Antibodies To Lrp6

Abstract

Anti-LRP6 antibodies and antigen-binding fragments thereof, as well as pharmaceutical compositions comprising such antibodies and antigen-binding fragments are described. These anti-LRP6 antibodies can be used to enhance Wnt activity and/or antagonize Dkk1 activity. Also described are methods of therapy using such antibodies and antigen-binding regions to bind modulate Wnt/LRP6 signaling to promote tissue homeostasis, regeneration and repair in diseases such as, but not limited to, bone disorders, such as osteoporosis, rheumatoid arthritis, and osteolytic lesions caused by osteoarthritis and multiple myeloma, gastrointestinal disease and wound healing.

Description

CROSS REFERENCE TO PRIOR APPLICATIONS

[0001] This application claims the benefit of priority under 35 U.S.C. .sctn.119(e)(1) of 5 provisional application Ser. No. 60/998,647 filed Nov. 16, 2007, which application is hereby incorporated by reference in its entirety.

TECHNICAL FIELD OF THE INVENTION

[0002] The present invention relates to anti-LRP6 antibodies and to binding epitopes of LRP6 used to produce such antibodies. The invention also relates to methods of using such antibodies to diagnose and treat Wnt-associated diseases, such as bone disorders.

SEQUENCE LISTING

[0003] The sequences of the polynucleotides and polypeptides of the invention are listed in the Sequence Listing and are submitted electronically in the file labeled "NUV0-31 PCT_ST25.txt"--263 KB (269,747 bytes) which was created on an IBM PC, Windows 2000 operating system on Oct. 8, 2008 at 9:06:02 AM. The Sequence Listing entitled "NUV0-31 PCT_ST25.txt" is herein incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

[0004] The Wnt/13-catenin cell signaling pathway is implicated in a variety of developmental processes including stem cell maintenance and growth, cellular differentiation, cell growth, oncogenesis and disease pathogenesis (Kirikoshi et al, nt. J. Oneal. 19:767-771 (2001); Munroe et al, Proc. Nat/. Acad. Sci. USA 96:1569-1573 (1999); Reya and Clevers, Nature 434:843-850 (2005); Sher et al, FEBS Lett. 522:150-154 (2003)). The activation and regulation of the Wnt/13-catenin pathway therefore appears to be critical for tissue homeostasis, regeneration and repair. The "canonical" Wnt cell signaling pathway has as its central player, the cytosolic protein 13-catenin (FIG. 1). When Wnt receptors are not engaged, the level of cytosolic 13-catenin is kept low through the action of an intracellular complex, known as the "destruction complex," composed of the tumor suppressor proteins axin and adenomatous polyposis coli (APC) and the serine kinase protein glycogen synthase 3.beta. (GSK3.beta.). The constitutive kinase activity of the destruction complex on .beta. catenin results in the targeted proteosomal degradation of phosphorylated .beta. catenin. Binding of Wnt to the receptor proteins LRP5 and/or LRP6, members of the 5 LDL receptor family, and Frizzled (FZD), a serpentine receptor, induces phorphorylation-dependent binding of Axin to the LRP6 cytoplasmic tail and recruitment of the cytoplasmic protein Dishevelled (Dvl) to the cytoplasmic tail of FZD, which together lead to the inactivation of the .beta. catenin destruction complex. As a consequence, .beta.-catenin accumulates in the cytoplasm and translocates to the nucleus where it is thought to interact with members of the lymphoid enhancer factor (LEF)/T-cell factor (TCF) family of transcription factors and activate target gene expression.

[0005] The Wnt coreceptors LRP5/6 are modulated by the secreted ligands Dkk1, Dkk2 and SOST/Sclerostin, a ligand for LRP5/6 and a Wnt signaling inhibitor. 15 Interaction of SOST or Dkk1/2 wth LRP5/6 antagonizes Wnt/.beta.catenin signaling. Dkk1 is a high affinity ligand for LRP5/6 and disrupts the formation of the FZD-LRP complex. Dkk1 also binds Kremen-1 and -2 which are single-pass transmembrane proteins that cooperate with Dkk1 to inhibit Wnt-FZD-LRP6 function. Upon binding of Dkk1 to LRP6 and Kremen-1, receptor complex internalization occurs thereby dampening the Wnt signal due to a decrease of the Wnt coreceptors available for signaling indicating that the cell surface levels of LRP5/6 may limit cellular responses to Wnt ligands (reviewed in He et al, Development 131:1663-1677 (2004) and Semenov et al, J. Biol. Chem. 283:21427-21432 (2008)).

[0006] An area in which Wnt signaling has been implicated is the regulation of bone mass in homeostasis and bone disease. Bone mass appears to be influenced by the balance achieved between bone forming cells (osteoblasts) and bone resorbing cells (osteoclasts). Mutations in LRP5 and LRP6 receptors have been reported that either decrease or increase bone density, indicating that the level of Wnt and/or LRP5/6 signaling is critical for maintaining normal bone homeostasis. Consistant with these findings, it has been reported that elevated levels of the LRP5/6 inhibitor Dkk1 in diseases such as rheumatoid arthritis and multiple myeloma, result in osteolytic bone lesions, which can be reversed by Dkk1 antagonists, indicating that Dkk1 may be a regulator of bone density (reviewed in Krishnan et al, J Clin Invest 116:1202-1209 (2006)).

[0007] Several studies have also implicated the Wnt signaling pathway in tissue homeostasis and repair in a variety of systems including intestinal, epidermal, and hematopoietic systems. In the intestine, continuous renewal of absorptive epithelium is driven by proliferation of stem cells residing in the intestinal crypts. Current evidence suggests that the Wnt signaling cascade is important in controlling stem cell function in the intestinal crypt since deletion of the .beta.-catenin-dependent transcription factor TCF4 in mice results in depletion of intestinal crypts and loss of intestinal function (Korinek et al., Nat Genet. 19:379-83 (1998); Barker and Clevers, Nature Rev. 5:997 (2006)). Similarly, overexpression of Dkk1 in the intestine in transgenic mice or in mice injected with adenovirus expressing Dkk1, resulted in a complete loss of crypts in adult mice (Kormek et al, Nature Gen. 19:1-5 (1998); Kuhnert et al., Proc Nat/ Acad Sci USA. 101:266-71 (2004)). Intestinal diseases, such as inflammatory bowel disease, ulcerative colitis and radiation- or chemotherapy-induced mucositis, are associated with intestinal lesions and loss of intestinal absorptive epithelium, suggesting that modulation of Wnt signaling in intestinal crypts could have therapeutic benefit in treating such diseases.

[0008] A similar mechanism of Wnt signaling regulating stem cell function, tissue homeostasis and repair is found in the skin. Hair follicle density and the hair cycle are regulated by Wnt-dependent hair follicle epithelial stem cells (van Genderen et al, Genes Dev. 8:2691-2703 (1994); Lo Celso et al, Development 131:1787-1799 (2004)). Interestingly, the LRP5/6 inhibitor Dkk1 is expressed adjacent to hair follicle buds and over-expression of Dkk1 reduces hair follicle density, indicating that the level of LRP/Wnt signaling is important for regulation of hair follicle density and that Dkk1 may be a regulator of this process (Sick et al., Science. 314:1447-50 (2006)). Recently it was shown that hair follicle stem cells contribute to re-epithelialization during wound healing (Ito et al, Nat. Med. 11:1351-4 (2005)), indicating that modulation of Wnt signaling in hair follicle stem cells could be beneficial for wound repair.

[0009] In addition to the examples described above, Wnt signaling has also been shown to be important for regulation of stem cells in other tissues and organs, including hematopoietic stem cells (Reya et al, Nature 423:409-414 (2003); Xu et al., Nature Immunol. 4:1177-1182 (2003)), neuronal progenitor stem cells (Zecher et al, Dev. Bioi. 258:406-418 (2003)), and even embryonic stem cells (Sato et al, Nature Med. 10:55-63 (2004)) suggesting that modulation of Wnt signaling could also have therapeutic benefits in these systems.

[0010] Thus, molecules that modulate Wnt signaling can be useful targets for a broad range of conditions where proliferation, differentiation, tissue regeneration and repair are important to disease processes. The present invention provides anti-LRP6 antibodies that enhance LRP6 activity and antagonize Dkk1 activity for treatment of diseases such as, but not limited to bone disorders such as osteoporosis and osteolytic lesions caused by osteoarthritis and multiple myeloma as well as gastrointestinal disease and wound healing.

[0011] The Wnt/.beta.-catenin cell signaling pathway is implicated in a variety of developmental processes including stem cell maintenance and growth, cellular differentiation, cell growth, oncogenesis and disease pathogenesis (Kirikoshi et al, Int. J. Oncol. 19:767-771 (2001); Munroe et al, Proc. Natl. Acad. Sci. USA 96:1569-1573 (1999); Reya and Clevers, Nature 434:843-850 (2005); Sher et al, FEBS Lett. 522:150-154 (2003)). The activation and regulation of the Wnt/.beta.-catenin pathway therefore appears to be critical for tissue homeostasis, regeneration and repair. The "canonical" Wnt cell signaling pathway has as its central player, the cytosolic protein .beta.-catenin (FIG. 1). When Wnt receptors are not engaged, the level of cytosolic .beta.-catenin is kept low through the action of an intracellular complex, known as the "destruction complex," composed of the tumor suppressor proteins axin and adenomatous polyposis coli (APC) and the serine kinase protein glycogen synthase 3.beta. (GSK3.beta.). The constitutive kinase activity of the destruction complex on .beta.-catenin results in the targeted proteosomal degradation of phosphorylated .beta.-catenin. Binding of Wnt to the receptor proteins LRP5 and/or LRP6, members of the LDL receptor family, and Frizzled (FZD), a serpentine receptor, induces phorphorylation-dependent binding of Axin to the LRP6 cytoplasmic tail and recruitment of the cytoplasmic protein Dishevelled (Dvl) to the cytoplasmic tail of FZD, which together lead to the inactivation of the .beta.-catenin destruction complex. As a consequence, .beta.-catenin accumulates in the cytoplasm and translocates to the nucleus where it is thought to interact with members of the lymphoid enhancer factor (LEF)/T-cell factor (TCF) family of transcription factors and activate target gene expression.

[0012] The Wnt coreceptors LRP5/6 are modulated by the secreted ligands Dkk1, Dkk2 and SOST/Sclerostin, a ligand for LRP5/6 and a Wnt signaling inhibitor. Interaction of SOST or Dkk1/2 wth LRP5/6 antagonizes Wnt/.beta.-catenin signaling. Dkk1 is a high affinity ligand for LRP5/6 and disrupts the formation of the FZD-LRP complex. Dkk1 also binds Kremen-1 and -2 which are single-pass transmembrane proteins that cooperate with Dkk1 to inhibit Wnt-FZD-LRP6 function. Upon binding of Dkk1 to LRP6 and Kremen-1, receptor complex internalization occurs thereby dampening the Wnt signal due to a decrease of the Wnt coreceptors available for signaling indicating that the cell surface levels of LRP5/6 may limit cellular responses to Wnt ligands (reviewed in He et al, Development 131:1663-1677 (2004) and Semenov et al, J. Biol. Chem. 283:21427-21432 (2008)).

[0013] An area in which Wnt signaling has been implicated is the regulation of bone mass in homeostasis and bone disease. Bone mass appears to be influenced by the balance achieved between bone forming cells (osteoblasts) and bone resorbing cells (osteoclasts). Mutations in LRP5 and LRP6 receptors have been reported that either decrease or increase bone density, indicating that the level of Wnt and/or LRP5/6 signaling is critical for maintaining normal bone homeostasis. Consistant with these findings, it has been reported that elevated levels of the LRP5/6 inhibitor Dkk1 in diseases such as rheumatoid arthritis and multiple myeloma, result in osteolytic bone lesions, which can be reversed by Dkk1 antagonists, indicating that Dkk1 may be a regulator of bone density (reviewed in Krishnan et al, J Clin Invest 116:1202-1209 (2006)).

[0014] Several studies have also implicated the Wnt signaling pathway in tissue homeostasis and repair in a variety of systems including intestinal, epidermal, and hematopoietic systems. In the intestine, continuous renewal of absorptive epithelium is driven by proliferation of stem cells residing in the intestinal crypts. Current evidence suggests that the Wnt signaling cascade is important in controlling stem cell function in the intestinal crypt since deletion of the .beta.-catenin-dependent transcription factor TCF4 in mice results in depletion of intestinal crypts and loss of intestinal function (Korinek et al., Nat Genet. 19:379-83 (1998); Barker and Clevers, Nature Rev. 5:997 (2006)). Similarly, overexpression of Dkk1 in the intestine in transgenic mice or in mice injected with adenovirus expressing Dkk1, resulted in a complete loss of crypts in adult mice (Kormek et al, Nature Gen. 19:1-5 (1998); Kuhnert et al., Proc Natl Acad Sci USA. 101:266-71 (2004)). Intestinal diseases, such as inflammatory bowel disease, ulcerative colitis and radiation- or chemotherapy-induced mucositis, are associated with intestinal lesions and loss of intestinal absorptive epithelium, suggesting that modulation of Wnt signaling in intestinal crypts could have therapeutic benefit in treating such diseases.

[0015] A similar mechanism of Wnt signaling regulating stem cell function, tissue homeostasis and repair is found in the skin. Hair follicle density and the hair cycle are regulated by Wnt-dependent hair follicle epithelial stem cells (van Genderen et al, Genes Dev. 8:2691-2703 (1994); Lo Celso et al, Development 131:1787-1799 (2004)). Interestingly, the LRP5/6 inhibitor Dkk1 is expressed adjacent to hair follicle buds and over-expression of Dkk1 reduces hair follicle density, indicating that the level of LRP/Wnt signaling is important for regulation of hair follicle density and that Dkk1 may be a regulator of this process (Sick et al., Science. 314:1447-50 (2006)). Recently it was shown that hair follicle stem cells contribute to re-epithelialization during wound healing (Ito et al, Nat. Med. 11:1351-4 (2005)), indicating that modulation of Wnt signaling in hair follicle stem cells could be beneficial for wound repair.

[0016] In addition to the examples described above, Wnt signaling has also been shown to be important for regulation of stem cells in other tissues and organs, including hematopoietic stem cells (Reya et al, Nature 423:409-414 (2003); Xu et al., Nature Immunol. 4:1177-1182 (2003)), neuronal progenitor stem cells (Zecher et al, Dev. Biol. 258:406-418 (2003)), and even embryonic stem cells (Sato et al, Nature Med. 10:55-63 (2004)) suggesting that modulation of Wnt signaling could also have therapeutic benefits in these systems.

[0017] Thus, molecules that modulate Wnt signaling can be useful targets for a broad range of conditions where proliferation, differentiation, tissue regeneration and repair are important to disease processes. The present invention provides anti-LRP6 antibodies that enhance LRP6 activity and antagonize Dkk1 activity for treatment of diseases such as, but not limited to bone disorders such as osteoporosis and osteolytic lesions caused by osteoarthritis and multiple myeloma as well as gastrointestinal disease and wound healing.

