randomization is often carried out to produce libraries of proteins from which desired products may be selected. For example, an antibody mimic can comprise a chimeric non-immunoglobulin binding polypeptide having an immunoglobulin-like domain containing scaffold having two or more solvent exposed loops containing a different CDR from a parent antibody inserted into each of the loops and exhibiting selective binding activity toward a ligand bound by the parent antibody. Non-immunoglobulin protein scaffolds have been proposed for obtaining proteins with novel binding properties. (Tramontano et al, J. Mol. Recognit. 7:9, 1994; McConnell and Hoess, J. Mol. Biol. 250:460, 1995). Other proteins have been tested as frameworks and have been used to display randomized residues on alpha helical surfaces (Nord et al, Nat. Biotechnol. 15:772, 1997; Nord et al, Protein Eng. 8:601, 1995), loops between alpha helices in alpha helix bundles (Ku and Schultz, Proc. Natl. Acad. Sci. USA 92:6552, 1995), and loops constrained by disulfide bridges, such as those of the Attorney Docket No.: 27527-0106WO1 small protease inhibitors (Markland et al, Biochemistry 35:8045, 1996; Markland et al, Biochemistry 35:8058, 1996; Rottgen and Collins, Gene 164:243, 1995; Wang et al, J. Biol. Chem. 270: 12250, 1995). Methods for employing scaffolds for antibody mimics are disclosed in U.S. Pat. No. 5,770,380 and US Patent Publications 2004/01711 16, 2004/0266993, and 2005/0038229.

[00185] An LRP6 LDLRR antibody and binding fragment thereof may be highly specific for LRP6 LDLRR domain and not cross-react with (i.e., also bind to) the LDLRR domain of LRP5. However, in certain embodiments, an LRP6 LDLRR binding antibody or binding fragment thereof may cross-react with the LDLRR domain of LRP5. Similarly, an LRP5 LDLRR binding antibody or binding fragment thereof may or may not cross-react with the LDLRR domain of LRP6.

[00186] While LDLRR binding antibodies and binding fragments thereof (e.g., LRP5 and/or LRP6 binding antibodies and fragments) are the preferred inhibitors and agonists of the present invention, any inhibitor and/or agonist that specifically targets the LDLRR domain, e.g., of LRP6 and/or LRP5, are encompassed by the present invention. Examples of other such inhibitors and agonists are provided, below.

Small Molecules

[00187] By way of non-limiting example, the skilled artisan can develop small molecule inhibitors or agonists of the LDLRR domain to modulate autocrine canonical Wnt signaling in a cell (e.g., cancer cell). Such compositions include small molecule inhibitors of signaling molecules in the canonical Wnt signaling pathway. Chemical agents, referred to in the art as "small molecule" compounds, are typically organic, non-peptide molecules, having a molecular weight less than 10,000 Da, preferably less than 5,000 Da, more preferably less than 1,000 Da, and most preferably less than 500 Da. This class of modulators includes chemically synthesized molecules, for instance, compounds from combinatorial chemical libraries. Synthetic compounds may be rationally designed or identified utilizing the screening methods described below for characterizing the inhibitory or agonistic activity of a test compound on canonical Wnt signaling, and, in particular, on activated autocrine Wnt signaling, e.g., in a cancer cell. Alternative appropriate modulators of this class are natural products, particularly secondary metabolites from organisms such as plants or fungi, which can also be identified by screening compound libraries for canonical Wnt signaling inhibitory or agonistic activity. Methods for generating and obtaining small molecules are well known Attorney Docket No.: 27527-0106WO1 in the art (Schreiber, Science 2000; 151 : 1964-1969; Radmann et al, Science 2000; 151 : 1947- 1948; and Chen et al. Am J Physiol Gastrointest Liver Physiol. 2010 Aug;299(2):G293-300). A small molecule inhibitor or agonist of the invention can be determined to inhibit or be an agonist of (enhance) autocrine Wnt signaling in a cell using, e.g., the screening assays described below

Antisense Nucleic Acids

[00188] Antisense nucleic acids (including, e.g., TFOs, ribozymes, siRNA, shRNA) (oligonucleotide inhibitors), can also be used to inhibit expression of a target protein of the invention by targeting, specifically the LDLRR domain (e.g., of LRP5 and/or LRP6). An "antisense nucleic acid" is a single stranded nucleic acid molecule which, on hybridizing under cytoplasmic conditions with complementary bases in an RNA or DNA molecule, inhibits the latter's role. If the RNA is a messenger RNA transcript, the antisense nucleic acid is a countertranscript or mRNA-interfering complementary nucleic acid. As presently used, "antisense" broadly includes RNA-RNA interactions, RNA-DNA interactions, ribozymes and RNase-H mediated arrest. Antisense nucleic acid molecules can be encoded by a recombinant gene for expression in a cell (e.g., U.S. Patent No. 5,814,500; U.S. Patent No. 5,81 1,234), or alternatively they can be prepared synthetically (e.g., U.S. Patent No. 5,780,607).

[00189] Specific non-limiting examples of synthetic oligonucleotides envisioned for this invention include oligonucleotides that contain phosphorothioates, phosphotriesters, methyl phosphonates, short chain alkyl, or cycloalkl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages. Most preferred are those with CH2-NH-0-CH2, CH2- N(CH3)-0-CH2, CH2-0-N(CH3)-CH2, CH2-N(CH3)-N(CH3)-CH2 and 0-N(CH3)-CH2- CH2 backbones (where phosphodiester is 0-P02-0-CH2). US Patent No. 5,677,437 describes heteroaromatic olignucleoside linkages. Nitrogen linkers or groups containing nitrogen can also be used to prepare oligonucleotide mimics (U.S. Patents No. 5,792,844 and No. 5,783,682). U.S. Patent No. 5,637,684 describes phosphoramidate and phosphorothioamidate oligomeric compounds. Also envisioned are oligonucleotides having morpholino backbone structures (U.S. Patent No. 5,034,506). In other embodiments, the phosphodiester backbone of the oligonucleotide may be replaced with a polyamide backbone, the bases being bound directly or indirectly to the aza nitrogen atoms of the polyamide backbone (Nielsen, P.E., Egholm, M., Berg, R.H., and Buchardt, O. (1991) Science 254, Attorney Docket No.: 27527-0106WO1

1497-1500). Other synthetic oligonucleotides may contain substituted sugar moieties comprising one of the following at the 2' position: OH, SH, SCH3, F, OCN, 0(CH2)nNH2 or 0(CH2)nCH3 where n is from 1 to about 10; CI to CIO lower alkyl, substituted lower alkyl, alkaryl or aralkyl; CI; Br; CN; CF3; OCF3; 0-; S-, or N-alkyl; 0-, S-, or N-alkenyl; SOCH3 ; S02CH3; ON02;N02; N3; NH2; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substitued silyl; a fluorescein moiety; an RNA cleaving group; a reporter group; an intercalator; a group for improving the pharmacokinetic properties of an oligonucleotide; or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. Oligonucleotides may also have sugar mimetics such as cyclobutyls or other carbocyclics in place of the pentofuranosyl group. Nucleotide units having nucleosides other than adenosine, cytidine, guanosine, thymidine and uridine, such as inosine, may be used in an oligonucleotide molecule.