SUMMARY OF THE INVENTION

[0018] The present invention provides isolated antibodies or immunologically functional antibody fragments (i.e. antigen-binding fragments) thereof that bind LRP6 epitopes with high affinity and can be used to enhance Wnt signaling and/or antagonize DKK1 activity. These antibodies can be used for treating a variety of diseases in which Wnt signaling is implicated, such as bone diseases and disorders and other cell proliferative-related disorders including wound healing and gastrointestinal diseases such as inflammatory bowel disease, ulcerative colitis and radiation- or chemotherapy-induced mucositis. Preferably the antibodies or antibody fragments thereof bind to primate and human LRP6. More preferably, the antibodies and antigen-binding fragments bind with high affinity to human LRP6. In particular embodiments, the antibodies or antigen-binding fragments thereof are chimeric, humanized, or human antibodies or antigen-binding fragments thereof. In other embodiments, the antibodies or antigen-binding fragments thereof are selected from the group consisting of scFv, Fab, Fab', F(ab').sub.2, Fv, and single chain antibodies. In another particular embodiment, the antibody or antigen-binding fragment thereof is an IgG isotype. Preferably the antibodies or antibody fragments enhance LRP6 activity. In one embodiment, the antibodies or antibody fragments enhance Wnt activity. In another embodiment, the antibodies or antibody fragments antagonize Dkk1 activity. In yet another embodiment, the antibodies or antibody fragments enhance LRP6 activity and antagonize Dkk1 activity.

[0019] One aspect of the present invention provides antibodies or antibody fragments thereof comprising a heavy chain variable region (V.sub.H) and/or a light chain variable region (V.sub.L) of anti-LRP6 antibodies 77.2, 135.16, 213.7, 240.8, 413.1, 421.1, 498.3, 537.2, 606.4, 620.1, 856.6, 923.3, 931.1, 993.9, 995.5, 1115.3, 1213.2, 1253.12, 1281.1, 1293.11, 1433.8, 1470.2, or 1903.1. In a particular embodiment, the antibodies comprise a heavy chain variable region of SEQ ID NO: 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 72, 74, 78, 82, 86, 90, 94, 98, 102 and/or a light chain variable region of SEQ ID NO: 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 76, 80, 84, 88, 92, 96, 100, or 104. In another embodiment, the antibodies comprise a heavy chain variable region comprising a sequence that has at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 72, 74, 78, 82, 86, 90, 94, 98, or 102 and/or a light chain variable region comprising a sequence that has at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 76, 80, 84, 88, 92, 96, 100, or 104.

[0020] Some of the antibodies and antigen-binding fragments that are provided include (a) one or more light chain (LC) complementarity determining regions (CDRs) selected from the group consisting of: [0021] (i) a LC CDR1 with at least 80% sequence identity to SEQ ID NO: 114, 126, 138, 150, 162, 174, 186, 198, 210, 222, 234, 246, 258, 282, 294, 306, 318, 330, 342, 354, or 366; [0022] (ii) a LC CDR2 with at least 80% sequence identity to SEQ ID NO: 115, 127, 139, 151, 163, 175, 187, 199, 211, 223, 235, 247, 259, 283, 295, 307, 319, 331, 343, 355, or 367; and [0023] (iii) a LC CDR3 with at least 80% sequence identity to SEQ ID NO: 116, 128, 140, 152, 164, 176, 188, 200, 212, 224, 236, 248, 260, 284, 296, 308, 320, 332, 344, 356, or 368; [0024] (b) one or more heavy chain (HC) CDRs selected from the group consisting of: [0025] (i) a HC CDR1 with at least 80% sequence identity to SEQ ID NO: 108, 120, 132, 144, 156, 168, 180, 192, 204, 216, 228, 240, 252, 264, 270, 276, 288, 300, 312, 324, 336, 348, or 360; [0026] (ii) a HC CDR2 with at least 80% sequence identity to SEQ ID NO: 109, 121, 133, 145, 157, 169, 181, 193, 205, 217, 229, 241, 253, 265, 271, 277, 289, 301, 313, 325, 337, 349, or 361; and [0027] (iii) a HC CDR3 with at least 80% sequence identity to SEQ ID NO: 110, 122, 134, 146, 158, 170, 182, 194, 206, 218, 230, 242, 254, 266, 272, 278, 290, 302, 314, 326, 338, 350, or 362; or [0028] (c) one or more LC CDRs of (a) and one or more HC CDRs of (b).

[0029] Such antibodies or antigen-binding fragments thereof can specifically bind an LRP6 polypeptide. Certain antibodies or antigen-binding fragments thereof include one, two, three, four, five or six of the foregoing CDRs in any combination thereof.

[0030] The light chain and heavy chains of other antibodies or antigen-binding fragments thereof are as described above but have at least 90% sequence identity to the foregoing sequences. Still other antibodies or antigen-binding fragments thereof have a light chain in which CDR1 has the amino acid sequence as set forth in SEQ ID NO: 114, 126, 138, 150, 162, 174, 186, 198, 210, 222, 234, 246, 258, 282, 294, 306, 318, 330, 342, 354, or 366, CDR2 has the amino acid sequence as set forth in SEQ ID NO: 115, 127, 139, 151, 163, 175, 187, 199, 211, 223, 235, 247, 259, 283, 295, 307, 319, 331, 343, 355, or 367, and/or CDR3 has the amino acid sequence as set forth in SEQ ID NO: 116, 128, 140, 152, 164, 176, 188, 200, 212, 224, 236, 248, 260, 284, 296, 308, 320, 332, 344, 356 or 368. Some antibodies or antigen-binding fragments thereof may also have a heavy chain in which CDR1 has the amino acid sequence as set forth in SEQ ID NO: 108, 120, 132, 144, 156, 168, 180, 192, 204, 216, 228, 240, 252, 264, 270, 276, 288, 300, 312, 324, 336, 348, or 360, CDR2 has the amino acid sequence as set forth in SEQ ID NO: 109, 121, 133, 145, 157, 169, 181, 193, 205, 217, 229, 241, 253, 265, 271, 277, 289, 301, 313, 325, 337, 349, or 361, and/or CDR3 has the amino acid sequence as set forth in SEQ ID NO: 110, 122, 134, 146, 158, 170, 182, 194, 206, 218, 230, 242, 254, 266, 272, 278, 290, 302, 314, 326, 338, 350, or 362.

[0031] Another aspect of the present invention provides isolated antibodies or antigen-binding fragments thereof that bind to LRP6 or an LRP6 epitope. In a particular embodiment, the antibodies include isolated antibodies or antigen-binding fragments thereof bind with high affinity to a human LRP6 epitope defined by amino acids 43-324 of SEQ ID NO: 2 (i.e., SEQ ID NO: 13 or 16). In another embodiment, the antibodies include isolated antibodies or antigen-binding fragments thereof that bind with high affinity to a human LRP6 epitope defined by amino acids 43-627 of SEQ ID NO: 2 (i.e., SEQ ID NO: 15) or as defined by amino acids 352-627 of SEQ ID NO: 2 (i.e. SEQ ID NO: 370). In yet another embodiment, the antibodies include isolated antibodies or antigen-binding fragments thereof that bind with high affinity to a human LRP6 epitope defined by amino acids 236-283 of SEQ ID NO: 2 (i.e. SEQ ID NO: 371). Examples of such antibodies include monoclonal antibodies 77.2, 135.16, 213.7, 240.8, 413.1, 421.1, 498.3, 537.2, 606.4, 620.1, 856.6, 923.3, 931.1, 993.9, 995.5, 1115.3, 1213.2, 1253.12, 1281.1, 1293.11, 1433.8, 1470.2, or 1903.1.

[0032] The invention provides a pharmaceutical composition comprising the antibody and a pharmaceutically acceptable carrier. The pharmaceutical composition may further comprise another pharmaceutically active ingredient, such as an anti-tumor agent or an imaging reagent. A particular embodiment provides an antibody or antigen-binding fragment thereof present in a therapeutically effective amount, such as in a concentration of at least about 10 .mu.g/ml.

[0033] Another aspect of the invention provides LRP6 epitopes, which epitopes include isolated polypeptides comprising amino acids 43-324 of SEQ ID NO: 2 (i.e., SEQ ID NO: 16), or amino acids 236-283 of SEQ ID NO: 2 (i.e., SEQ ID NO: 371), or any fragment thereof that binds to an anti-LRP6 antibody or antigen-binding fragment thereof.

[0034] Another aspect of the invention provides LRP6 epitopes, which epitopes include isolated polypeptides comprising amino acids 352-627 of SEQ ID NO: 2 (i.e., SEQ ID NO: 370), or any fragment thereof that binds to an anti-LRP6 antibody or antigen-binding fragment thereof.

[0035] Diagnostic and therapeutic methods are also provided by the invention. A particular embodiment provides a method for diagnosing the presence or location of an LRP6-expressing tissue or cells using an anti-LRP6 antibody. In yet another embodiment, a therapeutic method comprises administering the antibody to a subject in need thereof. In yet a further embodiment, a therapeutic method comprises administering the antibody to a subject in need thereof in conjunction with administration of another therapeutic agent.

[0036] The invention provides isolated cell lines, such as hybridoma cells and/or host cells that have been transfected to express LRP6 antibodies or antigen-binding fragments thereof, that produce the anti-LRP6 antibody or antigen-binding fragment thereof, and antibodies or antigen-binding fragments thereof produced by such cell lines. A hybridoma may include B cells obtained from a transgenic non-human animal having a genome comprising a human heavy chain transgene and a human light chain transgene fused to an immortalized cell. In another aspect, a hybridoma may include B cells obtained from a non-transgenic, non-human animal. Such transformed host cells may include nucleic acids encoding a human heavy chain and a human light chain.

[0037] Another aspect of the present invention provides a method of producing an antibody or antigen-binding fragment thereof that binds with high affinity to a human LRP6 epitope defined by amino acids 43-324 of SEQ ID NO: 2 (i.e. SEQ ID NO: 16), comprising immunizing a non-human animal with a human LRP6 epitope defined by amino acids 43-324 of SEQ ID NO: 2, such that antibodies are produced by B cells of the animal; isolating the B cells of the animal; and fusing the B cells with myeloma cells to form immortal, hybridoma cells that secrete the antibody or antigen binding region thereof.

[0038] Another aspect of the present invention provides a method of producing an antibody or antigen-binding fragment thereof that binds with high affinity to a human LRP6 epitope defined by amino acids 263-283 of SEQ ID NO: 2 (i.e. SEQ ID NO: 371), comprising immunizing a non-human animal with a human LRP6 epitope defined by amino acids 263-283 of SEQ ID NO: 2, such that antibodies are produced by B cells of the animal; isolating the B cells of the animal; and fusing the B cells with myeloma cells to form immortal, hybridoma cells that secrete the antibody or antigen binding region thereof.

[0039] Yet another aspect of the present invention provides a method of producing an antibody or antigen-binding fragment thereof that binds with high affinity to a human LRP6 epitope defined by amino acids 352-627 of SEQ ID NO: 2 (i.e. SEQ ID NO: 370), comprising immunizing a non-human animal with a human LRP6 epitope defined by amino acids 352-627 of SEQ ID NO: 2, such that antibodies are produced by B cells of the animal; isolating the B cells of the animal; and fusing the B cells with myeloma cells to form immortal, hybridoma cells that secrete the anti-LRP6 antibody or antigen binding region thereof.

[0040] The invention also provides nucleic acid molecules encoding the heavy and/or light chain or antigen-binding portions thereof of an anti-LRP6 antibody.

[0041] The invention provides vectors and host cells comprising the nucleic acid molecules, as well as methods of recombinantly producing the polypeptides encoded by the nucleic acid molecules.

BRIEF DESCRIPTION OF THE DRAWINGS

[0042] FIG. 1: A model of the Wnt signaling pathway. A) In the absence of Wnt signaling, beta-catenin is targeted for degradation by a beta-catenin destruction complex consisting of GSK3.beta., Axin and APC. Upon binding of a canonical Wnt ligand to frizzled (FZD) and LRP5/6 receptors, recruitment of axin to the phosphorylated cytoplasmic tail of LRP5/6 and Dishevelled (Dsh) to the cytoplasmic tail of frizzled, lead to inactivation of the beta-catenin destruction complex, allowing beta-catenin to accumulate and initiate TCF mediated transcription. B) Wnt signaling is limited by the amount of LRP6 on the cell surface, which is kept low by the LRP6 inhibitor Dkk1. Dkk1 inhibits binding of Wnt ligands to the LRP6 receptor and targets LRP6 for internalization through formation of a ternary complex with Kremen1/2 receptors.

[0043] FIG. 2: Affinity measurements and KD determination for anti-LRP6 mAbs: A) 77.2, B) 213.7, C) 240.8, D) 421.1, E) 498.3, F) 606.4, G) 856.6, H) 923.3, I) 931.1, J) 993.9, K) 995.5, L) 21115.3, M) 1213.2, N) 1253, 12, O) 1281.1, P) 1293.11, Q) 1433.8, R) 1470.2, S) 1903.1, T) 135.16, U) 413.1, V) 620.1, W) 537.2.

[0044] FIG. 3: A) Schematic of constructs used to map the LRP6 epitope that binds anti-LRP6 mAbs; B) FACS analysis of anti-LRP6 mAb binding to the LRP6 deletion constructs; C) Schematic of constructs used to map the C-terminal region of propeller domain 1 of LRP6 that binds anti-LRP mAb 135.16; D) FACS analysis of anti-LRP6 mAb 135.16 binding to propeller domain 1 of LRP6; E) Schematic of LRP6 propeller domain 1, amino acid sequence of mouse and human C-terminal region of LRP6 propeller domain 1 (residues 236-283), and ribbon model of LRP6 indicating position of Ser.sup.243; F) FACS analysis of anti-LRP6 mAb 135.16 binding to LRP6 propeller domain 1 with the indicated amino acid substitutions.

[0045] FIG. 4: Multiple amino acid sequence alignment of the heavy chain variable regions for anti-LRP6 mAbs (77.2 (SEQ ID NO: 18), 135.16 (SEQ ID NO: 22), 213.7 (SEQ ID NO: 26), 240.8 (SEQ ID NO: 30), 413.1 (SEQ ID NO: 34), 421.1 (SEQ ID NO: 38), 498.3 (SEQ ID NO: 42), 537.2 (SEQ ID NO: 46), 606.4 (SEQ ID NO: 50), 620.1 (SEQ ID NO: 54), 856.6 (SEQ ID NO: 58), 923.3 (SEQ ID NO: 62), 931.1 (SEQ ID NO: 66), 993.9 (SEQ ID NO: 70), 995.5 (SEQ ID NO: 72), 1115.3 (SEQ ID NO: 74), 1213.2 (SEQ ID NO: 78), 1253.12 (SEQ ID NO: 82), 1281.1 (SEQ ID NO: 86), 1293.11 (SEQ ID NO: 90), 1433.8 (SEQ ID NO: 94), 1470.2 (SEQ ID NO: 98), and 1903.1 (SEQ ID NO: 102)).

[0046] FIG. 5: Multiple amino acid sequence alignment of the light chain variable regions for anti-LRP6 mAbs (77.2 (SEQ ID NO: 20), 135.16 (SEQ ID NO: 24), 213.7 (SEQ ID NO: 28), 240.8 (SEQ ID NO: 32), 413.1 (SEQ ID NO: 36), 421.1 (SEQ ID NO: 40), 498.3 (SEQ ID NO: 44), 537.2 (SEQ ID NO: 48), 606.4 (SEQ ID NO: 52), 620.1 (SEQ ID NO: 56), 856.6 (SEQ ID NO: 60), 923.3 (SEQ ID NO: 64), 931.1 (SEQ ID NO: 68), 1115.3 (SEQ ID NO: 76), 1213.2 (SEQ ID NO: 80), 1253.12 (SEQ ID NO: 84), 1281.1 (SEQ ID NO: 88), 1293.11 (SEQ ID NO: 92), 1433.8 (SEQ ID NO: 96), 1470.2 (SEQ ID NO: 100), and 1903.1 (SEQ ID NO: 104)).