[00190] Preferred vectors in vitro, in vivo, and ex vivo are viral vectors, such as lentiviruses, retroviruses, herpes viruses, adenoviruses, adeno-associated viruses, vaccinia virus, baculovirus, and other recombinant viruses with desirable cellular tropism. Thus, a gene encoding a functional or mutant protein or polypeptide domain fragment thereof can be introduced in vivo, ex vivo, or in vitro using a viral vector or through direct introduction of DNA. Expression in targeted tissues can be effected by targeting the transgenic vector to specific cells, such as with a viral vector or a receptor ligand, or by using a tissue-specific promoter, or both. Targeted gene delivery is described in PCT Publication WO 95/28494.

[00191] Various companies produce viral vectors commercially, including but by no means limited to Avigen, Inc. (Alameda, CA; AAV vectors), Cell Genesys (Foster City, CA; retroviral, adenoviral, AAV vectors, and lentiviral vectors), Clontech (retroviral and baculoviral vectors), Genovo, Inc. (Sharon Hill, PA; adenoviral and AAV vectors), Genvec (adenoviral vectors), IntroGene (Leiden, Netherlands; adenoviral vectors), Molecular Medicine (retroviral, adenoviral, AAV, and herpes viral vectors), Norgen (adenoviral vectors), Oxford BioMedica (Oxford, United Kingdom; lentiviral vectors), and Transgene (Strasbourg, France; adenoviral, vaccinia, retroviral, and lentiviral vectors).

[00192] In another embodiment, the vector can be non- viral. Such vectors include "naked" DNA, and transfection facilitating agents (peptides, polymers, etc.). Synthetic cationic lipids can be used to prepare liposomes for transfection of a gene encoding (Feigner et al. (1987) Proc. Nat'l. Acad. Sci. U.S.A. 84, 7413-7417; Feigner and Ringold (1989) Attorney Docket No.: 27527-0106WO1

Science 337, 387-388; see Mackey et al. (1988) Proc. Nat'l. Acad. Sci. U.S.A. 85, 8027- 8031 ; Ulmer et al. (1993) Science 259, 1745-1748). Useful lipid compounds and compositions for transfer of nucleic acids are described in International Patent Publications W095/18863 and W096/17823, and in U.S. Patent No. 5,459, 127. Lipids may be chemically coupled to other molecules for the purpose of targeting (see Mackey et. al. (1988) Proc. Nat'l. Acad. Sci. U.S.A. 85, 8027-8031). Targeted peptides, e.g., hormones or neurotransmitters, and proteins such as antibodies, or non-peptide molecules could be coupled to liposomes chemically. Other molecules are also useful for facilitating transfection of a nucleic acid in vivo, such as a cationic oligopeptide (e.g., International Patent Publication W095/21931), peptides derived from DNA binding proteins (e.g., International Patent Publication WO96/25508), or a cationic polymer (e.g., International Patent Publication W095/21931).

[00193] It is also possible to introduce the vector as a naked DNA plasmid. Naked DNA vectors for gene therapy can be introduced into the desired host cells by methods known in the art, e.g., electroporation, microinjection, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun, or use of a DNA vector transporter (see, e.g., Wu et al.(l 992) J. Biol. Chem. 267, 963-967; Wu and Wu (1988) J. Biol. Chem. 263, 14621-14624; Hartmut et al, Canadian Patent Application No. 2,012,31 1, filed March 15, 1990; and Williams et al. (1991) Proc. Nat'l. Acad. Sci. USA 88, 2726-2730). Receptor-mediated DNA delivery approaches can also be used (Curiel et al. (1992) Hum. Gene Ther. 3, 147-154; and Wu and Wu (1987) J. Biol. Chem. 262, 4429-4432). U.S. Patent Nos. 5,580,859 and 5,589,466 disclose delivery of exogenous DNA sequences, free of transfection facilitating agents, in a mammal. Recently, a relatively low voltage, high efficiency in vivo DNA transfer technique, termed electrotransfer, has been described (Mir et al. (1998) CP. Acad. Sci. 321, 893; WO 99/01 157; WO 99/01158; and WO 99/01175).

[00194] In some embodiments, the oligonucleotide inhibitor is at least 12 nucleobases in length. In other embodiments, the oligonucleotide inhibitor is at least 15 nucleobases in length. The oligonucleotide inhibitor may also be less than 22 nucleobases in length. Thus, in some embodiments, the oligonucleotide inhibitor is from 7 to 21 nucleobases in length. In other embodiments, the oligonucleotide inhibitor is from 8 to 21, 9 to 21, to 21, 11 to 21, 12 to 21, 13 to 21, 14 to 21, 15 to 21, 16 to 21, 17 to 21, or 18 to 21 nucleobases in length. In some instances, the oligonucleotide inhibitor is 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 nucleobases in length. Attorney Docket No.: 27527-0106WO1

[00195] As stated above, the oligonucleotide inhibitor may hybridize under stringent conditions to its target nucleic acid molecule (e.g., the nucleic acid sequence encoding the LDLRR domain of LRP 5 and/or LRP6). In some embodiment, the oligonucleotide inhibitor hybridizes under low stringency hybridization conditions to its target nucleic acid molecule. In other embodiments, the oligonucleotide inhibitor hybridizes under moderately stringent hybridization conditions to its target nucleic acid molecule. In other embodiments, the oligonucleotide inhibitor hybridizes under highly stringent hybridization conditions to its target nucleic acid molecule.

[00196] In some embodiments, the oligonucleotide inhibitor is substantially complementary to its target nucleic acid molecule (e.g., the nucleic acid sequence encoding the LDLRR domain of LRP 5 and/or LRP6 (SEQ ID NOs: 3 and 9, respectively)). The oligonucleotide inhibitor may have at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, or at least 85% sequence complementarity to a target region (e.g. seed region) within its target nucleic acid molecule. In other embodiments, an oligonucleotide inhibitor includes at least 90% sequence complementarity to a target region (e.g. seed region) within its target nucleic acid molecule. In other embodiments, the oligonucleotide inhibitor includes at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence complementarity to a target region (e.g. seed region) within its target nucleic acid molecule. For example, an oligonucleotide inhibitor in which 18 of 20 of its nucleobases are complementary to a target sequence (e.g. seed region) would represent 90 percent complementarity. Where an oligonucleotide inhibitor is substantially complementary to its target nucleic acid molecule, the remaining non-complementary nucleobases may be clustered or interspersed with complementary nucleobases and need not be contiguous to each other or to complementary nucleobases. Thus, an oligonucleotide inhibitor which is 22 nucleobases in length having 6 (six) non-complementary nucleobases which are flanked by two regions of complete complementarity with its target nucleic acid molecule would have 72.7% overall complementarity with the its target nucleic acid molecule. Percent complementarity of an oligonucleotide inhibitor with a region of its target nucleic acid molecule can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art (Altschul et al, J. Mol. Biol, 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656). In some embodiments, the oligonucleotide inhibitor is perfectly complementary to its target nucleic acid molecule (e.g., Attorney Docket No.: 27527-0106WO1 the nucleic acid sequence encoding the LDLRR domain of LRP 5 and/or LRP6 (SEQ ID NOs: 3 and 9, respectively)).

[00197] An antisense nucleic acid can be determined to inhibit autocrine Wnt signaling in a cell using, e.g., the screening assays described below.