[0047] FIG. 6: Effect of anti-LRP6 mAbs on Wnt3A-dependent 16TCF luciferase reporter activation.

[0048] FIG. 7: Effect of anti-LRP6 mAbs on Dkk1-dependent inhibition of Wnt3A-induced 16TCF luciferase reporter activation.

[0049] FIG. 8: Characterization of the dose response of anti-LRP6 mAb 135.16 on Wnt3A dependent 16TCF luciferase reporter activation in the absence (squares) or presence of Dkk1 (triangles).

[0050] FIG. 9: Effect of anti-LRP6 mAb 135.16 whole antibody (squares) or Fab fragment (triangles) on the dose response of Wnt3a-dependent 16TCF luciferase reporter activation.

[0051] FIG. 10: Effect of anti-LRP6 mAb 135.16 Fab fragment on Dkk1-dependent inhibition of Wnt3A-induced 16TCF luciferase reporter activity.

[0052] FIG. 11: Anti-LRP6 mAb 135.16 antagonizes Dkk1-dependent internalization of LRP6. A) Immunofluorescence microscopy of LRP6 internalization in HEK293 cells transfected with HA-tagged LRP6 and wildtype Kremen1 and treated with mAb 135.16 alone, Dkk1 alone, mAb 135.16 followed by Dkk1, or no treatment (NTC). B) Quantitative analysis of the immunofluorescence results as described in A.

DETAILED DESCRIPTION OF THE INVENTION

[0053] Section titles are used herein for convenience purposes only and are not to be construed in any way as limiting the invention.

I. Definitions

[0054] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation.

[0055] Standard techniques are used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g. electroporation, lipofection). Enzymatic reactions and purification techniques are performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures are as generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992), and Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1990), which are incorporated herein by reference in their entirety for all purposes. The nomenclatures utilized in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0056] As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:

[0057] The terms "a," "an," and "the" mean one or more and include the plural unless the context is inappropriate.

[0058] The term "polynucleotide" as referred to herein means a polymeric form of nucleotides of at least 10 bases in length, either ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide. The term includes single and double stranded forms of DNA.

[0059] The term "oligonucleotide" referred to herein includes naturally occurring, and modified nucleotides linked together by naturally occurring and non-naturally occurring oligonucleotide linkages. Oligonucleotides are a polynucleotide subset generally comprising a length of 200 bases or fewer. Preferably oligonucleotides are 10 to 60 bases in length and most preferably 12, 13, 14, 15, 16, 17, 18, 19, or 20 to 40 bases in length. Oligonucleotides are usually single stranded, e.g., for probes; although oligonucleotides may be double stranded, e.g., for use in the construction of a gene mutant. Oligonucleotides of the invention can be either sense or antisense oligonucleotides.

[0060] "Operably linked" sequences include both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest. The term "expression control sequence" as used herein refers to polynucleotide sequences which are necessary to effect the expression and processing of coding sequences to which they are ligated. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance protein secretion. The nature of such control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include promoters and transcription termination sequence. The term "control sequences" as referred to herein includes, at a minimum, all components whose presence is essential for expression and processing, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.

[0061] The term "vector" as used herein, refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" may be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

[0062] The term "recombinant host cell" (or simply "host cell"), as used herein, refers to a cell that has been transformed, or is capable of being transformed, with a nucleic acid sequence and thereby expresses a gene of interest. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. Host cells may be prokaryotic or eukaryotic cells that are capable of expressing exogenous nucleic acid sequences. Examples of host cells include bacteria such as E. coli, yeast, plant cells, Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK)-293 cells and insect cells.

[0063] The term "transduction" means the transfer of genes from one bacterium to another, usually by bacteriophage. "Transduction" also refers to the acquisition and transfer of eukaryotic cellular sequences by retroviruses.

[0064] The term "transfection" means the uptake of foreign or exogenous DNA by a cell, and a cell has been "transfected" when the exogenous DNA has been introduced inside the cell membrane. A number of transfection techniques are well known in the art and are disclosed herein. See, e.g., Graham et al., Virology 52:456 (1973); Sambrook et al., Molecular Cloning: A Laboratory Manual, Id. (2001); Davis et al., Basic Methods in Molecular Biology, Elsevier (1986); and Chu et al., Gene 13:197 (1981). Such techniques can be used to introduce one or more exogenous DNA moieties into suitable host cells.

[0065] The term "transformation" refers to a change in a cell's genetic characteristics, and a cell has been transformed when it has been modified to contain new DNA or RNA. For example, a cell is transformed wherein it is genetically modified from its native state by introducing new genetic material via transfection, transduction, or other techniques. Following transfection or transduction, the transforming DNA may recombine with that of the cell by physically integrating into a chromosome of the cell, or may be maintained transiently as an episomal element without being replicated, or may replicate independently as a plasmid. A cell is considered to have been "stably transformed" when the transforming DNA is replicated with the division of the cell.

[0066] The term "percent sequence identity" in the context of nucleic acid sequences refers to the residues in two sequences which are the same when aligned for maximum correspondence. The length of sequence identity comparison may be over a stretch of at least about nine nucleotides, usually at least about 18 nucleotides, more usually at least about 24 nucleotides, typically at least about 28 nucleotides, more typically at least about 32 nucleotides, and preferably at least about 36, 48 or more nucleotides. There are a number of different algorithms known in the art which can be used to measure nucleotide sequence identity. For instance, polynucleotide sequences can be compared using FASTA, GAP or BESTFIT, which are programs in Wisconsin Package Version 10.0, Genetics Computer Group (GCG), Madison, Wis. FASTA, which includes, e.g., the programs FASTA2 and FASTA3, provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson, Meth. Enzymol. 183:63-98 (1990); Pearson, Meth. Mol. Biol. 132:185-219 (2000); Pearson, Meth. Enzymol. 266:227-258 (1996); Pearson, J. Mol. Biol. 276:71-84 (1998); herein incorporated by reference). Unless specified otherwise, default parameters for a particular program or algorithm are used. For instance, percent sequence identity between nucleic acid sequences can be determined using FASTA with its default parameters (a word size of 6 and the NOPAM factor for the scoring matrix) or using GAP with its default parameters as provided in GCG Version 6.1, herein incorporated by reference.

[0067] A reference to a nucleic acid sequence encompasses its complement unless otherwise specified. Thus, a reference to a nucleic acid molecule having a particular sequence should be understood to encompass its complementary strand, with its complementary sequence.

[0068] The term "substantial similarity" or "substantial sequence similarity" when referring to a nucleic acid or fragment thereof, indicates that, when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 85%, preferably at least about 90%, and more preferably at least about 95%, at least 96%, at least 97%, at least 98% or at least 99% of the nucleotide bases, as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST, or GAP as discussed above.

[0069] The terms "polypeptide" or "protein" means a macromolecule having the amino acid sequence of a native protein, that is a protein produced by a naturally-occurring and non-recombinant cell, or produced by a genetically-engineered or recombinant cell, and comprise molecules having the amino acid sequence of the native protein, or molecules having deletions from, additions to, and/or substitutions of one or more amino acids of the native sequence. The terms "polypeptide" and "protein" specifically encompass anti-LRP6 antibodies antigen-binding fragments, or sequences that have deletions from, additions to, and/or substitutions of one or more amino acid of anti-LRP6 antibodies or antigen-binding fragments. The term "polypeptide fragment" refers to a polypeptide that has an amino-terminal deletion, a carboxyl-terminal deletion, and/or an internal deletion as compared with the full-length native protein. Such fragments may also contain modified amino acids as compared with the native protein. In certain embodiments, fragments are about 5 to 500 amino acids long. For example, fragments may be at least 5, 6, 8, 10, 14, 20, 50, 70, 100, 110, 150, 200, 250, 300, 350, 400, 450 or 500 amino acids long. Useful polypeptide fragments include immunologically functional fragments of antibodies, including binding domains. In the case of anti-LRP6 antibodies, useful fragments include but are not limited to a CDR region, a variable domain of a heavy or light chain, a portion of an antibody chain or just its variable region including two CDRs, and the like.

[0070] The term "isolated protein" referred to herein, means that a subject protein (1) is free of at least some other proteins with which it would normally be found, (2) is essentially free of other proteins from the same source, e.g., from the same species, (3) is expressed by a cell from a different species, (4) has been separated from at least about 50% of polynucleotides, lipids, carbohydrates, or other materials with which it is associated in nature, (5) is operably associated (by covalent or noncovalent interaction) with a polypeptide with which it is not associated in nature, or (6) does not occur in nature. Genomic DNA, cDNA, mRNA or other RNA, of synthetic origin, or any combination thereof may encode such an isolated protein. Preferably, the isolated protein is substantially free from proteins or polypeptides or other contaminants that are found in its natural environment that would interfere with its therapeutic, diagnostic, prophylactic, research or other use.

[0071] A "variant" of a polypeptide comprises an amino acid sequence wherein one or more amino acid residues are inserted into, deleted from and/or substituted into the amino acid sequence relative to another polypeptide sequence. Unless otherwise indicated, the term "variants" includes fusion proteins.

[0072] A "derivative" of a polypeptide is a polypeptide (e.g., an antibody) that has been chemically modified in some manner distinct from insertion, deletion, or substitution variants, e.g., via conjugation to another chemical moiety.

[0073] The term "antibody" refers to an intact immunoglobulin of any isotype, or a fragment thereof, that can compete with the intact antibody for specific binding to the target antigen, and includes chimeric, humanized, fully human, and bispecific antibodies. An intact antibody generally will comprise at least two full-length heavy chains and two full-length light chains, but in some instances may include fewer chains such as antibodies naturally occurring in camelids which may comprise only heavy chains. Antibodies may be derived solely from a single source, or may be "chimeric," that is, different portions of the antibody may be derived from two different antibodies. For example, the CDR regions may be derived from a rat or murine source, while the framework region of the V region is derived from a different animal source, such as a human. The antibodies or binding fragments thereof may be produced in hybridomas, by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. Unless otherwise indicated, the term "antibody" includes, in addition to antibodies comprising two full-length heavy chains and two full-length light chains, derivatives, variants, fragments, and muteins thereof, examples of which are described below.

[0074] The term "light chain" includes a full-length light chain and fragments thereof having sufficient variable region sequence to confer binding specificity. A full-length light chain includes a variable region domain (abbreviated herein as V.sub.L), and a constant region domain (abbreviated herein as C.sub.L). The variable region domain of the light chain is at the amino-terminus of the polypeptide. Light chains include kappa chains and lambda chains.

[0075] The term "heavy chain" includes a full-length heavy chain and fragments thereof having sufficient variable region sequence to confer binding specificity. A full-length heavy chain includes a variable region domain (abbreviated herein as V.sub.H), and three constant region domains (abbreviated herein as C.sub.H1, C.sub.H2, and C.sub.H3). The V.sub.H domain is at the amino-terminus of the polypeptide, and the C.sub.H domains are at the carboxy-terminus, with the C.sub.H3 being closest to the --COOH end. Heavy chains may be of any isotype, including IgG (including IgG.sub.1, IgG.sub.2, IgG.sub.3, and IgG.sub.4 subtypes), IgA (including IgA.sub.1 and IgA.sub.2 subtypes), IgM, and IgE.

[0076] The V.sub.H and V.sub.L regions can be further subdivided into regions of hypervariability, termed "complementarity determining regions" or "CDR", interspersed with regions that are more conserved, termed framework regions (FR). Each V.sub.H and V.sub.L is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3. CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. An amino acid sequence which is substantially the same as a heavy or light chain CDR exhibits a considerable amount or extent of sequence identity when compared to a reference sequence and contributes favorably to specific binding of an antigen bound specifically by an antibody having the reference sequence. Such identity is definitively known or recognizable as representing the amino acid sequence of the particular human monoclonal antibody. Substantially the same heavy and light chain CDR amino acid sequence can have, for example, minor modifications or conservative substitutions of amino acids so long as the ability to bind a particular antigen is maintained.

[0077] The term "CDR" or "complementarity determining region" means the non-contiguous antigen combining sites found within the variable region of both heavy and light chain polypeptides. This particular region has been described by Kabat et al., U.S. Dept. of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983) and by Chothia et al., J. Mol. Biol. 196:901-917 (1987) and additionally by MacCallum et al., J. Mol. Biol. 262:732-745 (1996), which are incorporated herein by reference, where the definitions include overlapping or subsets of amino acid residues when compared against each other. Nevertheless, application of either definition to refer to a CDR of an antibody or functional fragment thereof is intended to be within the scope of the term as defined and used herein. The exact amino acid residue numbers which encompass a particular CDR will vary depending on the structure of the CDR. Those skilled in the art can routinely determine which residues comprise a particular CDR given the variable region amino acid sequence of the antibody. Those skilled in the art can compare two or more antibody sequences by defining regions or individual amino acid positions of the respective sequences with the same CDR definition.

[0078] The term "antibody" includes both glycosylated and non-glycosylated immunoglobulins of any isotype or subclass or combination thereof, including human (including CDR-grafted antibodies), humanized, chimeric, multi-specific, monoclonal, polyclonal, and oligomers thereof, irrespective of whether such antibodies are produced, in whole or in part, via immunization, through recombinant technology, by way of in vitro synthetic means, or otherwise. Thus, the term "antibody" includes those that are prepared, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes or a hybridoma prepared therefrom, (b) antibodies isolated from a host cell transfected to express the antibody, (c) antibodies isolated from a recombinant, combinatorial library, and (d) antibodies prepared, expressed, created or isolated by any other means that involve splicing of immunoglobulin gene sequences of two distinct species of animals. In certain embodiments, however, such antibodies can be subjected to in vitro mutagenesis (or, when an animal transgenic for human immunoglobulin sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the V.sub.H and V.sub.L regions of the antibodies are sequences that, while derived from and related to the germline V.sub.H and V.sub.L sequences of a particular species (e.g., human), may not naturally exist within that species' antibody germline repertoire in vivo.

[0079] The term "antigen-binding fragment" of an antibody means one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., LRP6) that is specifically bound by a reference antibody, as disclosed herein. An "antigen-binding fragment" of an antibody may include, for example, polypeptides comprising individual heavy or light chains and fragments thereof, such as V.sub.L, V.sub.H, and Fd regions (consisting of the V.sub.H and C.sub.H1 domains); monovalent fragments, such as Fv, Fab, and Fab' regions; bivalent fragments, such as F(ab').sub.2; single chain antibodies, such as single chain Fv (scFv) regions; Fc fragments; diabodies; maxibodies (bivalent scFv fused to the amino terminus of the Fc (C.sub.H2-C.sub.H3 domains)) and complementary determining region (CDR) domains. Such terms are described, for example, in Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, NY (1989); Molec. Biology and Biotechnology: A Comprehensive Desk Reference (Myers, R. A. (ed.), New York: VCH Publisher, Inc.); Huston et al., Cell Biophysics, 22:189-224 (1993); Pluckthun and Skerra, Meth. Enzymol., 178:497-515 (1989) and in Day, E. D., Advanced Immunochemistry, 2d ed., Wiley-Liss, Inc. New York, N.Y. (1990), which are incorporated herein by reference.