Peptide Inhibitors and Agonists

[00198] Peptides may also be used as inhibitors or agonists of autocrine Wnt signaling by specifically targeting the LDLRR domain, e.g., of LRP5 and/or LRP6. Methods for the design of such peptides, based on the known amino acid sequences and structures of LRP5 and LRP6, and, in particular, the LDLRR domain, are known in the art. See, e.g., Nerbo et al. J Pept Sci. 2007 Dec; 13(12):822-32; Caputo et al. Biochemistry (2008) 47(33): 8600-8606; Kosugi et al. Chemistry & Biology (2008) Volume: 15, Issue: 9, Pages: 940-949; Hruby, V.J. (2002) Nat Rev Drug Discov; 1 1 :847-58. A peptide inhibitor or agonist of the invention can be determined to inhibit or be an agonist of (enhance) autocrine Wnt signaling in a cell using, e.g., the screening assays described below.

Methods of Screening Anti-LDLR Antibodies and Binding Fragments

[00199] The LDLRR binding specificity of antibodies and antigen-binding fragments thereof and functional activity (e.g., ability to inhibit or act as agonists of canonical Wnt signaling through LRP6 and/or LRP5) can be determined using in vitro assays known in the art. Such assays may also be used to assess the functional activity of other inhibitors and/or agonists of the invention, such as, e.g., small molecules, peptides and antisense nucleic acids.

[00200] For example, and without limitation, to determine if an LDLRR binding antibody or antigen-binding fragment thereof binds to an LDLRR domain, an antigen-specific ELISA may be performed, to detect binding to LDLRR (e.g. of LRP6 and/or LRP5) and or specific epitopes located partially or completely within the LDLRR domain.

[00201] Preferred LDLRR binding antibodies or antigen-binding fragments thereof for use in accordance with the invention generally bind to human LRP6 and/or LRP5 LDLRR with high affinity (e.g., as determined with BIACORE™ instrument for kinetic analysis of binding interactions). For example, but not necessarily, a preferred antibody or fragment will bind LDLRR domain with an equilibrium binding dissociation constant (KD) for LDLRR of about 10 nM or less, about 5 nM or less, about 1 nM or less, about 500 pM or less, or more preferably about 250 pM or less, about 100 pM or less, about 50 pM or less, about 25 pM or Attorney Docket No.: 27527-0106WO1 less, about 10 pM or less, about 5 pM or less, about 3 pM or less about 1 pM or less, about 0.75 pM or less, about 0.5 pM or less, or about 0.3 pM or less.

[00202] Further, in vitro and cell based screening assays for determining the functional activity of an LDLRR antibody or binding fragment thereof, or of a small molecule and/or antisense nucleic acid (e.g., inhibitory or agonistic activity) are well described in the art. By way of non-limiting example, the Wnt inhibitory and/or agonist activity of an antibody or antigen-binding fragment thereof directed against, e.g., the LRP6 LDLRR domain can be assayed in Wnt autocrine activated tumor cells that preferentially express LRP6 and/or LRP5, for example, but not limited to sarcoma cells, ovarian, breast and lung carcinoma cells, and glioma cells (see, e.g., Vijayakumar et al, Cancer Cell, 19:601-12, 2011 (disclosing autocrine Wnt signaling in HOS and A204 sarcoma cells) and Akiri et al., Oncogene, 28:2163-2172, 2009 (disclosing active autocrine Wnt signaling in lung carcinoma cells). Assays for screening the modulatory activity of an antisense nucleic acid are described in PCT Publication No. WO/2006/055635 and such methods may also be used to screen the activity of the small molecules described herein.

[00203] Wnt autocrine signaling in the cells following treatment with a candidate inhibitor or agonist can be quantified by measuring the levels of uncomplexed β-catenin in cell lysates and/or by quantifying Wnt signaling levels using a TCF luciferase reporter, which is exogenously expressed in the cells by lentiviral-mediated transduction (Vijayakumar et al, supra, and Akiri et al, supra.) The cells may be stimulated or not with one or more Wnt canonical ligands including, e.g., Wntl, Wnt2, Wnt3, Wnt3a, Wnt6, Wnt7a, Wnt7b, Wnt9a, WntlOa, and/or WntlOb, in the presence or absence of the candidate inhibitor or agonist (e.g., LRP 5 and/or LRP6 LDLLR domain binding antibody or antigen-binding fragment, small molecule and/or antisense nucleic acid) at different concentrations and at different time points (e.g., ranging from overnight to 72 hours). LDLRR binding antibodies and fragments thereof, small molecules and/or antisense nucleic acids that inhibit LRP6 and/or LRP5 signaling will significantly reduce the level of TCF luciferase reporter activity and decrease the level of uncomplexed β-catenin in treated cell lysates. Positive controls in such assays can include treatment of cells with a Wnt cell surface antagonist such as DKK1 or FRP or transducing such cells with a DNTCF transcription factor (see Vijayakumar et al, supra, and Akiri et al, supra). Test substances which are agonists of LRP5 and/or LRP6 signaling will increase the level of TCF luciferase reporter activity and increase the level of uncomplexed b-catenin in treated cell lysates. Attorney Docket No.: 27527-0106WO1

[00204] Other functional assays can include the following assessments: reduced levels of Axin2, LEF1, DKK1, DKK2 and CDC25A; ability to induce LRP6 and/or LRP5 internalization (e.g., by quantifying cell surface levels of the protein before and after treatment); ability to block LRP6 and/or LRP5 phosphorylation (as determined using specific antibodies recognizing phosphorylated LRP 5 or 6 and total LRP 5 or 6); and ability to inhibit cell growth/proliferation (e.g., tumor cells having activated autocrine Wnt signaling) (see, e.g., Vijayakumar, et al. (2011) Cancer Cell; 19(5):601-12).

[00205] Further analysis of efficacy of the LDLRR antibody or binding fragment thereof can include analysis of the in vitro and in vivo inhibition of tumor cell growth alone or in combination with chemo/irradiation in in vivo models as described in Vijayakumar et al, supra.

[00206] It certain embodiments, an LDLRR binding antibody or antigen-binding fragment thereof is an agonist of canonical Wnt signaling. Enhancement of canonical Wnt signaling can be screened for using the same approaches as detailed above, e.g., by detecting increased levels of uncomplexed β-catenin in cell lysates and/or increased levels of TCF luciferase reporter activity.

Pharmaceutical Formulations

[00207] Antibodies or antibody fragments described herein may be formulated for delivery by any available route including, but not limited to parenteral (e.g., intravenous), intradermal, subcutaneous, oral, nasal, bronchial, opthalmic, transdermal (topical), transmucosal, rectal, and vaginal routes. Antibodies or antibody fragments may include a delivery agent (e.g., a cationic polymer, peptide molecular transporter, surfactant, etc., as described above) in combination with a pharmaceutically acceptable carrier. As used herein the term "pharmaceutically acceptable carrier" includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into pharmaceutical formulations comprises an antibody or fragment thereof as described herein.

[00208] A pharmaceutical composition is formulated to be compatible with its intended route of administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene Attorney Docket No.: 27527-0106WO1 glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[00209] Pharmaceutical compositions suitable for injectable use typically include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition should be sterile and should be fluid to the extent that easy syringability exists. Pharmaceutical formulations are ideally stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms such as bacteria and fungi. In general, the relevant carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The 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 the case of dispersion 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 advantageous to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[00210] Sterile injectable solutions can be prepared by incorporating the antibody or antibody fragment in the required amount 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 purified antibody or antibody fragment into a sterile vehicle which contains 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, exemplary methods of preparation are vacuum drying and freeze-drying Attorney Docket No.: 27527-0106WO1 which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[00211 ] Oral compositions generally include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the antibody or antibody fragment can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring. Formulations for oral delivery may advantageously incorporate agents to improve stability within the gastrointestinal tract and/or to enhance absorption.

[00212] For administration by inhalation, the antibody or antibody fragment and a delivery agent are preferably delivered in the form of an aerosol spray from a pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer. The present disclosure particularly contemplates delivery of the compositions using a nasal spray, inhaler, or other direct delivery to the upper and/or lower airway. Intranasal administration of DNA vaccines directed against influenza viruses has been shown to induce CD8 T cell responses, indicating that at least some cells in the respiratory tract can take up DNA when delivered by this route, and the delivery agents of the invention will enhance cellular uptake. According to certain embodiments, antibody or antibody fragment and a delivery agent are formulated as large porous particles for aerosol administration.

[00213] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the purified polypeptide or protein and delivery agents are formulated into ointments, salves, gels, or creams as generally known in the art. Attorney Docket No.: 27527-0106WO1

[00214] In certain embodiments, compositions are prepared with carriers that will protect the antibody or antibody fragment against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,81 1.

[00215] Micro encapsulation of recombinant proteins for sustained release has been performed successfully with human growth hormone (rhGH), interferon-(rhlFN-), interleukin-2, and MN rgpl20. Johnson et al, Nat. Med., 2:795-799 (1996); Yasuda, Biomed. Ther., 27: 1221-1223 (1993); Hora et al, Bio/Technologv. 8:755-758 (1990); Cleland, "Design and Production of Single Immunization Vaccines Using Polylactide Polyglycolide Microsphere Systems", in Vaccine Design: The Subunit and Adjuvant Approach, Powell and Newman, eds, (Plenum Press: New York, 1995), pp. 439-462; WO 97/03692, WO 96/40072, WO 96/07399; and U.S. Pat. No. 5,654,010. The sustained-release formulations of these proteins were developed using poly-lactic-coglycolic acid (PLGA) polymer due to its biocompatibility and wide range of biodegradable properties. The degradation products of PLGA, lactic and glycolic acids can be cleared quickly within the human body. Moreover, the degradability of this polymer can be depending on its molecular weight and composition. Lewis, "Controlled release of bioactive agents from lactide/glycolide polymer", in: M. Chasin and R. Langer (Eds.), Biodegradable Polymers as Drug Delivery Systems (Marcel Dekker: New York, 1990), pp. 1-41. Additional examples of sustained release compositions include, for example, EP 58,481A, U.S. Pat. No. 3,887,699, EP 158,277A, Canadian Patent No. 1 176565, U. Sidman et al., Biopolymers 22, 547 [1983], R. Langer et al, Chem. Tech. 12, 98 [1982], Sinha et al, J. Control. Release 90, 261 [2003], Zhu et al, Nat. Biotechnol. 18, 24 [2000], and Dai et al, Colloids Surf B Biointerfaces 41, 1 17 [2005].

[00216] Bioadhesive polymers are also contemplated for use in or with compositions of the present invention. Bioadhesives are synthetic and naturally occurring materials able to adhere to biological substrates for extended time periods. For example, Carbopol and Attorney Docket No.: 27527-0106WO1 polycarbophil are both synthetic cross-linked derivatives of poly(acrylic acid). Bioadhesive delivery systems based on naturally occurring substances include for example hyaluronic acid, also known as hyaluronan. Hyaluronic acid is a naturally occurring mucopolysaccharide consisting of residues of D-glucuronic and N-acetyl-D-glucosamine. Hyaluronic acid is found in the extracellular tissue matrix of vertebrates, including in connective tissues, as well as in synovial fluid and in the vitreous and aqueous humour of the eye. Esterified derivatives of hyaluronic acid have been used to produce microspheres for use in delivery that are biocompatible and biodegrable (see for example, Cortivo et al, Biomaterials (1991) 12:727- 730; European Publication No. 517,565; International Publication No. WO 96/29998; Mum et al, J. Controlled Rel. (1994) 29: 133-141). Exemplary hyaluronic acid containing compositions of the present invention comprise a hyaluronic acid ester polymer in an amount of approximately 0.1% to about 40% (w/w) of an LDLRR binding antibody or fragment to hyaluronic acid polymer.

[00217] Both biodegradable and non-biodegradable polymeric matrices can be used to deliver compositions in accordance with the invention, and such polymeric matrices may comprise natural or synthetic polymers. Biodegradable matrices are preferred. The period of time over which release occurs is based on selection of the polymer. Typically, release over a period ranging from between a few hours and three to twelve months is most desirable. Exemplary synthetic polymers which can be used to form the biodegradable delivery system include: polymers of lactic acid and glycolic acid, polyamides, polycarbonates, polyalkylenes, polyalkylene glycols, polyalkylene oxides, polyalkylene terepthalates, polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, poly-vinyl halides, polyvinylpyrrolidone, polyglycolides, polysiloxanes, polyanhydrides, polyurethanes and copolymers thereof, poly(butic acid), poly(valeric acid), alkyl cellulose, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, intro celluloses, polymers of acrylic and methacrylic esters, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxy- propyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxylethyl cellulose, cellulose triacetate, cellulose sulphate sodium salt, poly(methyl methacrylate), poly(ethyl methacrylate), poly(butylmethacrylate), poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate), polyethylene, polypropylene, poly(ethylene glycol), poly(ethylene Attorney Docket No.: 27527-0106WO1 oxide), poly(ethylene terephthalate), poly(vinyl alcohols), polyvinyl acetate, poly vinyl chloride, polystyrene and polyvinylpyrrolidone. Exemplary natural polymers include alginate and other polysaccharides including dextran and cellulose, collagen, chemical derivatives thereof (substitutions, additions of chemical groups, for example, alkyl, alkylene, hydroxylations, oxidations, and other modifications routinely made by those skilled in the art), albumin and other hydrophilic proteins, zein and other prolamines and hydrophobic proteins, copolymers and mixtures thereof. In general, these materials degrade either by enzymatic hydrolysis or exposure to water in vivo, by surface or bulk erosion. The polymer optionally is in the form of a hydrogel (see for example WO 04/009664, WO 05/087201, Sawhney, et al, Macromolecules, 1993, 26, 581-587,) that can absorb up to about 90% of its weight in water and further, optionally is cross-linked with multi-valent ions or other polymers.

[00218] Delivery systems also include non-polymer systems that are lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono- di- and tri-glycerides; hydrogel release systems; silastic systems; peptide based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants; and the like. Specific examples include, but are not limited to: (a) erosional systems in which the product is contained in a form within a matrix such as those described in U.S. Pat. Nos. 4,452,775, 4,675, 189 and 5,736, 152 and (b) diffusional systems in which a product permeates at a controlled rate from a polymer such as described in U.S. Pat. Nos. 3,854,480, 5, 133,974 and 5,407,686. Liposomes containing the product may be prepared by methods known methods, such as for example (DE 3,218, 121 ; Epstein et al, Proc. Natl. Acad. Sci. USA, 82: 3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA, 77: 4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641 ; Japanese patent application 83-1 18008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324).