[0080] The term "antigen-binding fragment" also includes, for example, fragments produced by protease digestion or reduction of a human monoclonal antibody and by recombinant DNA methods known to those skilled in the art. One skilled in the art knows that the exact boundaries of a fragment of a human monoclonal antibody can be variable, so long as the fragment maintains a functional activity. Using well-known recombinant methods, one skilled in the art can engineer a nucleic acid to express a functional fragment with any endpoints desired for a particular application. Furthermore, although the two domains of the Fv fragment, V.sub.L and V.sub.H, 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 V.sub.L and V.sub.H regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al., Science 242:423-426 (1988); and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988). Such single chain antibodies are also intended to be encompassed within the term "antigen-binding fragment" of an antibody. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies. Such fragments include those obtained by amino-terminal and/or carboxy-terminal deletions, but where the remaining amino acid sequence is substantially identical to the corresponding positions in the naturally-occurring sequence deduced, for example, from a full-length cDNA sequence. Antigen-binding fragments also include fragments of an antibody which retain at least one (e.g., 1, 2, 3 or more) light chain sequences for a particular complementarity determining region (CDR) (e.g., at least one or more of CDR1, CDR2, and/or CDR3 from the heavy and/or light chain). Fusions of CDR containing sequences to an Fc region (or a C.sub.H2 or C.sub.H3 region thereof) are included within the scope of this definition including, for example, scFv fused, directly or indirectly, to an Fc region are included herein. An antigen-binding fragment is inclusive of, but not limited to, those derived from an antibody or fragment thereof (e.g., by enzymatic digestion or reduction of disulfide bonds), produced synthetically using recombinant methods, created via in vitro synthetic means (e.g., Merrifield resins), combinations thereof, or through other methods. Antigen-binding fragments may also comprise multiple fragments, such as CDR fragments, linked together synthetically, chemically, or otherwise, in the form of oligomers. Thus, antigen-binding fragments include polypeptides produced by any number of methods which comprise at least one CDR from a V.sub.H or V.sub.L chain of an anti-LRP6 antibody (e.g., derived from monoclonal antibodies 77.2, 135.16, 213.7, 240.8, 413.1, 421.1, 498.3, 537.2, 606.4, 620.1, 856.6, 923.3, 931.1, 993.9, 995.5, 1115.3, 1213.2, 1253.12, 1281.1, 1293.11, 1433.8, 1470.2, or 1903.1).

[0081] The term "V.sub.L fragment" means a fragment of the light chain of a monoclonal antibody which includes all or part of the light chain variable region, including the CDRs. A V.sub.L fragment can further include light chain constant region sequences.

[0082] The term "V.sub.H fragment" means a fragment of the heavy chain of a monoclonal antibody which includes all or part of the heavy chain variable region, including the CDRs. A V.sub.H fragment can further include heavy chain constant region sequences.

[0083] The term "Fd fragment" means a fragment of the heavy chain of a monoclonal antibody which includes all or part of the V.sub.H heavy chain variable region, including the CDRs. An Fd fragment can further include C.sub.H1 heavy chain constant region sequences.

[0084] An "Fc" region contains two heavy chain fragments comprising the C.sub.H1 and C.sub.H2 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the C.sub.H3 domain.

[0085] The term "Fv fragment" means a monovalent antigen-binding fragment of a monoclonal antibody, including all or part of the variable regions of the heavy and light chains, and absent of the constant regions of the heavy and light chains. The variable regions of the heavy and light chains include, for example, the CDRs.

[0086] The term "Fab fragment" means a monovalent antigen-binding fragment of an antibody consisting of the V.sub.L, V.sub.H, C.sub.L and C.sub.H1 domains, which is larger than an Fv fragment. For example, a Fab fragment includes the variable regions, and all or part of the first constant domain of the heavy and light chains.

[0087] The term "Fab' fragment" means a monovalent antigen-binding fragment of a monoclonal antibody that is larger than a Fab fragment. For example, a Fab' fragment includes all of the light chain, all of the variable region of the heavy chain, and all or part of the first and second constant domains of the heavy chain.

[0088] The term "F(ab').sub.2 fragment" means a bivalent antigen-binding fragment of a monoclonal antibody comprising two Fab fragments linked by a disulfide bridge at the hinge region. An F(ab').sub.2 fragment includes, for example, all or part of the variable regions of two heavy chains and two light chains, and can further include all or part of the first constant domains of two heavy chains and two light chains.

[0089] "Single-chain antibodies" are Fv molecules in which the heavy and light chain variable regions have been connected by a flexible linker to form a single polypeptide chain, which forms an antigen-binding fragment. Single chain antibodies are discussed in detail in International Patent Application Publication No. WO 88/01649 and U.S. Pat. Nos. 4,946,778 and 5,260,203, the disclosures of which are herein incorporated by reference.

[0090] A "domain antibody" is an antigen-binding fragment containing only the variable region of a heavy chain or the variable region of a light chain. In some instances, two or more V.sub.H regions are covalently joined with a peptide linker to create a bivalent domain antibody. The two V.sub.H regions of a bivalent domain antibody may target the same or different antigens.

[0091] The term "bivalent antibody" means an antibody that comprises two antigen binding sites. In some instances, the two binding sites have the same antigen specificities. However, bivalent antibodies may be bispecific (see below).

[0092] The term "bispecific antibody" means an antibody that binds to two or more distinct epitopes. For example, the antibody may bind to, or interact with, (a) a cell surface antigen and (b) an Fc receptor on the surface of an effector cell. The term "multispecific antibody" or "heterospecific antibody" means an antibody that binds to more than two distinct epitopes. For example, the antibody may bind to, or interact with, (a) a cell surface antigen, (b) an Fc receptor on the surface of an effector cell, and (c) at least one other component. Accordingly, the invention includes, but is not limited to, bispecific, trispecific, tetraspecific, and other multispecific antibodies or antigen-binding fragments thereof which are directed to LRP6 epitopes and to other targets, such as Fc receptors on effector cells. Bispecific antibodies are a species of multispecific antibody and may be produced by a variety of methods including, but not limited to, fusion of hybridomas or linking of Fab' fragments. See, e.g., Songsivilai and Lachmann, Clin. Exp. Immunol. 79:315 (1990); Kostelny et al., J. Immunol. 148:1547 (1992). The two binding sites of a bispecific antibody will bind to two different epitopes, which may reside on the same or different protein targets.

[0093] The term "bispecific antibodies" also includes diabodies. Diabodies are bivalent, bispecific antibodies in which the V.sub.H and V.sub.L 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., Hollinger et al., Proc Natl. Acad. Sci. USA 90:6444-6448 (1993); Polijak et al., Structure 2:1121-1123 (1994).

[0094] The term "monoclonal antibody" or "mAb," as used herein, refers to an antibody obtained from a population of substantially homogeneous antibodies, e.g., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. In contrast to polyclonal antibody preparations that typically include different antibodies against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The term is not limited regarding the species or source of the antibody, nor is it intended to be limited by the manner in which it is made. The term encompasses whole immunoglobulins as well as fragments such as Fab, F(ab').sub.2, Fv, and other fragments, as well as chimeric and humanized homogeneous antibody populations, that exhibit immunological binding properties of the parent monoclonal antibody molecule.

[0095] The term "mouse monoclonal antibody" means a monoclonal antibody, as defined above, produced by immunizing a mouse, with an antigen of interest (e.g., LRP6). A "mouse monoclonal antibody" is produced using conventional methods well known in the art, from mouse-mouse hybridomas, described more fully below.

[0096] The term "rabbit monoclonal antibody" as used herein means a monoclonal antibody, as defined above, produced by immunizing a rabbit with an antigen of interest (e.g., LRP6). A "rabbit monoclonal antibody" can be produced using rabbit-rabbit hybridomas (e.g., fusions between an antibody-producing cell from the immunized rabbit with an immortalized cell from a rabbit), rabbit-mouse hybridomas (e.g., fusions between an antibody-producing cell from the immunized rabbit with an immortalized cell from a mouse), and the like.

[0097] The term "human monoclonal antibody" means a monoclonal antibody with substantially human CDR amino acid sequences produced, for example, by recombinant methods, by lymphocytes or by hybridoma cells.

[0098] The monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851 (1984).

[0099] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit, or nonhuman primate having the desired specificity, affinity, and capacity. In some instances, framework region (FR) residues of the immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. See, e.g., Jones et al., Nature 321:522 (1986); Riechmann et al., Nature 332:323 (1988); Presta, Curr. Op. Struct. Biol. 2:593 (1992); Vaswani and Hamilton, Ann. Allergy, Asthma and Immunol. 1:105 (1998); Harris, Biochem. Soc. Transactions 23; 1035 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428 (1994).

[0100] A "human antibody" is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human and/or has been made using any of the techniques for making human antibodies as disclosed herein. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding regions.

[0101] An "affinity matured" antibody is one with one or more alterations in one or more CDRs thereof which result in an improvement in the affinity of the antibody for antigen, compared to a parent antibody which does not possess those alteration(s). Preferred affinity matured antibodies will have nanomolar or even picomolar affinities for the target antigen. Affinity matured antibodies are produced by procedures known in the art. Marks et al., Bio/Technology 10:779 (1992) describes affinity maturation by V.sub.H and V.sub.L domain shuffling. Random mutagenesis of CDR and/or framework residues is described by: Barbas et al., Proc. Natl. Acad. Sci. USA 91:3809 (1994); Schier et al., Gene 169:147 (1995); Yelton et al., J. Immunol. 155:1994 (1995); Jackson et al., J. Immunol. 154:3310 (1995); and Hawkins et al., J. Mol. Biol. 226:889 (1992).

[0102] "Immunoadhesions" or "immunoadhesins" are antibody-like molecules that combine the binding domain of a non-antibody polypeptide with the effector functions of an antibody or an antibody constant domain. The binding domain of the non-antibody polypeptide can be, for example, a ligand or cell surface receptor having ligand binding activity. Immunoadhesions for use as anti-LRP6 antibodies can contain at least the Fc receptor binding effector functions of the antibody constant domain.

[0103] "Immunologically reactive" means that the antibody of interest will bind with LRP6 antigens present in a biological sample.

[0104] The term "immunogenic sequence of an LRP6" means an LRP6 molecule that includes an amino acid sequence with at least one epitope such that the molecule is capable of stimulating the production of antibodies in an appropriate host.

[0105] The term "immunogenic composition" means a composition that comprises at least one immunogenic polypeptide (e.g., an LRP6 antigen or antibody).

[0106] The term "antigen" refers to a molecule or a portion of a molecule capable of being bound by a selective binding agent, such as an antibody, and additionally capable of being used in an animal to produce antibodies capable of binding to that antigen. An antigen may possess one or more epitopes that are capable of interacting with different antibodies.

[0107] The term "selective binding agent" refers to a molecule that binds to an antigen. Non-limiting examples include antibodies, antigen-binding fragments, scFv, Fab, Fab', F(ab').sub.2, single chain antibodies, peptides, peptide fragments and proteins.

[0108] The term "epitope" includes any determinant capable of binding with high affinity to an immunoglobulin or to a T-cell receptor. An epitope is a region of an antigen that is bound by an antibody that specifically targets that antigen, and when the antigen is a protein, includes specific amino acids that directly contact the antibody. Most often, epitopes reside on proteins, but in some instances, may reside on other kinds of molecules, such as nucleic acids. Epitope determinants may include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and may have specific three dimensional structural characteristics, and/or specific charge characteristics. Generally, antibodies specific for a particular target antigen will preferentially recognize an epitope on the target antigen in a complex mixture of proteins and/or macromolecules.

[0109] Regions of a given polypeptide that include an epitope can be identified using any number of epitope mapping techniques, well known in the art. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66 (Glenn E. Morris, Ed., 1996) Humana Press, Totowa, N.J. For example, linear epitopes may be determined by e.g., concurrently synthesizing large numbers of peptides on solid supports, the peptides corresponding to portions of the protein molecule, and reacting the peptides with antibodies while the peptides are still attached to the supports. Such techniques are known in the art and described in, e.g., U.S. Pat. No. 4,708,871; Geysen et al. Proc. Natl. Acad. Sci. USA 81:3998-4002 (1984); Geysen et al. Proc. Natl. Acad. Sci. USA 82:178-182 (1985); Geysen et al. Molec. Immunol. 23:709-715 (1986). Similarly, conformational epitopes are readily identified by determining spatial conformation of amino acids such as by, e.g., x-ray crystallography and two-dimensional nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols, supra. Antigenic regions of proteins can also be identified using standard antigenicity and hydropathy plots, such as those calculated using, e.g., the Omiga version 1.0 software program available from the Oxford Molecular Group. This computer program employs the Hopp/Woods method, Hopp et al., Proc. Natl. Acad. Sci USA 78:3824-3828 (1981) for determining antigenicity profiles, and the Kyte-Doolittle technique, Kyte et al., J. Mol. Biol. 157:105-132 (1982) for hydropathy plots.

[0110] An antibody is said to "specifically bind" its target antigen when the dissociation constant (K.sub.D) is .ltoreq.10.sup.-8 M. The antibody specifically binds antigen with "high affinity" when the K.sub.D is .ltoreq.5.times.10.sup.-9 M, and with "very high affinity" when the K.sub.D is .ltoreq.5.times.10.sup.-10 M. In one embodiment of the invention, the antibody has a K.sub.D of .ltoreq.10.sup.-9M and an off rate (k.sub.d) of about 1.times.10.sup.-4/sec. In one embodiment of the invention, the off rate if <10.sup.-5/sec. In another embodiment of the invention, the antibody will bind LRP6 with a KD of between 10.sup.-8 and 10.sup.-10 M.

[0111] The term "surface plasmon resonance," as used herein, refers to an optical phenomenon that allows for the analysis of real-time biospecific interactions by detection of alterations in protein concentrations within a biosensor matrix, for example using the BIAcore system (Biacore International AB, Uppsala, Sweden). For further descriptions, see Jonsson et al., Ann. Biol. Clin. 51:19-26 (1993); Jonsson et al., Biotechniques 11:620-627 (1991); Johnsson et al., J. Mol. Recognit. 8:125-131 (1995); and Johnsson et al., Anal. Biochem. 198:268-277 (1991).

[0112] It is understood that the antibodies of the present invention may be modified, such that they are substantially identical to the antibody polypeptide sequences, or fragments thereof, and still bind the LRP6 epitopes provided herein. Polypeptide sequences are "substantially identical" when optimally aligned using such programs as GAP or BESTFIT using default gap weights, they share at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, or at least 99% sequence identity.