[00219] It is advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active antibody or antibody fragment calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

[00220] One preparation can involve an effective quantity of an LDLRR (e.g., LRP6 and/or LRP6 LDLRR domain) binding antibody or fragment in a mixture with non-toxic Attorney Docket No.: 27527-0106WO1 excipients which are suitable for the manufacture of tablets. By dissolving the tablets in sterile water, or another appropriate vehicle, solutions can be prepared in unit dose form. Suitable excipients include, but are not limited to, inert diluents, such as calcium carbonate, sodium carbonate or bicarbonate, lactose, or calcium phosphate; or binding agents, such as starch, gelatin, or acacia; or lubricating agents such as magnesium stearate, stearic acid, or talc.

[00221 ] Pharmaceutical formulations as described herein can be included in a container, pack, or dispenser together with instructions for administration.

Uses of LDLRR Inhibitors

[00222] In one embodiment, the invention provides a method of inhibiting growth of a cancer cell comprising contacting the cell with an isolated LDLRR binding antibody or antigen-binding fragment of the invention. Cancer cells that are suitable for treatment with an LDLRR antibody of the invention are cancer cells that have activated autocrine Wnt signaling. Cancer cells that have activated autocrine Wnt signaling include, but are not limited to, breast cancer, ovarian cancer and lung (e.g., non-small cell lung cancer (NSCLC)) cells, sarcoma cells, and glioma cells (e.g., astrocytomas and glioblastomas). One of skill in the art can readily determine whether a cell has activated autocrine Wnt signaling by assays well described in the art (see, e.g., Bafico, A., et al. Cancer Cell 6, 497-506 (2004)). Briefly, for example and without limitation, it can be determined if the cancer cell has upregulation of uncomplexed β-catenin and/or Axin2. Such methods are also described in detail in international patent publication no. WO 2010/121269. In one embodiment, the cancer cell does not have genetic alterations of β-catenin and/or Adenomatous Polyposis Coli (APC) (see, Giles, R. H., van Es, J. H., and Clevers, H. (2003) Biochim Biophys Acta 1653, 1-24).

[00223] Thus, in conjunction with inhibiting growth of a cancer cell, the invention provides a method of treating a cancer characterized by activated autocrine Wnt signaling in a subject by administering to a subject in need thereof an effective amount for inhibiting autocrine Wnt signaling of an LDLRR binding antibody or antigen binding fragment of such treatment.

[00224] It is further contemplated that an LDLRR (e.g., LRP6 and/or LRP6 LDLRR domain) binding antibody or fragment administered to a subject in accordance with the invention may be administered in combination with treatment with at least one additional Attorney Docket No.: 27527-0106WO1 active agent or additional therapy. The different treatments (agent or therapy) can be administered simultaneously with the LDLRR binding antibody or fragment or sequentially and are said to be "coadministered." In a specific embodiment, for example, a cancer patient is co-administered an LDLRR binding antibody according to the present invention and a chemotherapy or radiotherapy. While, as specified above, inhibition of canonical Wnt signaling by an LDLRR binding antibody or binding fragment thereof can inhibit cancer cell growth where such signaling is activated in the absence of other therapeutic modalities, the present invention also provides that the inhibition of activate autocrine Wnt signaling can cooperate with other therapeutic modalities (e.g., chemotherapeutics and/or radiation therapy) to enhance cancer cell killing. Further, in another embodiment, the invention provides a method for sensitizing a cancer cell to a treatment such as chemotherapy or radiotherapy comprising contacting the cancer cell with an isolated LDLRR binding antibody or antigen- binding fragment thereof of the invention.

[00225] The combination therapy method of the present invention comprises combining inhibiting activated canonical Wnt signaling with any chemotherapeutics and/or radiation therapy method useful for a given type of tumor. Examples of chemotherapeutic agents useful in the combination treatments of the invention include, but are not limited to, agents which induce apoptosis, necrosis, mitotic cell death, alkylating agents, purine antagonists, pyrimidine antagonists, plant alkaloids, intercalating antibiotics, aromatase inhibitors, antimetabolites, mitotic inhibitors, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, biological response modifiers, steroid hormones, and anti- androgens. Some non-limiting specific examples of such useful chemotherapeutic agents include, e.g., cisplatin, erlotinib, Navelbine, gemcitabine (2'-2'-difluorodeoxycytidine), methotrexate, 5-fluorouracil (5FU), taxol, doxorubicin, paclitaxel, mitomycin C, etoposide, carmustine, and Gliadel Wafer.

[00226] In the therapeutic methods of the present invention, the Wnt signaling inhibitors can be administered alone or in combination with one or more chemotherapeutic agents and/or radiation treatment to the individual in need thereof, either locally or systemically. Depending on the severity and responsiveness of the cancer to be treated, dosing can be of a single or a plurality of administrations, with course of treatment lasting until a cure is effected or diminution of the disease state is achieved. The amount of compounds and radiation to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the Attorney Docket No.: 27527-0106WO1 prescribing physician, etc. Evaluation of effectiveness of Wnt signaling inhibition and combination treatments of the present invention can be performed using any method acceptable in the art. For example, for solid tumors, tumor volumes can be measured two to three times a week. Tumor volumes can be calculated using the length and width of the tumor (in millimeters). The effect of the treatment can be evaluated by comparing the tumor volume using statistical analyses such as Student's t test. In addition, histological analyses can be performed using markers typical for each type of cancer.

[00227] Compositions or therapies that are co-administered may be administered at the same time, or at different times, e.g., 1 hour apart, 2 hours apart, 3 hours apart, 5 hours apart, 7 hours apart, 9 hours apart, 12 hours apart, 18 hours apart, 24 hours apart, 1 day apart, 2 days apart, 3 days parts, 5 days apart, 7 days part, 10 days apart, 2 weeks apart or longer, depending on the specific combinations of compositions or therapies being administered, and the response of the subject being treated to the treatment.

[00228] In another embodiment, the invention provides agonists of the LDLRR domain in Wnt receptors such as LRP6 and/or LRP5, which increase autocrine Wnt signaling. Such agonists are useful, e.g., for treating diseases such as, but not limited to, osteoporosis, where there is evidence that increased Wnt canonical signaling would be beneficial (see, Liu G, et al. J Cell Biol. 2009 Apr 6; 185(l):67-75).

Dosage and Administration

[00229] The compounds of the invention and/or other therapies can be administered to a subject by any suitable route known to one of skill in the art. Exemplary compositions are suitable for injection or infusion into an animal by any route available to the skilled worker, such as intraarticular, subcutaneous, intravenous, intramuscular, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, intralesional, intrarectal, transdermal, oral, and inhaled routes. A parenteral formulation typically will be a sterile, pyrogen-free, isotonic aqueous solution, optionally containing pharmaceutically acceptable preservatives. By way of example, and without limitation, examples of suitable methods for administration of an antisense nucleic acid molecule described herein include, for example, cell transfection methods such as chemical, biological or mechanical means. Other recognized methods include electroporation, use of a virus vector, lipofection, gene guns, and microinjection. Non-limiting examples of suitable Attorney Docket No.: 27527-0106WO1 methods of administration of an antibody or binding fragment, small molecule or peptide of the invention includes intravenous administration.

[00230] Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringers' dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present, such as, for example, anti-microbials, anti-oxidants, chelating agents, inert gases and the like. See generally, Remington's Pharmaceutical Science, 16th Ed., Mack Eds., 1980, which is incorporated herein by reference. The LDLRR antibody and antibody fragment can be administered at various intervals and over different periods of time as required, e.g., one time per week for between about 1 to 10 weeks, between 2 to 8 weeks, between about 3 to 7 weeks, about 4, 5, or 6 weeks, etc. Those of ordinary skill in the art will appreciate that certain factors can influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Generally, treatment of a subject with an LDLRR binding antibody or antigen-binding fragment thereof as described herein can include a single treatment or, in many cases, can include a series of treatments. It is furthermore understood that appropriate doses may depend upon the potency of the antibody or antigen-binding fragment thereof and may optionally be tailored to the particular recipient, for example, through administration of increasing doses until a preselected desired response is achieved. It is understood that the specific dose level for any particular animal subject may depend upon a variety of factors including the activity of the specific polypeptide or protein employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drug combination, and the degree of expression or activity to be modulated.

[00231] In certain embodiments, the LDLRR binding antibody or an LDLRR-binding antibody fragment is administered in a single daily dose on at least one day of a dosing regimen, e.g., on each day of the dosing regimen, for at least one day, at least two days, at least three days, at least four days, at least five days, at least six days, or at least seven days, or longer. In certain embodiments, the LDLRR binding antibody or an LDLRR-binding Attorney Docket No.: 27527-0106WO1 antibody fragment is administered more than once a day on at least one day of the dosing regimen, e.g., on each day of the dosing regimen. In certain embodiments, the interval between administrations is at least one hour. In certain embodiments, the LDLRR binding antibody or an LDLRR-binding antibody fragment is administered over a period of time on at least one day of the dosing regimen, e.g., over a period of at least fifteen minutes.

[00232] In certain embodiments, the amount of the LDLRR binding antibody or LDLRR- binding antibody fragment administered is about 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 1 mg, 2 mg, 3 mg 5 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg or more. In other embodiments, the amount of the LDLRR binding antibody or an LDLRR-binding antibody fragment administered does not exceed about 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 1 mg, 2 mg, 3 mg 5 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, or 100 mg per day, although higher or lower doses are possible, and different doses may be administered on different days of the dosing regimen (e.g., 0.1 mg on day 1, 0.2 mg on day 2, 0.3 mg on day 3, and so on).

[00233] Moreover, doses and methods of administration may be selected in accordance with known principles of veterinary pharmacology and medicine. Guidance may be found, for example, in Adams, R. (ed.), Veterinary Pharmacology and Therapeutics, 8.sup.th edition, Iowa State University Press; ISBN: 0813817439; 2001.

[00234] Certain embodiments of methods and compositions provided herein are further illustrated by the following examples. The examples are provided for illustrative purposes only. They are not to be construed as limiting the scope or content of the invention in any way.

EXAMPLES

Example 1 : Mutations in the LDLRR of LRP6 inhibit autocrine Wnt signaling

[00235] Functional analysis of a previously unexplored LRP6 domain designated as the low-density lipoprotein receptor related (LDLRR) domain, which shares homology with a domain of the low density lipoprotein receptor (LDLR), was performed. The LDLRR has three subdomains (also termed in the art as "repeats", though their sequences are not identical) that are homologous with an LDLR domain, which is completely unrelated in its Attorney Docket No.: 27527-0106WO1 function to that of the LRP6 LDLRR domain. A sequence alignment of the relevant domain of human LDLR (amino acid residues 139-270 of the sequence having GenBank® Accession No. AAP36025.1) (SEQ ID NO: 10) and a fragment containing the LDLRR domain of LRP6 (SEQ ID NO: 11) (corresponding to residues 1239-1360 of SEQ ID NO: 2) is shown in Figure 2.

[00236] Point mutations in the homologous domain in LDLR can be associated with markedly diminished LDLR function, leading to high levels of serum cholesterol in affected individuals. For example, a D— >N substitution at position 72 of the LDLR amino acid sequence or a C— >R mutation at position 73 results in an 85-95% reduction in LDLR activity, and a C— >Y mutation at position 197 and a S— »P mutation at position 226 of the LDLR amino acid sequence results in >98% reduction in LDLR activity (Hobbs et al, Human Mutation, 1 : 445-466, 1996).

[00237] To determine whether the LDLRR domain might also play a critical role in LRP6 function, and, in particular, autocrine Wnt signaling, single point mutations within the LDLRR domain were generated at amino acid positions 1288 and 1317 of SEQ ID NO: 2 (LRP6 amino acid sequence), positions which are homologous to positions 197 and 226, respectively, in the LDLR domain, and which, as discussed above, have been determined to interfere with LDLR function when mutated. Site directed mutagenesis was performed as described by the manufacturer's protocol (Stratagene, Santa Clara, CA). Specifically, in one mutant LRP6, a C— >Y mutation was introduced at position 1288 of SEQ ID NO: 2 ("LRP6C1288Y"), and, in another, an S— >P mutation was introduced at position 1317 of SEQ ID NO: 2 ("LRP6S1317P").

[00238] Next, the mutant LRP6 proteins were tested in a canonical Wnt signaling TCF luciferase reporter assay as described in Bafico, A., et al. Cancer Cell 6, 497-506 (2004), to determine if the LDLRR domain was critical for Wnt signaling. Luciferase activity in the presence or absence of canonical Wnt3a in 293 T cells overexpressing wild-type (WT) LRP6, the LRP6 mutant C1288Y, or the LRP6 mutant S1317P, or in control 293 T cells (no exogenous LRP6 or LRP5) cells was determined. As shown in Figure 3A and Figure 3B, the LDLRR domain point mutants C1288Y and S1317P markedly diminished Wnt signaling by the LRP6 receptor in the absence (Fig. 3A) or presence (Fig. 3B) of added canonical Wnt3a. Significantly decreased signaling activities of both point mutant receptors indicated that the Attorney Docket No.: 27527-0106WO1

LDLRR domain has important functional significance for LRP6 signaling and is thus an important target for inhibition of canonical Wnt signaling.

Example 2: Generation of additional mutations in the LDLRR of LRP6 and analysis of their effect on autocrine Wnt signaling

[00239] Several additional point mutants (C1288F, W1268L, F 1293Y, E1319D, D1315N, S 1317P, E1319K) were generated within the LDLRR of LRP6 to further test the importance of this domain in Wnt signaling. Mutations in each case involved conserved regions with the homologous domain of the LDLR. Site directed mutagenesis was used to create single base substitutions on wild type human LRP6 (SEQ ID NO: 2). Each LDLRR mutant was transfected in 293T cells and 36 hours later cells were processed for luciferase assay (Akiri et al, Oncogene, 28:2163-2172, 2009; Vijayakumar et al., Cancer Cell, 201 1, 19:601-612). Whole cell lysates were used for immunoblotting.

[00240] Point mutants at positions analogous to known LDLR disease mutations (C1288Y;S1317P; D1315N; E1319K) showed in each case markedly decreased Wnt signaling (Fig. 4). However, two other point mutants (F1293Y; E1319D) also showed strikingly reduced Wnt signaling as measured by the TCF reporter assay (Bafico, A., et al. Cancer Cell 6, 497-506 (2004)). One mutant (W1268L), however, showed if anything increased Wnt signaling function (Fig. 4), arguing that not all conserved residues within this domain were essential for normal function of the LRP6 receptor. Analysis of total LRP6 protein expression levels, revealed similar levels for each of the receptors analyzed (Fig. 4).