[0113] As discussed herein, minor variations in the amino acid sequences of antibodies or antigen-binding regions thereof are contemplated as being encompassed by the present invention, providing that the variations in the amino acid sequence maintain at least 75%, more preferably at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% and most preferably at least 99% sequence identity. In particular, conservative amino acid replacements are contemplated. Conservative replacements are those that take place within a family of amino acids that are related in their side chains. Genetically encoded amino acids are generally divided into families: (1) acidic (aspartate, glutamate); (2) basic (lysine, arginine, histidine); (3) nonpolar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan); and (4) uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). More preferred families are: (1) aliphatic-hydroxy (serine, threonine); (2) amide-containing (asparagine, glutamine); (3) aliphatic (alanine, valine, leucine, isoleucine); and (4) aromatic (phenylalanine, tryptophan). For example, it is reasonable to expect that an isolated replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid will not have a major effect on the binding or properties of the resulting molecule, especially if the replacement does not involve an amino acid within a framework site. Whether an amino acid change results in a functional peptide can readily be determined by assaying the specific activity of the polypeptide derivative. Assays are described in detail herein. Fragments or analogs of antibodies or immunoglobulin molecules can be readily prepared by those of ordinary skill in the art.

[0114] Preferred amino- and carboxy-termini of fragments or analogs occur near boundaries of functional domains. Structural and functional domains can be identified by comparison of the nucleotide and/or amino acid sequence data to public or proprietary sequence databases. Preferably, computerized comparison methods are used to identify sequence motifs or predicted protein conformation domains that occur in other proteins of known structure and/or function. Methods to identify protein sequences that fold into a known three-dimensional structure are known. Bowie et al., Science 253:164 (1991). Thus, the foregoing examples demonstrate that those of skill in the art can recognize sequence motifs and structural conformations that may be used to define structural and functional domains in accordance with the invention.

[0115] The anti-LRP6 antibodies may also be generated using peptide analogs of the epitopic determinants disclosed herein, which analogs may consist of non-peptide compounds having properties analogous to those of the template peptide. These types of non-peptide compound are termed "peptide mimetics" or "peptidomimetics". Fauchere, J. Adv. Drug Res. 15:29 (1986); Veber and Freidinger TINS p. 392 (1985); and Evans et al., J. Med. Chem. 30:1229 (1987).

[0116] The term "immune complex" refers to the combination formed when an antibody binds to an epitope on an antigen.

[0117] The term "effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

[0118] A "therapeutically effective amount" of a substance/molecule of the invention may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the substance/molecule to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the substance/molecule are outweighed by the therapeutically beneficial effects.

[0119] The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells. The term is intended to include radioactive isotopes (e.g., phosphorus-32, copper-67, arsenic-77, rhodium-105, palladium-109, silver-111, tin-121, iodine-125 or 131, holmium-166, lutetium-177, rhenium-186 or 188, iridium-194, gold-199, astatium-211, yttrium-90, samarium-153, or bismuth-212), chemotherapeutic agents, e.g., methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chloramucil, daunorubicin, or other intercalating agents, enzymes and fragments thereof such as nucleolytic enzymes, antibiotics, and toxins such as small molecule toxins or enzymatically active toxins of bacterial (e.g., Diptheria toxin, Pseudomonas endotoxin and exotoxin, Staphylococcal enterotoxin A), fungal (e.g., .alpha.-sarcin, restrictocin), plant (e.g., abrin, ricin, modeccin, viscumin, pokeweed anti-viral protein, saporin, gelonin, momoridin, trichosanthin, barley toxin) or animal origin, e.g., cytotoxic RNases, such as extracellular pancreatic RNases; DNase I, including fragments and/or variants thereof, and the various antitumor or anticancer agents disclosed below. Other cytotoxic agents are described below. A tumoricidal agent causes destruction of tumor cells.

[0120] The term "chemotherapeutic agent" means a chemical compound that non-specifically decreases or inhibits the growth, proliferation, and/or survival of cancer cells. Such chemical agents are often directed to intracellular processes necessary for cell growth or division, and are thus particularly effective against cancerous cells, which generally grow and divide rapidly. For example, vincristine depolymerizes microtubules, and thus inhibits cells from entering mitosis. In general, chemotherapeutic agents can include any chemical agent that inhibits, or is designed to inhibit, a cancerous cell or a cell likely to become cancerous. Such agents are often administered, and are often most effective, in combination, e.g., in the formulation CHOP.

[0121] Examples of chemotherapeutic agents contemplated by the present invention include, but are not limited to, alkylating agents such as thiotepa and CYTOXAN.RTM. cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL.RTM.); beta-lapachone; lapachol; colchicines; betulinic acid; a camptothecin (including the synthetic analogue topotecan (HYCAMTIN.RTM.), CPT-11 (irinotecan, CAMPTOSAR.RTM.), acetylcamptothecin, scopolectin, and 9-aminocamptothecin); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); podophyllotoxin; podophyllinic acid; teniposide; cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gamma1 and calicheamicin omega1 (see, e.g., Agnew, Chem Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, including dynemicin A; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including ADRIAMYCIN.RTM., morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, doxorubicin HCl liposome injection (DOXIL.RTM.) and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate, gemcitabine (GEMZAR.RTM.), tegafur (UFTORAL.RTM.), capecitabine (XELODA.RTM.), an epothilone, and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSK.RTM. polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine (ELDISINE.RTM., FILDESIN.RTM.); dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); thiotepa; taxoids, e.g., paclitaxel (TAXOL.RTM.), albumin-engineered nanoparticle formulation of paclitaxel (ABRAXANE.RTM.), and doxetaxel (TAXOTERE.RTM.); chloranbucil; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine (VELBAN.RTM.); platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine (ONCOVIN.RTM.); oxaliplatin; leucovovin; vinorelbine (NAVELBINE.RTM.); novantrone; edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0122] Also useful are combinations of two or more of the above such as CHOP (a combination of cyclophosphamide, doxorubicin, vincristine and prednisone) as well as the use of the constituents of CHOP either alone or in various combinations such as CO, CH, CP, COP, CHO, CHP, HO, HP, HOP, OP, etc.; CHOP and bleomycin (CHOP-BLED); cyclophosphamide and fludarabine; cyclophosphamide, mitoxantrone, prednisone and vincristine; cyclophosphamide, dexamethasone, doxorubicin and vincristine (CAVD); CAV; cyclophosphamide, doxorubicin and prednisone; cyclophosphamide, mitoxantrone, prednisone and vincristine (CNOP); cyclophosphamide, methotrexate, leucovorin and cytarabine (COMLA); cyclophosphamide, dexamethasone, doxorubicin and prednisone; cylophosphamide, prednisone, procarbazine and vincristine (COPP); cylophosphamide, prednisone and vincristine (COP and CVP-1); cyclophosphamide and mitoxantrone; etoposide; mitoxantrone, ifosfamide and etoposide (MIV); cytarabine; methylprednisolone and cisplatin (ESHAP); methylprednisolone, cytarabine and cisplatin (ESAP); fludarabine, cytosine arabinoside (Ara-C) and G-CSF (FLAG); irinotecan, 5-FU (IFL); oxaliplatin, 5-FU, leucovorin (FOLFOX); oxaliplatin, irinotecan (IROX); leucovorin, 5-FU, irinotecan (FOLFIRI); methotrexate, leucovorin, doxorubicin, cyclophosphamide, vincristine, bleomycin and prednisone (MACOP-B); methotrexate, bleomycin, doxorubicin, cyclophosphamide, vincristine, and dexamethasone (m-BACOD); prednisone, cyclophosphamide, etopo side, cytarabine, bleomycin, vincristine, methotrexate and leucovorin (PROMACE-CYTABOM); etoposide, cyclophosphamide, vincristine, prednisone and bleomycin (VACOP-B); fludarabine and mitoxantrone; cisplatine, cytarabine and etoposide; desamethasone, fludarabine and mitoxantrone; chlorambucil and prednisone; busulfan and fludarabine; ICE; DVP; ATRA; Idarubicin, hoelzer chemotherapy regime; La La chemotherapy regime; ABVD; CEOP; 2-CdA; FLAG and IDA (with or without subsequent G-CSF treatment); VAD; M and P; C-Weekly; ABCM; MOPP; cisplatin, cytarabine and dexamethasone (DHAP), as well as the additional known chemotherapeutic regimens. Preparation and dosing schedules for such chemotherapeutic agents are also described in Chemotherapy Service Ed., M. C. Perry, Williams and Wilkins, Baltimore, Md. (1992).

[0123] Also included in this definition are anti-hormonal agents that act to regulate, reduce, block, or inhibit the effects of hormones that can promote the growth of cancer, and are often in the form of systemic or whole-body treatment. They may be hormones themselves. Examples include anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX.RTM. tamoxifen), raloxifene (EVISTA.RTM.), droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY 117018, onapristone, and toremifene (FARESTON.RTM.); anti-progesterones; estrogen receptor down-regulators (ERDs); estrogen receptor antagonists such as fulvestrant (FASLODEX.RTM.); agents that function to suppress or shut down the ovaries, for example, leutinizing hormone-releasing hormone (LHRH) agonists such as leuprolide acetate (LUPRON.RTM. and ELIGARD.RTM.), goserelin acetate, buserelin acetate and tripterelin; other anti-androgens such as flutamide, nilutamide and bicalutamide; and aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, megestrol acetate (MEGASE.RTM.), exemestane (AROMASIN.RTM.), formestanie, fadrozole, vorozole (RIVISOR.RTM.), letrozole (FEMARA.RTM.), and anastrozole (ARIMIDEX.RTM.). In addition, such definition of chemotherapeutic agents includes bisphosphonates such as clodronate (for example, BONEFOS.RTM. or OSTAC.RTM.), etidronate (DIDROCAL.RTM.), NE-58095, zoledronic acid/zoledronate (ZOMETA.RTM.), alendronate (FOSAMAX.RTM.), pamidronate (AREDIA.RTM.), tiludronate (SKELID.RTM.), or risedronate (ACTONEL.RTM.); as well as troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those that inhibit expression of genes in signaling pathways implicated in aberrant cell proliferation, such as, for example, PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); vaccines such as THERATOPE.RTM. vaccine and gene therapy vaccines, for example, ALLOVECTIN.RTM. vaccine, LEUVECTIN.RTM. vaccine, and VAXID.RTM. vaccine; topoisomerase 1 inhibitor (e.g., LURTOTECAN.RTM.); rmRH (e.g., ABARELIX.RTM.); lapatinib ditosylate (an ErbB-2 and EGFR dual tyrosine kinase small-molecule inhibitor also known as GW572016); COX-2 inhibitors such as celecoxib (CELEBREX.RTM.; 4-(5-(4-methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl)benzene sulfonamide; and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0124] The terms "radiation therapy" or "radiotherapeutic agents" mean the administration of radioactivity or radioactive compounds to a subject with cancer. Radiation decreases or inhibits the growth of dividing cells, such as cancer cells. Such therapy may include radiation from radioactive isotopes (e.g., phosphorous-32, copper-67, arsenic-77, rhodium-105, palladium-109, silver-111, tin-121, iodine-125 or 131, holmium-166, lutetium-177, rhenium-186 or 188, iridium-194, gold-199, astatium-21, yttrium-90, samarium-153, or bismuth-212). The radiation therapy may be whole body irradiation, or may be directed locally to a specific site or tissue in the body, such as the colon or small intestine.

[0125] The term "targeted anti-cancer agents" means molecules directed to specific proteins, lipids, or other cellular components. Such targeted anti-cancer agents include monoclonal antibodies or other types of antibodies (i.e., fragments, single chain antibodies, bi-specific antibodies) or molecules (such as peptibodies) that target antigens. Examples of such immunotherapeutic targeted antibodies include without limitation bevacizumab (AVASTIN.RTM., Genentech, South San Francisco, Calif.), tositumomab (BEXXAR.RTM., GlaxoSmithKline, United Kingdom), alemtuzumab (CAMPATH.RTM., Genzyme, Cambridge, Mass.), cetuximab (ERBITUX.RTM., ImClone Systems Inc., New York), trastuzumab (HERCEPTIN.RTM., Genentech), gemtuzumab ozogamicin (MYLOTARG.RTM., Wyeth, Madison, N.J.), rituximab (RITUXAN.RTM., Biogen Idec, San Diego, Calif.), ibritumomab tiuxetan (ZEVALIN.RTM., Biogen Idec), mitomomab (BEC2), C225, OncoLym, epratuzumab (Lymphocide), oregovomab (OVAREX.RTM., ViRexx, Edmonton, Alberta, Canada), lintuzumab (SMART M195), apolizumab (SMART 1D10), VITAXIN.RTM. (Medimmune, Inc., Gaithersburg, Md.). Also captured by the term "targeted anti-cancer agents" are immunotoxins. By "immunotoxin" is meant an antibody- or antibody-like-toxin conjugate intended to destroy specific target cells (e.g., tumor cells) that bear antigens homologous to the antibody. Examples of toxins that are coupled to such antibodies include but are not limited to ricin A chain (RTA), blocked ricin (blR), saporin (SAP), pokeweed antiviral protein (PAP) and Pseudomonas exotoxin (PE), and other toxic compounds, such as radioisotopes and other chemotherapeutic drugs, as described above.

[0126] The term "immunotherapeutic agent" is used herein to denote an agent that is an immunopotentiator or an immunosuppressant and is useful for treating diseases and disorders including cancer. Such agents include, without limitation, various cytokines and lymphokines, such as a number of interleukins, including IL-1, IL-2, IL-3, IL-4, IL-5, IL-12 and muteins of these molecules; interferons, such as but not limited to IFN-.alpha., IFN-.beta., IFN-.gamma. and muteins thereof; colony stimulating factors such as GM-CSF and muteins of GM-CSF; tumor necrosis factors, such as TNF-.alpha. and TNF-.beta. and muteins of these molecules. Also captured by the term "immunotherapeutic agent" are immunotoxins. By "immunotoxin" is meant an antibody-toxin conjugate intended to destroy specific target cells (e.g., tumor cells) which bear antigens homologous to the antibody. Examples of toxins that are coupled to such antibodies include but are not limited to ricin A chain (RTA), blocked ricin (blR), saporin (SAP), pokeweed antiviral protein (PAP) and Pseudomonas exotoxin (PE), and other toxic compounds, such as radioisotopes and other chemotherapeutic drugs, described further below.

[0127] The term "immunoconjugate" refers to the association of an antibody with another agent, such as a chemotherapeutic agent, a toxin, an immunotherapeutic agent, and the like. In this way, the agent of interest can be targeted directly to cells bearing the LRP6 cell surface receptor. The mode of association between the antibody and the agent of interest is immaterial. Thus, the antibody and agent may be associated through non-covalent interactions such as through electrostatic forces, or by covalent bonds. Various linkers, known in the art, can be employed in order to form the immunoconjugate. Additionally, the immunoconjugate can be provided in the form of a fusion protein that may be expressed from a polynucleotide encoding the immunoconjugate.

[0128] The term "agent" means any substance, naturally occurring or synthetic, and includes, without limitation, small molecules, single or double stranded oligonucleotide molecules such as aptamers, polynucleotides (DNA or RNA) interfering nucleic acid molecules (shRNA, siRNA, double stranded RNA, or microRNA), lipids, simple or complex sugars or other carbohydrates, peptide-nucleic acids, peptomimetics, peptides, single or multi chain polypeptides, antibodies, antibody fragments such as Fabs or Fc-fusion molecules, or peptibodies. Also included as agents are those substances that are chimeras, hybrids, or fusions of any of the foregoing, such as, for example, a peptide-lipid fusion molecule, a polypeptide linked to a sugar molecule such as polyethylene glycol, an aptamer fused to a lipid, and the like.

[0129] The term "anti-cancer agent" means any agent that can be used to treat a cell proliferative disorder such as cancer, including cytotoxic agents, chemotherapeutic agents, radiotherapy and radiotherapeutic agents, targeted anti-cancer agents, and immunotherapeutic agents.

[0130] As used herein, the terms "label" and "detectable label" refer to a molecule capable of detection, including, but not limited to, radioactive isotopes, fluorescers, semiconductor nanocrystals, chemiluminescers, chromophores, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, dyes, metal ions, metal sols, ligands (e.g., biotin, streptavidin or haptens) and the like. The term "fluorescer" refers to a substance or a portion thereof which is capable of exhibiting fluorescence in the detectable range. Particular examples of labels which may be used under the invention include, but are not limited to, horseradish peroxidase (HRP), fluorescein, FITC, rhodamine, dansyl, umbelliferone, dimethyl acridinium ester (DMAE), Texas red, luminol, NADPH and .alpha.- or .beta.-galactosidase.

[0131] The term "anti-tumor activity" means a reduction in the rate of cell proliferation and hence a decline in growth rate of abnormal cells that arises during therapy. Such activity can be assessed using accepted animal models.

[0132] The term "subject" as used herein means a mammal, such as, but not limited to, domestic and farm animals and zoo, sports or pet animals, such as cow, monkey, horse, sheep, pig, cat, dog, mouse, rat, rabbit, guinea pig or human. Preferably the mammal is a human. A subject can be a human patient.

[0133] The term "biological sample" as used herein refers to a sample of tissue or fluid isolated from a subject such as, but not limited to, blood, plasma, platelets, serum, fecal matter, urine, bone marrow, bile, spinal fluid, lymph fluid, cerebrospinal fluid, samples of the skin, secretions of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, blood cells, organs, biopsies and also samples of in vitro cell culture constituents including but not limited to conditioned media resulting from the growth of cells and tissues in culture medium, e.g., recombinant cells, and cell components. The samples detailed above need not necessarily be in the form obtained directly from the source. For example, the sample can be treated prior to use, such as, for example, by heating, centrifuging, etc. prior to analysis.

[0134] The term "Wnt signaling pathway" means the canonical Wnt pathway in which members of the Wnt family of secreted protein ligands bind a receptor complex of LRP and Frizzled (FZD) allowing .beta.-catenin to be translocated into the nucleus, interact with the LEF/TCF transcription factors and activate target gene expression.

[0135] The phrase "cell proliferation related disease or disorder" means those diseases or disorders in which cell proliferation is altered, i.e., either increased or decreased as compared with the homeostatic state.

[0136] The phrase "pharmaceutically acceptable" vehicle, carrier or adjuvant means a non-toxic agent that can be tolerated by a recipient patient at the dosages and concentrations employed. Often the pharmaceutical carrier is an aqueous pH buffered solution. Representative non-limiting examples of such agents include human serum albumin, gelatin, ion exchangers, alumina, lecithin, buffer substances such as phosphates, citrate, glycine, antioxidants such as ascorbic acid, potassium sorbate and other organic acids, and salts or electrolytes such as protamine sulfate. Suitable vehicles are, for example, water, saline, phosphate-buffered saline, dextrose, glycerol, ethanol, hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN.TM. (ICI Americas, Inc., Bridgewater, N.J.), polyethylene glycol (PEG), and PLURONIC.RTM. (BASF, Florham Park, N.J.). Other suitable agents are well known to those in the art. See, for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 19th edition, 1995. Actual methods of preparing such compositions are also known, or will be apparent, to those skilled in the art. See, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 19th edition, 1995.

[0137] Various aspects of the invention are described in further detail in the following sections and subsections.

II. LRP6 and the Wnt-Signaling Pathway

[0138] The Wnt signaling pathway is important in embryonic development and postnatal tissue maintenance. This is achieved by directing a specific set of genes that control temporal and spatial regulation of cell growth, movement and cell survival (reviewed in Barker and Clevers, Nature Rev. 5:997 (2006) herein incorporated by reference in its entirety). Proper regulation of this pathway is important for maintaining tissue homeostasis. Chronic activation of this pathway promotes uncontrolled cell growth and survival and can consequently drive the development of cell proliferative diseases, such as cancer. Alternatively, abnormal inhibition of this pathway can result in many disease states, for example loss of bone mass and other bone diseases. Wnt proteins initiate downstream signaling by interacting with a Frizzled receptor and one of two cell-surface receptors, which are members of the low-density-lipoprotein receptor (LDLR)-related proteins (LRPs): LRP5 and LRP6 (reviewed in He et al, Development 131:1663-1677 (2004), herein incorporated by reference in its entirety).

[0139] The role of LRP6 in canonical Wnt signaling was discovered via genetic studies. Mutant mice lacking LRP6 exhibited composite phenotypes similar to mutations in several individual Wnt genes (Pinson et al, Nature 407:535-538 (2000)). In Xenopus embryos, dominant-negative LRP6 blocked signaling by several Wnt proteins, whereas overexpression of LRP6 activated Wnt/.beta.-catenin signaling (Tamai et al, Nature 407:530-535 (2000)). Furthermore, it has been shown that expression of either LRP6 or LRP5 is necessary for cells to respond to canonical Wnt signaling (reviewed in He et al., supra, 2004).

[0140] LRP5 and LRP6 are highly homologous and share 73% and 64% identity in their extra- and intracellular domains, respectively. They are widely co-expressed during embryogenesis and in adult tissues and share some functional redundancy. The extracellular domains of LRP5 and LRP6 comprise three basic domains: 1) a YWTD (tyrosine, tryptophan, threonine, aspartic acid)-type .beta.-propeller domain, 2) an EGF (epidermal growth factor)-like domain, and 3) an LDLR type A (LA) domain. The YWTD-type .beta.-propeller domain contains six YWTD repeats of 43-50 amino acid residues each and forms a six-bladed .beta.-propeller structure. In LRP5 and LRP6, there are four YWTD-type .beta.-propeller domains that are each followed by an EGF-like domain, which comprises about 40 amino acid residues with conserved cysteine residues, which in turn are followed by three LA domains. (Springer et al, J. Mol. Biol. 283:837-862 (1998); Jeon et al, Nat. Struct. Biol. 8:499-504 (2001)). The .beta.-propeller-EGF-like domains appear to bind extracellular ligands. The extracellular domain of LRP6 is defined by amino acid residues 20 to 1375 and contains four propeller domains at amino acid residues 43-324, 352-627, 654-929, and 957-1250. Amino acid residues 32-1386 of LRP5 comprise the extracellular domain which contains four propeller domains at amino acid residues 75-336, 365-639, 667-941, and 969-1253.

[0141] LRP5 and LRP6 purportedly bind Axin directly via their intracellular domains thereby regulating .beta.-catenin phosphorylation and degradation. LRP5/6 activity is modulated by secreted ligands Dkk1, Dkk2 and SOST/Sclerostin, which through their interaction with LRP5/6 antagonize Wnt activity. Dkk1 is a high affinity ligand for LRP5/6 and disrupts the binding of the FZD-LRP complex. Dkk1 appears to bind LRP6 via its C-terminal cysteine-rich domain which is also suggested to be required for Wnt antagonism (He et al, supra, 2004). Dkk1 has been demonstrated to interact with the region of LRP6 encompassing the third and fourth propeller domains which is distinct from the Wnt binding region of LRP6. Dkk1 also binds Kremen-1 and -2 which are single-pass transmembrane proteins. The interaction of Dkk1 and LRP5/6 with Kremen-1 internalizes the complex for degradation thereby reducing the number of Wnt coreceptors available for signaling.

[0142] Wnt signaling has been shown to be involved in normal skeletogenesis and cancer-related bone diseases. Activating mutations in LRP5 have been demonstrated to cause osteoporosis-pseudoglioma syndrome which is characterized by low bone mineral density and skeletal fragility (Gong et al, Cell 107:513-523 (2001)). On the other hand, mutations in LRP5 that prevent binding of Dkk1 have been implicated in the syndrome of hereditary high bone density (Boyden et al, New Engl J Med 346:1513-1521 (2002)). Dkk1 has also been implicated in normal skeletal development. Mice lacking Dkk1 grow extra digits, while increased expression of Dkk1 results in a loss of bony structures (Mukhopadhyay et al, Dev Cell 1:423-434 (2001)). In addition, plasma cells from multiple myeloma patients express Dkk1 whereas those from normal patients do not. The expression of Dkk1 positively correlates with the presence of bone lesions in multiple myeloma. Osteolytic lesions have also been found in prostate cancer patients (Tian et al, New Engl J Med 349:2483-2494 (2003); Politou et al, Int. J Cancer 119:1728-1731 (2006)).

[0143] Maintenance of bone mass is influenced by the balance achieved between bone forming cells (osteoblasts) and bone resorbing cells (osteoclasts). According to Diana et al (Nat Med 13:156-163 (2007)), Dkk1 appears to be involved in bone loss in inflammatory joint disease such as rheumatoid arthritis, osteoarthritis and ankylosing spondylitis by inhibiting differentiation of osteoblasts and promoting the activity of osteoclasts. In situations where higher than normal levels of Dkk1 are present, Dkk1 appears to be involved in the bone destructive phenotype entailing joint instability common to diseases such as rheumatoid arthritis. In situations where lower than normal levels of Dkk1 are present, the bone anabolic reaction in the joint may be enhanced, leading to joint ankylosis in osteoarthritis and ankylosing spondylosis (Diarra et al, supra, 2007).

[0144] Modulation of LRP6 and/or Dkk1 resulting in activation of Wnt signaling may be useful to treat conditions such as bone disorders including, but not limited to osteoarthritis, rheumatoid arthritis, ankylosing spondylosis, and osteolytic lesions caused by a variety of diseases including osteoarthritis and multiple myeloma. Additional conditions that may benefit from these treatments include, but are not limited to, gastrointestinal disorders, such as irritable bowel disease, peptic ulcers, and mucositis, and wound healing, as Wnt/LRP6 signaling has been shown to regulate tissue homeostasis and repair in these tissues.

III. Anti-LRP6 Antibodies and Antigen-Binding Fragments

[0145] A variety of selective binding agents useful for regulating the activity of LRP6 are provided. These agents include, for instance, antibodies and antigen-binding fragments thereof that contain an antigen binding domain (e.g., single chain antibodies, domain antibodies, immunoadhesions, and polypeptides with an antigen-binding region) that specifically bind to an LRP6 polypeptide (e.g., a human, rat and/or murine LRP6 polypeptide).

[0146] The present invention provides isolated anti-LRP6 antibodies that bind to human LRP6 epitopes. In a preferred embodiment, the LRP6 epitope is substantially the same epitope as a human LRP6 epitope defined by amino acids 43-324 of SEQ ID NO: 2 (e.g., SEQ ID NO: 13 or 16). In another embodiment, the isolated anti-LRP6 antibodies and antigen-binding fragments thereof bind to a human LRP6 epitope, or substantially the same epitope, defined by amino acids 43-324 of SEQ ID NO: 2. In another embodiment, an isolated antibody or antigen-binding fragment thereof specifically binds to a human LRP6 epitope, or substantially the same epitope, defined by amino acids 43-324 of SEQ ID NO: 2. In another embodiment, a monoclonal antibody or antigen-binding fragment thereof specifically binds to a human LRP6 epitope, or substantially the same epitope, defined by amino acids 43-324 of SEQ ID NO: 2. Such antibodies or antigen-binding fragments thereof can be prepared by any one of a number of processes disclosed below, for example, by immunizing an animal with at least a first LRP6 antigenic composition and selecting from the immunized animal an antibody that substantially cross-reacts with the anti-LRP6 monoclonal antibodies provided herein.

[0147] In another embodiment, the LRP6 epitope is substantially the same epitope as a human LRP6 epitope defined by amino acids 263-283 of SEQ ID NO: 2 (e.g., SEQ ID NO: 371). In another embodiment, isolated anti-LRP6 antibodies and antigen-binding fragments thereof bind to a human LRP6 epitope, or substantially the same epitope, defined by amino acids 263-283 of SEQ ID NO: 2. In another embodiment, an isolated antibody or antigen-binding fragment thereof specifically binds to a human LRP6 epitope, or substantially the same epitope, defined by amino acids 263-283 of SEQ ID NO: 2. In another embodiment, a monoclonal antibody or antigen-binding fragment thereof specifically binds to a human LRP6 epitope, or substantially the same epitope, defined by amino acids 263-283 of SEQ ID NO: 2. Such antibodies or antigen-binding fragments thereof can be prepared by any one of a number of processes disclosed below, for example, by immunizing an animal with at least a first LRP6 antigenic composition and selecting from the immunized animal an antibody that substantially cross-reacts with the anti-LRP6 monoclonal antibodies provided herein.

[0148] In another embodiment, the LRP6 epitope is substantially the same epitope as a human LRP6 epitope defined by amino acids 352-627 of SEQ ID NO: 2 (e.g., SEQ ID NO: 370). In another embodiment, isolated anti-LRP6 antibodies and antigen-binding fragments thereof bind to a human LRP6 epitope, or substantially the same epitope, defined by amino acids 352-627 of SEQ ID NO: 2. In another embodiment, an isolated antibody or antigen-binding fragment thereof specifically binds to a human LRP6 epitope, or substantially the same epitope, defined by amino acids 352-627 of SEQ ID NO: 2. In another embodiment, a monoclonal antibody or antigen-binding fragment thereof specifically binds to a human LRP6 epitope, or substantially the same epitope, defined by amino acids 352-627 of SEQ ID NO: 2. Such antibodies or antigen-binding fragments thereof can be prepared by any one of a number of processes disclosed below, for example, by immunizing an animal with at least a first LRP6 antigenic composition and selecting from the immunized animal an antibody that substantially cross-reacts with the anti-LRP6 monoclonal antibodies provided herein.

[0149] Some of the antibodies and antigen-binding fragments that are provided include (a) one or more light chain (LC) complementary determining regions (CDRs) selected from the group consisting of: [0150] (i) a LC CDR1 with at least 80% sequence identity to SEQ ID NO: 114, 126, 138, 150, 162, 174, 186, 198, 210, 222, 234, 246, 258, 282, 294, 306, 318, 330, 342, 354, or 366; [0151] (ii) a LC CDR2 with at least 80% sequence identity to SEQ ID NO: 115, 127, 139, 151, 163, 175, 187, 199, 211, 223, 235, 247, 259, 283, 295, 307, 319, 331, 343, 355, or 367; and [0152] (iii) a LC CDR3 with at least 80% sequence identity to SEQ ID NO: 116, 128, 140, 152, 164, 176, 188, 200, 212, 224, 236, 248, 260, 284, 296, 308, 320, 332, 344, 356, or 368; [0153] (b) one or more heavy chain (HC) CDRs selected from the group consisting of: [0154] (i) a HC CDR1 with at least 80% sequence identity to SEQ ID NO: 108, 120, 132, 144, 156, 168, 180, 192, 204, 216, 228, 240, 252, 264, 270, 276, 288, 300, 312, 324, 336, 348, or 360; [0155] (ii) a HC CDR2 with at least 80% sequence identity to SEQ ID NO: 109, 121, 133, 145, 157, 169, 181, 193, 205, 217, 229, 241, 253, 265, 271, 277, 289, 301, 313, 325, 337, 349, or 361; and [0156] (iii) a HC CDR3 with at least 80% sequence identity to SEQ ID NO: 110, 122, 134, 146, 158, 170, 182, 194, 206, 218, 230, 242, 254, 266, 272, 278, 290, 302, 314, 326, 338, 350, or 362; or [0157] (c) one or more LC CDRs of (a) and one or more HC CDRs of (b).