Example 3 : Investigation of the role of the LRP6 LDLRR domain in transduction of the Wnt signal

[00241] The role of the LRP6 LDLRR domain in transduction of the Wnt signal by LRP was investigated. It has been reported that wild-type LRP receptors are internalized in response to Wnt stimulation and that receptor internalization is important for its signaling functions (Komekado and Kikuchi, Dev. Cell, 2006, 1 1 :213-213). The kinetics of LRP6 internalization in response to Wnt3a stimulation were compared for wild-type (WT) and a representative LDLRR domain mutant, S1317P. HeLa cells were transfected with WT or S 1317P LDLRR mutant LRP6 and 24 hours later treated with Wnt3a culture medium from Attorney Docket No.: 27527-0106WO1

Wnt3 a producing L cells (ATCC CRL-2647) (Vijayakumar et al, Cancer Cell, 2011, 19:601- 612) for various times. Cells were fixed and stained for LRP6 using flag antibody. Fig 5 shows that within 60 minutes following Wnt ligand addition, there was little remaining cell surface expression of the WT receptor. In striking contrast cell surface levels of the mutant receptor remained similar to levels observed at zero time. Under the conditions of exogenous expression in response to transient transfection of Hela cells, not all cells expressed the receptor serving as an internal control for the specificity of the LRP6 antibody utilized in these experiments.

[00242] In an independent experiment, WT LRP6 and a wild-type functioning mutant, W1268L (see Example 2, above), showed similar effective internalization associated also with intracellular localization of receptors in discrete puncti, whereas two signaling defective LDLRR mutants (D1315N and E1319K) showed little if any receptor internalization or intercellular puncti under the same conditions (Fig. 6). These results strongly correlated defective signaling with LDLRR mutations exhibiting impaired receptor internalization.

[00243] To confirm and extend these findings, an independent approach was utilized. 293T cells were transfected with various LRP6 constructs (WT, C1288F, D1315N, S 1317P, and E1319K) and 36 hours later processed for internalization assay. Cells were first labeled with cleavable biotin and then treated with Wnt3a for lhr. After the Wnt3a treatment, cells were rinsed with PBS and were incubated with reduced glutathione to remove any remaining cell surface biotinylated proteins. Only the internalized biotinylated proteins will be protected from cleavage with glutathione. Internalized biotinylated proteins including LRP6 were immunoprecipitated with avidin-conjugated beads (cells were lysed in NP40 buffer and 500 μg WCL was used for immunoprecipitation with avidin-conjugated beads). Immunoprecipitated proteins were resolved by PAGE. LRP6 was detected using a flag antibody. E-cadherin and tubulin were used as controls. This experiment showed that Wnt3a increased internalization of only the wild type LRP6, while none of the Wnt signaling defective LDLRR mutants showed any significant increase in the levels of internalized LRP6 (Fig. 7).

Example 4: Preparation of LDLRR Binding Antibodies Using Phage Display

[00244] Transgenic mice comprising human immunoglobulin loci are immunized with LRP6 LDLRR polypeptide having the amino acid sequence Attorney Docket No.: 27527-0106WO1

PTCSPQQFTCFTGEIDCIPVAWRCDGFTECEDHSDELNCPVCSESQFQCASGQCIDGA LRCNGDANCQDKSDEKNCEVLCLIDQFRCANGQCIGKHKKCDHNVDCSDKSDELD CY (SEQ ID NO: 3), or an antigenic portion thereof, or with recombinant LRP6 polypeptide (see, Brown et al, (1998) Biochem Biophys Res Commun; 248(3):879-88), and Complete Freund's Adjuvant (CFA) to induce an anti-LDLRR domain humoral immune response, followed by antigen boosts to enhance the LDLRR-specific immune response. Antibody- producing B cells are isolated from the spleens and/or lymph nodes of the immunized mice and RNA is isolated from the extracted cells. The RNA is then reverse transcribed to produce cDNA according to methods well known in the art, which is then polymerase chain reaction (PCR) amplified using a specific primer.

[00245] Genes encoding single chain Fv (scFv) fragments are made by randomly combining heavy and light chain V-genes using PCR, and the combinatorial library is cloned for display on the surface of a bacteriophage lambda vector, as described in Clackson et al, (Nature 352:624-628, 1991) and Marks et al, (J. Mol. Biol. 222:581-597, 1991). Phage displaying F(ab) with "antigen binding" activities are selected by four rounds of growth and panning with antigen (LDLRR polypeptide or fragment thereof), and the encoding heavy and light chain genes are sequenced. Soluble antibody fragments are prepared and tested to identify antibodies with LDLRR domain-binding specificity and good or high affinities. Affinity maturation may be performed. Candidate F(ab)s are converted to IgG and screened for ability to bind to the LDLRR domain of LRP6 in LDLRR binding assays (ELISA and/or FACS) Binding affinity to LRP6 LDLRR epitope is determined (e.g., using BIACORE™). Cross-reactivity with homologous polypeptides in cynomolgus and mouse is also determined by ELISA and/or FACS. Cross-reactivity with human, mouse, and cynomolgus LRP5 LDLRR domain is determined.

[00246] Candidate antibodies are next tested in in vitro functional assays for the ability to inhibit canonical Wnt signaling in cells having activated autocrine Wnt signaling. Functional assays can include the following assessments: ability to decrease uncomplexed β-catenin levels in sarcoma cells (method described in Bafico et al, supra); ability to induce LRP6 internalization; ability to block LRP6 phosphorylation; and ability to block cell proliferation (see, e.g., Vijayakumar, et al. (2011) Cancer Cell; 19(5):601-12).

[00247] For example, Wnt signaling activates TCF dependent transcription, which can be monitored by reporters containing TCF responsive elements (Morin et al, (1997) Science Attorney Docket No.: 27527-0106WO1

275: 1787-1790). TCF responsive elements operably linked to a reporter (TOP-Glow, wild type, or FOP-Glow, mutant) are analyzed for transcriptional activity in sarcoma cells in the presence or absence of the candidate antibody. Sarcoma cells are plated at 3xl0⁵ per well in 6-well plates and co-transfected with 1 μg of either the TOP-glow or FOP-glow plasmids (Upstate Biotechnology, Waltham, MA) and 0.001 μg of the Renilla control plasmid (pRL- CMV) utilizing Fugene (Roche) according to the manufacturer's instructions. After 48 hours cells are lysed and analyzed utilizing the Dual Luciferase Reporter Assay system (Promega, Madison, WI). The assay is also described in PCT publication no. WO/2006/055635 by Bafico et al.

[00248] Candidate antibodies which inhibit canonical Wnt signaling are selected for further study in in vivo animal models for evaluation of safety.

Example 5: Characterization of Candidate LDLRR Binding Antibodies in Tumor

Cells

[00249] The ability of the candidate antibodies to inhibit tumor growth in A204 sarcoma cell line is assessed. A good candidate antibody is expected to inhibit growth by at least 40%. Inhibition of Wnt signaling in tumor tissues is assessed by measuring total and phospho-LRP6, Axin2, LEFl, DKKl, DKK2 and CDC25A using commercially available antibodies, e.g., Axin 2: 5863S (Cell Signaling Technologies, Inc. (Danvers, MA)), Lefl : C12A5 (Cell Signaling Technologies, Inc.), Dkkl : SK-custom-made polyclonal rabbit) also available from Novus Biologicals (Littleton, CO), Dkk2:ab38594 (Abeam (Cambridge, MA)), and CDC25a: sc-7389 (Santa Cruz Biotechnology (Santa Cruz, CA)). Immunohistochemistry is performed to assess cell proliferation, apoptosis, and differentiation markers in tumor tissue. Toxicity is assessed (e.g., sign of body weight loss, skin rash). Pharmacokinetic and pharmacodynamics properties are assessed (PK/PD).