[0158] Such antibodies or antigen-binding fragments thereof may specifically bind an LRP6 polypeptide. Certain antibodies or fragments include one, two, three, four, five or six of the foregoing CDRs. In a particular embodiment, the CDRs are arranged as in monoclonal antibodies 77.2, 135.16, 213.7, 240.8, 413.1, 421.1, 498.3, 537.2, 606.4, 620.1, 856.6, 923.3, 931.1, 993.9, 995.5, 1115.3, 1213.2, 1253.12, 1281.1, 1293.11, 1433.8, 1470.2, or 1903.1.

[0159] The light chain and heavy chains of other antibodies or fragments are as described above but have at least 90% sequence identity to the foregoing sequences. Still other antibodies or antigen-binding fragments thereof have a light chain in which CDR1 has the amino acid sequence as set forth in SEQ ID NO: 114, 126, 138, 150, 162, 174, 186, 198, 210, 222, 234, 246, 258, 282, 294, 306, 318, 330, 342, 354 or 366, CDR2 has the amino acid sequence as set forth in SEQ ID NO: 115, 127, 139, 151, 163, 175, 187, 199, 211, 223, 235, 247, 259, 283, 295, 307, 319, 331, 343, 355 or 367, and/or CDR3 has the amino acid sequence as set forth in SEQ ID NO: 116, 128, 140, 152, 164, 176, 188, 200, 212, 224, 236, 248, 260, 284, 296, 308, 320, 332, 344, 356, or 368. Some antibodies or antigen-binding fragments thereof may also have a heavy chain in which CDR1 has the amino acid sequence as set forth in SEQ ID NO: 108, 120, 132, 144, 156, 168, 180, 192, 204, 216, 228, 240, 252, 264, 270, 276, 288, 300, 312, 324, 336, 348, or 360, CDR2 has the amino acid sequence as set forth in SEQ ID NO: 109, 121, 133, 145, 157, 169, 181, 193, 205, 217, 229, 241, 253, 265, 271, 277, 289, 301, 313, 325, 337, 349, or 361, and/or CDR3 has the amino acid sequence as set forth in SEQ ID NO: 110, 122, 134, 146, 158, 170, 182, 194, 206, 218, 230, 242, 254, 266, 272, 278, 290, 302, 314, 326, 338, 350, or 362.

[0160] The antibodies encompassed by the present invention include IgA, IgG.sub.1-4, IgE, IgM, and IgD antibodies. In a preferred embodiment, the antibody is an IgG and is an IgG.sub.1, IgG.sub.2, IgG.sub.3, or IgG.sub.4 subtype. In another preferred embodiment, the anti-LRP6 antibody is the same class and subclass as antibodies 77.2, 135.16, 213.7, 240.8, 413.1, 421.1, 498.3, 537.2, 606.4, 620.1, 856.6, 923.3, 931.1, 993.9, 995.5, 1115.3, 1213.2, 1253.12, 1281.1, 1293.11, 1433.8, 1470.2, or 1903.1.

[0161] The class and subclass of anti-LRP6 antibodies may be identified by any method known in the art. In general, the class and subclass of an antibody may be identified using antibodies that are specific for a particular class and subclass of antibody. Such antibodies are available commercially. The class and subclass can be determined by ELISA, Western blot, as well as other techniques. Alternatively, the class and subclass may be determined by sequencing all or a portion of the constant domains of the heavy and/or light chains of the antibodies, comparing their amino acid sequences to the known amino acid sequences of various classes and subclasses of immunoglobulins, and determining the class and subclass of the antibodies.

[0162] In another aspect of the invention, the anti-LRP6 antibody demonstrates both species and molecule selectivity. In one embodiment, the anti-LRP6 antibody binds to human, cynomologous, rhesus or chimpanzee LRP6. Following the teachings of the specification, one may determine the species selectivity for the anti-LRP6 antibody using methods well known in the art. For instance, one may determine species selectivity using Western blot, FACS, ELISA or RIA.

A. Naturally Occurring Antibody Structure

[0163] Some of the selective binding agents that are provided have the structure typically associated with naturally occurring antibodies. The structural units of these antibodies typically comprise one or more tetramers, each composed of two identical couplets of polypeptide chains, though some species of mammals also produce antibodies having only a single heavy chain. In a typical antibody, each pair or couplet includes one full-length "light" chain (in certain embodiments, about 25 kD) and one full-length "heavy" chain (in certain embodiments, about 50-70 kD). Each individual immunoglobulin chain is composed of several "immunoglobulin (Ig) domains," each consisting of roughly 90 to 110 amino acids and expressing a characteristic folding pattern. These domains are the basic units of which antibody polypeptides are composed. The amino-terminal portion of each chain typically includes a variable domain that is responsible for antigen recognition. The carboxy-terminal portion is more conserved evolutionarily than the other end of the chain and is referred to as the "constant region" or "C region". Human light chains generally are classified as kappa (.kappa.) and lambda (.lamda.) light chains, and each of these contains one variable domain and one constant domain. Heavy chains are typically classified as mu (.mu.), delta (.delta.), gamma (.gamma.), alpha (.alpha.), or epsilon (.epsilon.) chains and these define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subtypes, including, but not limited to, IgG.sub.1, IgG.sub.2, IgG.sub.3, and IgG.sub.4. IgM subtypes include IgM.sub.1 and IgM.sub.2. IgA subtypes include IgA, and IgA.sub.2. In humans, the IgA and IgD isotypes contain four heavy chains and four light chains; the IgG and IgE isotypes contain two heavy chains and two light chains; and the IgM isotype contains five heavy chains and five light chains. The heavy chain C region typically comprises one or more domains that may be responsible for effector function. The number of heavy chain constant region domains will depend on the isotype. IgG heavy chains, for example, each contains three C region domains known as C.sub.H1, C.sub.H2, and C.sub.H3. The antibodies that are provided may have any of these isotypes and subtypes. In certain embodiments of the invention, the anti-LRP6 antibodies are of the IgG.sub.1, IgG.sub.2a or IgG.sub.2b subtypes.

[0164] In full-length light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 10 or more amino acids. See, e.g., Fundamental Immunology, 2.sup.nd ed., Ch. 7 (Paul, W., ed) 1989, New York: Raven Press (herein incorporated by reference in its entirety for all purposes). The variable regions of each light/heavy chain pair typically form the antigen binding site.

[0165] Variable regions of immunoglobulin chains generally exhibit the same overall structure, comprising relatively conserved framework regions (FR) joined by three hypervariable regions, more often called "complementarity determining regions" or CDRs. The CDRs from the two chains of each heavy chain/light chain pair mentioned above typically are aligned by the framework regions to form a structure that binds specifically with a specific epitope on the target protein (e.g., LRP6). From N-terminal to C-terminal, naturally occurring light and heavy chain variable regions both typically conform to the following order of these elements: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. A numbering system has been devised for assigning numbers to amino acids that occupy positions in each of these domains. This numbering system is defined in Kabat et al., Sequences of Proteins of Immunological Interest (1991, National Institutes of Health Publication No. 91-3242, 5.sup.th ed., U.S. Department of Health and Human Services, Bethesda, Md.) or Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987); Chothia et al., Nature 342:878-883 (1989).

[0166] As a specific example of such antibodies, in one embodiment, the anti-LRP6 antibody is a monoclonal antibody derived from mice. Exemplary antibodies capable of binding to the aforementioned epitope are the monoclonal antibodies 77.2, 135.16, 213.7, 240.8, 413.1, 421.1, 498.3, 537.2, 606.4, 620.1, 856.6, 923.3, 931.1, 993.9, 995.5, 1115.3, 1213.2, 1253.12, 1281.1, 1293.11, 1433.8, 1470.2, or 1903.1 (see, Examples below), each of which comprises a light chain and a heavy chain.

B. Variable Domains of Antibodies

[0167] Also provided are antibodies that comprise a light chain variable region selected from the group consisting of V.sub.L1, V.sub.L2, V.sub.L3, V.sub.L4, V.sub.L5, V.sub.L6, V.sub.L7, V.sub.L8, V.sub.L9, V.sub.L10, V.sub.L11, V.sub.L12, V.sub.L13, V.sub.L14, V.sub.L15, V.sub.L16, V.sub.L17, V.sub.L18, V.sub.L19, V.sub.L20, V.sub.L21, V.sub.L22, or V.sub.L23 and/or a heavy chain variable region selected from the group consisting of V.sub.H1 and V.sub.H2, V.sub.H3, V.sub.H4, V.sub.H5, V.sub.H6, V.sub.H7, V.sub.H8, V.sub.H9, V.sub.H10, V.sub.H11, V.sub.H12, V.sub.H13, V.sub.H14, V.sub.H15, V.sub.H16, V.sub.H17, V.sub.H18, V.sub.H19, V.sub.H20, V.sub.H21, V.sub.H22, or V.sub.H23 as shown in Table 1 below, and antigen-binding regions, derivatives, muteins and variants of these light and heavy chain variable regions.

[0168] Antibodies of this type can generally be designated by the formula "V.sub.LxV.sub.Hy," wherein "x" is the number of the light chain variable region and "y" corresponds to the number of the heavy chain variable region as listed in Table 1. In general, x and y are each 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23.

TABLE-US-00001 TABLE 1 Antibody Abbreviated Chain NT Sequence AA Sequence Designation Name Type (SEQ ID NO:) (SEQ ID NO:) 77.2 V.sub.H1 Heavy 17 18 77.2 V.sub.L1 Light 19 20 135.16 V.sub.H2 Heavy 21 22 135.16 V.sub.L2 Light 23 24 213.7 V.sub.H3 Heavy 25 26 213.7 V.sub.L3 Light 27 28 240.8 V.sub.H4 Heavy 29 30 240.8 V.sub.L4 Light 31 32 413.1 V.sub.H5 Heavy 33 34 413.1 V.sub.L5 Light 35 36 421.1 V.sub.H6 Heavy 37 38 421.1 V.sub.L6 Light 39 40 498.3 V.sub.H7 Heavy 41 42 498.3 V.sub.L7 Light 43 44 537.2 V.sub.H8 Heavy 45 46 537.2 V.sub.L8 Light 47 48 606.4 V.sub.H9 Heavy 49 50 606.4 V.sub.L9 Light 51 52 620.1 V.sub.H10 Heavy 53 54 620.1 V.sub.L10 Light 55 56 856.6 V.sub.H11 Heavy 57 58 856.6 V.sub.L11 Light 59 60 923.3 V.sub.H12 Heavy 61 62 923.3 V.sub.L12 Light 63 64 931.1 V.sub.H13 Heavy 65 66 931.1 V.sub.L13 Light 67 68 993.9 V.sub.H14 Heavy 69 70 993.9 V.sub.L14 Light N/D N/D 995.5 V.sub.H15 Heavy 71 72 995.5 V.sub.L15 Light N/D N/D 1115.3 V.sub.H16 Heavy 73 74 1115.3 V.sub.L16 Light 75 76 1213.2 V.sub.H17 Heavy 77 78 1213.2 V.sub.L17 Light 79 80 1253.12 V.sub.H18 Heavy 81 82 1253.12 V.sub.L18 Light 83 84 1281.1 V.sub.H19 Heavy 85 86 1281.1 V.sub.L19 Light 87 88 1293.11 V.sub.H20 Heavy 89 90 1293.11 V.sub.L20 Light 91 92 1433.8 V.sub.H21 Heavy 93 94 1433.8 V.sub.L21 Light 95 96 1470.2 V.sub.H22 Heavy 97 98 1470.2 V.sub.L22 Light 99 100 1903.1 V.sub.H23 Heavy 101 102 1903.1 V.sub.L23 Light 103 104 *N/D = not determined

[0169] Thus, V.sub.L2V.sub.H1 refers to an antibody with a light chain variable region domain comprising the amino acid sequence of V.sub.L2 and a heavy chain variable region comprising the amino acid sequence of V.sub.H1. In some instances, the foregoing antibodies include two light chain variable region domains and two heavy chain variable region domains (e.g., V.sub.L1.sub.2V.sub.H1.sub.2, etc.).

[0170] As a specific example of such antibodies, certain antibodies or antigen-binding fragments thereof comprise the variable region of the light chain or the variable region of the heavy chain of 77.2, 135.16, 213.7, 240.8, 413.1, 421.1, 498.3, 537.2, 606.4, 620.1, 856.6, 923.3, 931.1, 993.9, 995.5, 1115.3, 1213.2, 1253.12, 1281.1, 1293.11, 1433.8, 1470.2, or 1903.1, wherein the light chain variable region consists of the amino acids shown in SEQ ID NO: 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 76, 80, 84, 88, 92, 96, 100, or 104 and the heavy chain variable region consists of the amino acids shown in SEQ ID NO: 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 72, 74, 78, 82, 86, 90, 94, 98, or 102. In one aspect of this embodiment, the antibody consists of two identical heavy chains and two identical light chains.

[0171] Certain antibodies or antigen-binding fragments thereof comprise a light chain variable domain comprising a sequence of amino acids that differs from the sequence of a light chain variable domain selected from V.sub.L1, V.sub.L2, V.sub.L3, V.sub.L4, V.sub.L5, V.sub.L6, V.sub.L7, V.sub.L8, V.sub.L9, V.sub.L10, V.sub.L11, V.sub.L12, V.sub.L13, V.sub.L14, V.sub.L15, V.sub.L16, V.sub.L17, V.sub.L18, V.sub.L19, V.sub.L20, V.sub.L21, V.sub.L22, or V.sub.L23 at only 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid residues, wherein each such sequence difference is independently either a deletion, insertion, or substitution of one amino acid. The light chain variable region in some antibodies comprises a sequence of amino acids that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequences of the light chain variable regions of V.sub.L1, V.sub.L2, V.sub.L3, V.sub.L4, V.sub.L5, V.sub.L6, V.sub.L7, V.sub.L8, V.sub.L9, V.sub.L10, V.sub.L11, V.sub.L12, V.sub.L13, V.sub.L14, V.sub.L15, V.sub.L16, V.sub.L17, V.sub.L18, V.sub.L19, V.sub.L20, V.sub.L21, V.sub.L22, or V.sub.L23.

[0172] Some antibodies or antigen-binding fragments thereof that are provided comprise a heavy chain variable domain comprising a sequence of amino acids that differs from the sequence of a heavy chain variable domain selected from V.sub.H1 and V.sub.H2, V.sub.H3, V.sub.H4, V.sub.H5, V.sub.H6, V.sub.H7, V.sub.H8, V.sub.H9, V.sub.H10, V.sub.H11, V.sub.H12, V.sub.H13, V.sub.H14, V.sub.H15, V.sub.H16, V.sub.H17, V.sub.H18, V.sub.H19, V.sub.H20, V.sub.H21, V.sub.H22 or V.sub.H23 only at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acid residues, wherein each such sequence difference is independently either a deletion, insertion, or substitution of one amino acid. The heavy chain variable region in some antibodies comprises a sequence of amino acids that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequences of the heavy chain variable region of V.sub.H1 and V.sub.H2, V.sub.H3, V.sub.H4, V.sub.H5, V.sub.H6, V.sub.H7, V.sub.H8, V.sub.H9, V.sub.H10, V.sub.H11, V.sub.H12, V.sub.H13, V.sub.H14, V.sub.H15, V.sub.H16, V.sub.H17, V.sub.H18, V.sub.H19, V.sub.H20, V.sub.H21, V.sub.H22 or V.sub.H23. Still other antibodies or antigen-binding fragments thereof include variant forms of a variant light chain and a variant heavy chain as just described. An alignment of the variable domains of the antibodies listed in Table 1 is seen in FIGS. 4 (heavy chain alignment) and 5 (light chain alignment).