Example 6: Clinical Trial with Candidate LDLRR Antibody

[00250] The candidate antibodies identified in Prophetic Examples 4 and 5 are administered to sarcoma patients and the following parameters are evaluated as evidence of proof of mechanism (POM):

1. cDNAs isolated from pre- and post-treatment Wnt positive sarcoma tissues (as identified by the uncomplexed β-catenin assay, see, e.g., Bafico et al, Cancer Cell, Attorney Docket No.: 27527-0106WO1

6:497-506, 2004; Akiri et al., Oncogene, 28:2163-2172, 2009; Vijayakumar et al, Cancer Cell, 2011, 19:601-612) to compare by RT-PCR the expression levels of Wnt target genes: Axin2, LEF1 , DKK1, DKK2, and CDC25A.

2. Analysis of proliferation, apoptosis, and differentiation markers in pre- and post- treatment sarcoma tissues (e.g., proliferation - Ki67, PCNA; apoptosis - cleaved caspase 3, TdT positivity; differentiation - markers for bone, cartilage, see, e.g., Vijayakumar et al., Cancer Cell, 2011, 19:601-612).

3. Analysis of uncomplexed β-catenin levels in pre- and post-treatment sarcoma tissues (see, e.g., Bafico et al, Cancer Cell, 6:497-506, 2004; Akiri et al, Oncogene, 28:2163-2172, 2009; Vijayakumar et al, Cancer Cell, 201 1, 19:601-612).

4. CT scans prior to the first dose and after 6 weeks to detect tumor shrinkage.

Response rate exceeding 25% will be considered POM.

Preferred safety includes limited evidence of adverse toxicity in normal tissue with an appropriate therapeutic window. Sarcoma patients with APC or β- catenin activating mutations are excluded.

* * *

[00251] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

[00252] All patents, applications, publications, test methods, literature, and other materials cited herein are hereby incorporated by reference in their entirety as if physically present in this specification.

Claims

Attorney Docket No.: 27527-0106WO1 What is claimed is:

1. An isolated antibody or antigen-binding fragment thereof which specifically binds to the low density lipoprotein receptor related (LDLRR) domain of low density lipoprotein receptor-related protein (LRP) 6 and/or LRP5.

2. The antibody or antigen-binding fragment of claim 1, wherein the LDLRR domain consists of amino acids 1247-1361 of SEQ ID NO: 2 (LRP6).

3. The antibody or antigen-binding fragment of claim 1, wherein the LDLRR domain consists of amino acids 1257-1370 of SEQ ID NO: 8 (LRP5).

4. The antibody or antigen-binding fragment of claim 1, wherein the antibody or antigen-binding fragment has the property of inhibiting autocrine Wnt signaling in a cell.

5. The antibody or antigen-binding fragment of claim 1, wherein the antibody or antigen-binding fragment has the property of increasing Wnt ligand stimulation in a cell.

6. The antibody or antigen-binding fragment of claim 1, wherein the antibody or antigen-binding fragment is a monoclonal antibody.

7. The antibody or antigen-binding fragment of claim 1, wherein the antibody or antigen-binding fragment comprises a member selected from the group consisting of a single chain antibody, an scFv fragment, a Fab fragment, a F(ab)2 fragment, a humanized antibody, a monovalent antibody, a multispecific antibody, and a chimeric antibody.

8. The antibody or antigen-binding fragment of claim 1, wherein the antibody or antigen-binding fragment is a bispecific antibody which specifically binds to the LDLRR domain of LRP6 and the LDLRR domain of LRP5.

9. A pharmaceutical formulation comprising an effective amount for inhibiting autocrine Wnt signaling of the antibody or antigen-binding fragment of claim 4, and a pharmaceutically acceptable carrier.

10. A pharmaceutical formulation comprising an effective amount for increasing Wnt ligand stimulation of the antibody or antigen-binding fragment of claim 5, and a pharmaceutically acceptable carrier. Attorney Docket No.: 27527-0106WO1

11. A method of inhibiting growth of a cancer cell comprising contacting the cell with the antibody or antigen-binding fragment of claim 1.

12. The method of claim 1 1, wherein the cancer cell has activated autocrine Wnt signaling.

13. The method of claim 11, further comprising treating the cancer cell with a chemotherapeutic agent or radiation.

14. The method of claim 11, wherein the cancer cell is selected from the group consisting of breast carcinoma cell, ovarian carcinoma cell, non-small cell lung cancer (NSCLC) cell, sarcoma cell, and glioma cell.

15. The method of claim 11, wherein the cancer cell is in a patient.

16. A method for sensitizing a cancer cell to a treatment comprising contacting the cell with the antibody or antigen-binding fragment of claim 1.

17. The method of claim 16, wherein the cancer cell has activated autocrine Wnt signaling.

18. The method of claim 16, wherein the treatment is a chemotherapy or a radiation treatment.

19. The method of claim 16, wherein the cancer cell is selected from the group consisting of breast carcinoma cell, ovarian carcinoma cell, non-small cell lung cancer (NSCLC) cell, sarcoma cell, and glioma cell.

20. The method of claim 16, wherein the cancer cell is in a patient.

21. A method of inhibiting autocrine Wnt signaling mediated by canonical Wnt ligands in a cancer cell which comprises contacting the cell with the antibody or antigen binding fragment of claim 1.

22. A method of increasing Wnt ligand stimulation in a cell comprising contacting the cell with the antibody or antigen-binding fragment of claim 1.

23. The method of claim 22, wherein the cell is in a patient. Attorney Docket No.: 27527-0106WO1

24. The method of claim 23, wherein the patient is afflicted with osteoporosis.

25. The method of claim 23, wherein the patient has a disease treatable by increasing Wnt ligand stimulation.

26. A method of inhibiting growth of a cancer cell comprising contacting the cell with an LDLRR inhibitor of LRP6 and/or LRP5.

27. A method of sensitizing a cancer cell to a treatment comprising contacting the cell with an LDLRR inhibitor of LRP6 and/or LRP5.

28. The method of claim 27, wherein the treatment is a chemotherapy or a radiation treatment.

29. A method of inhibiting an autocrine Wnt signaling in a cancer cell comprising contacting the cell with an LDLRR inhibitor of LRP6 and/or LRP5.

30. The method of any one of claims 26-29, wherein the cancer cell has activated autocrine Wnt signaling.

31. The method of any one of claims 26-29, wherein the cancer cell is selected from the group consisting of breast carcinoma cell, ovarian carcinoma cell, non-small cell lung cancer ( SCLC) cell, sarcoma cell, and glioma cell.

32. The method of any one of claims 26-29, wherein the cancer cell is in a patient.

33. A method of increasing Wnt ligand stimulation in a cell comprising contacting the cell with an agonist of the LDLRR domain of LRP6 and/or LRP5.

34. The method of claim 33, wherein the cell is in a patient.

35. The method of claim 34, wherein the patient is afflicted with osteoporosis.

36. The method of claim 34, wherein the patient has a disease treatable by increasing Wnt ligand stimulation.