C. CDRs of Antibodies

[0173] Complementarity determining regions (CDRs) and framework regions (FR) of a given antibody may be identified using the system described by Kabat et al., 1991, supra. Certain antibodies that are disclosed herein comprise one or more amino acid sequences that are identical or have substantial sequence identity to the amino acid sequences of one or more of the CDRs as summarized in Table 2.

TABLE-US-00002 TABLE 2 Antibody NT Sequence AA Sequence Designation Chain CDR (SEQ ID NO:) (SEQ ID NO:) 77.2 Heavy CDR1 105 108 77.2 Heavy CDR2 106 109 77.2 Heavy CDR3 107 110 77.2 Light CDR1 111 114 77.2 Light CDR2 112 115 77.2 Light CDR3 113 116 135.16 Heavy CDR1 117 120 135.16 Heavy CDR2 118 121 135.16 Heavy CDR3 119 122 135.16 Light CDR1 123 126 135.16 Light CDR2 124 127 135.16 Light CDR3 125 128 213.7 Heavy CDR1 129 132 213.7 Heavy CDR2 130 133 213.7 Heavy CDR3 131 134 213.7 Light CDR1 135 138 213.7 Light CDR2 136 139 213.7 Light CDR3 137 140 240.8 Heavy CDR1 141 144 240.8 Heavy CDR2 142 145 240.8 Heavy CDR3 143 146 240.8 Light CDR1 147 150 240.8 Light CDR2 148 151 240.8 Light CDR3 149 152 413.1 Heavy CDR1 153 156 413.1 Heavy CDR2 154 157 413.1 Heavy CDR3 155 158 413.1 Light CDR1 159 162 413.1 Light CDR2 160 163 413.1 Light CDR3 161 164 421.1 Heavy CDR1 165 168 421.1 Heavy CDR2 166 169 421.1 Heavy CDR3 167 170 421.1 Light CDR1 171 174 421.1 Light CDR2 172 175 421.1 Light CDR3 173 176 498.3 Heavy CDR1 177 180 498.3 Heavy CDR2 178 181 498.3 Heavy CDR3 179 182 489.3 Light CDR1 183 186 489.3 Light CDR2 184 187 489.3 Light CDR3 185 188 537.2 Heavy CDR1 189 192 537.2 Heavy CDR2 190 193 537.2 Heavy CDR3 191 194 537.2 Light CDR1 195 198 537.2 Light CDR2 196 199 537.2 Light CDR3 197 200 606.4 Heavy CDR1 201 204 606.4 Heavy CDR2 202 205 606.4 Heavy CDR3 203 206 606.4 Light CDR1 207 210 606.4 Light CDR2 208 211 606.4 Light CDR3 209 212 620.1 Heavy CDR1 213 216 620.1 Heavy CDR2 214 217 620.1 Heavy CDR3 215 218 620.1 Light CDR1 219 222 620.1 Light CDR2 220 223 620.1 Light CDR3 221 224 856.6 Heavy CDR1 225 228 856.6 Heavy CDR2 226 229 856.6 Heavy CDR3 227 230 856.6 Light CDR1 231 234 856.6 Light CDR2 232 235 856.6 Light CDR3 233 236

[0174] The antibodies and antigen-binding fragments that are provided can each include one, two, three, four, five or six of the CDRs listed above. Certain antibodies have variant forms of the CDRs listed in Table 2, with one or more (e.g., 2, 3, 4, 5 or 6) of the CDRs each having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to a CDR sequence listed in Table 2. For example, the antibody or antigen-binding region may include both a light chain CDR3 and a heavy chain CDR3 that each have at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the light chain CDR3 and heavy chain CDR3, respectively, listed in Table 2. The invention also provides for antibodies that have CDR sequences that differ from the CDR sequences listed in Table 2 such that the amino acid sequence for any given CDR differs from the sequence listed in Table 2 by no more than 1, 2, 3, 4, or 5 amino acid residues. Differences from the listed sequences usually are conservative substitutions (see below).

[0175] As a specific example, the antibodies and antigen-binding fragments that are provided may comprise one or more of the following CDR sequences from the 77.2 light chain:

CDR1: amino acids 44-59 of SEQ ID NO: 20, which also corresponds to SEQ ID NO: 114 (encoded by nucleotides 130-177 of SEQ ID NO: 19 (SEQ ID NO: 111)); CDR2: amino acids 75-81 of SEQ ID NO: 20, which also corresponds to SEQ ID NO: 115 (encoded by nucleotides 223-243 of SEQ ID NO: 19 (SEQ ID NO: 112)); and CDR3: amino acids 114-122 of SEQ ID NO: 20, which also corresponds to SEQ ID NO: 116 (encoded by nucleotides 340-366 of SEQ ID NO: 19 (SEQ ID NO: 113)).

[0176] Additional antibodies and antigen-binding fragments of the invention may comprise one or more of the following CDR sequences from the 77.2 heavy chain:

CDR1: amino acids 45-54 of SEQ ID NO: 18, which also corresponds to SEQ ID NO: 108 (encoded by nucleotides 133-162 of SEQ ID NO: 17 (SEQ ID NO: 105)); CDR2: amino acids 69-85 of SEQ ID NO: 18, which also corresponds to SEQ ID NO: 109 (encoded by nucleotides 205-255 of SEQ ID NO: 17 (SEQ ID NO: 106)); and CDR3: amino acids 118-133 of SEQ ID NO: 18, which also corresponds to SEQ ID NO: 110 (encoded by nucleotides 352-399 of SEQ ID NO: 17 (SEQ ID NO: 107)).

[0177] As another specific example, the antibodies and antigen-binding fragments that are provided may comprise one or more of the following CDR sequences from the 135.16 light chain:

CDR1: amino acids 48-58 of SEQ ID NO: 24, which also corresponds to SEQ ID NO: 126 (encoded by nucleotides 142-174 of SEQ ID NO: 23 (SEQ ID NO: 123)); CDR2: amino acids 74-80 of SEQ ID NO: 24, which also corresponds to SEQ ID NO: 127 (encoded by nucleotides 222-240 of SEQ ID NO: 23 (SEQ ID NO: 124)); and CDR3: amino acids 113-121 of SEQ ID NO: 24, which also corresponds to SEQ ID NO: 128 (encoded by nucleotides 337-363 of SEQ ID NO: 23 (SEQ ID NO: 125)).

[0178] Additional antibodies and antigen-binding fragments of the invention may comprise one or more of the following CDR sequences from the 135.16 heavy chain:

CDR1: amino acids 45-54 of SEQ ID NO: 22, which also corresponds to SEQ ID NO: 120 (encoded by nucleotides 133-162 of SEQ ID NO: 21 (SEQ ID NO: 117)); CDR2: amino acids 69-85 of SEQ ID NO: 22, which also corresponds to SEQ ID NO: 121 (encoded by nucleotides 205-255 of SEQ ID NO: 21 (SEQ ID NO: 118)); and CDR3: amino acids 118-129 of SEQ ID NO: 22, which also corresponds to SEQ ID NO: 122 (encoded by nucleotides 352-387 of SEQ ID NO: 21 (SEQ ID NO: 119)).

[0179] As another specific example, the antibodies and antigen-binding fragments that are provided may comprise one or more of the following CDR sequences from the 213.7 light chain:

CDR1: amino acids 45-54 of SEQ ID NO: 28, which also corresponds to SEQ ID NO: 138 (encoded by nucleotides 133-162 of SEQ ID NO: 27 (SEQ ID NO: 135)); CDR2: amino acids 70-76 of SEQ ID NO: 28, which also corresponds to SEQ ID NO: 139 (encoded by nucleotides 208-228 of SEQ ID NO: 27 (SEQ ID NO: 136)); and CDR3: amino acids 109-117 of SEQ ID NO: 28, which also corresponds to SEQ ID NO: 140 (encoded by nucleotides 325-351 of SEQ ID NO: 27 (SEQ ID NO: 137)).

[0180] Additional antibodies and antigen-binding fragments of the invention may comprise one or more of the following CDR sequences from the 213.7 heavy chain:

CDR1: amino acids 45-54 of SEQ ID NO: 26, which also corresponds to SEQ ID NO: 132 (encoded by nucleotides 133-162 of SEQ ID NO: 25 (SEQ ID NO: 129)); CDR2: amino acids 69-85 of SEQ ID NO: 26, which also corresponds to SEQ ID NO: 133 (encoded by nucleotides 205-255 of SEQ ID NO: 25 (SEQ ID NO: 130)); and CDR3: amino acids 118-129 of SEQ ID NO: 26, which also corresponds to SEQ ID NO: 134 (encoded by nucleotides 352-387 of SEQ ID NO: 25 (SEQ ID NO: 131)).

[0181] As another specific example, the antibodies and antigen-binding fragments that are provided may comprise one or more of the following CDR sequences from the 240.8 light chain:

CDR1: amino acids 45-54 of SEQ ID NO: 32, which also corresponds to SEQ ID NO: 150 (encoded by nucleotides 130-162 of SEQ ID NO: 31 (SEQ ID NO: 147)); CDR2: amino acids 70-76 of SEQ ID NO: 32, which also corresponds to SEQ ID NO: 151 (encoded by nucleotides 208-228 of SEQ ID NO: 31 (SEQ ID NO: 148)); and CDR3: amino acids 109-117 of SEQ ID NO: 32, which also corresponds to SEQ ID NO: 152 (encoded by nucleotides 325-351 of SEQ ID NO: 31 (SEQ ID NO: 149)).

[0182] Additional antibodies and antigen-binding fragments of the invention may comprise one or more of the following CDR sequences from the 240.8 heavy chain:

CDR1: amino acids 45-54 of SEQ ID NO: 30, which also corresponds to SEQ ID NO: 144 (encoded by nucleotides 133-162 of SEQ ID NO: 29 (SEQ ID NO: 141)); CDR2: amino acids 69-85 of SEQ ID NO: 30, which also corresponds to SEQ ID NO: 145 (encoded by nucleotides 205-255 of SEQ ID NO: 29 (SEQ ID NO: 142)); and CDR3: amino acids 118-128 of SEQ ID NO: 30, which also corresponds to SEQ ID NO: 146 (encoded by nucleotides 352-384 of SEQ ID NO: 29 (SEQ ID NO: 143)).

[0183] As another specific example, the antibodies and antigen-binding fragments that are provided may comprise one or more of the following CDR sequences from the 413.1 light chain:

CDR1: amino acids 46-55 of SEQ ID NO: 36, which also corresponds to SEQ ID NO: 162 (encoded by nucleotides 139-165 of SEQ ID NO: 35 (SEQ ID NO: 159)); CDR2: amino acids 71-77 of SEQ ID NO: 36, which also corresponds to SEQ ID NO: 163 (encoded by nucleotides 211-231 of SEQ ID NO: 35 (SEQ ID NO: 160)); and CDR3: amino acids 110-118 of SEQ ID NO: 36, which also corresponds to SEQ ID NO: 164 (encoded by nucleotides 328-354 of SEQ ID NO: 35 (SEQ ID NO: 161)).

[0184] Additional antibodies and antigen-binding fragments of the invention may comprise one or more of the following CDR sequences from the 413.1 heavy chain:

CDR1: amino acids 58-67 of SEQ ID NO: 34, which also corresponds to SEQ ID NO: 156 (encoded by nucleotides 172-201 of SEQ ID NO: 33 (SEQ ID NO: 153)); CDR2: amino acids 82-98 of SEQ ID NO: 34, which also corresponds to SEQ ID NO: 157 (encoded by nucleotides 244-294 of SEQ ID NO: 33 (SEQ ID NO: 154)); and CDR3: amino acids 131-142 of SEQ ID NO: 34, which also corresponds to SEQ ID NO: 158 (encoded by nucleotides 391-426 of SEQ ID NO: 33 (SEQ ID NO: 155)).

[0185] As another specific example, the antibodies and antigen-binding fragments that are provided may comprise one or more of the following CDR sequences from the 421.1 light chain:

CDR1: amino acids 44-53 of SEQ ID NO: 40, which also corresponds to SEQ ID NO: 174 (encoded by nucleotides 130-159 of SEQ ID NO: 39 (SEQ ID NO: 171)); CDR2: amino acids 69-75 of SEQ ID NO: 40, which also corresponds to SEQ ID NO: 175 (encoded by nucleotides 205-225 of SEQ ID NO: 39 (SEQ ID NO: 172)); and CDR3: amino acids 108-116 of SEQ ID NO: 40, which also corresponds to SEQ ID NO: 176 (encoded by nucleotides 322-348 of SEQ ID NO: 39 (SEQ ID NO: 173)).

[0186] Additional antibodies and antigen-binding fragments of the invention may comprise one or more of the following CDR sequences from the 421.1 heavy chain:

CDR1: amino acids 45-54 of SEQ ID NO: 38, which also corresponds to SEQ ID NO: 168 (encoded by nucleotides 133-162 of SEQ ID NO: 37 (SEQ ID NO: 165)); CDR2: amino acids 69-75 of SEQ ID NO: 38, which also corresponds to SEQ ID NO: 169 (encoded by nucleotides 205-225 of SEQ ID NO: 37 (SEQ ID NO: 166)); and CDR3: amino acids 108-116 of SEQ ID NO: 38, which also corresponds to SEQ ID NO: 170 (encoded by nucleotides 322-348 of SEQ ID NO: 37 (SEQ ID NO: 167)).

[0187] As another specific example, the antibodies and antigen-binding fragments that are provided may comprise one or more of the following CDR sequences from the 498.3 light chain:

CDR1: amino acids 44-54 of SEQ ID NO: 44, which also corresponds to SEQ ID NO: 186 (encoded by nucleotides 130-162 of SEQ ID NO: 43 (SEQ ID NO: 183)); CDR2: amino acids 70-76 of SEQ ID NO: 44, which also corresponds to SEQ ID NO: 187 (encoded by nucleotides 208-228 of SEQ ID NO: 43 (SEQ ID NO: 184)); and CDR3: amino acids 109-117 of SEQ ID NO: 44, which also corresponds to SEQ ID NO: 188 (encoded by nucleotides 325-351 of SEQ ID NO: 43 (SEQ ID NO: 185)).

[0188] Additional antibodies and antigen-binding fragments of the invention may comprise one or more of the following CDR sequences from the 489.3 heavy chain:

CDR1: amino acids 45-54 of SEQ ID NO: 42, which also corresponds to SEQ ID NO: 180 (encoded by nucleotides 133-162 of SEQ ID NO: 41 (SEQ ID NO: 177)); CDR2: amino acids 69-85 of SEQ ID NO: 42, which also corresponds to SEQ ID NO: 181 (encoded by nucleotides 205-255 of SEQ ID NO: 41 (SEQ ID NO: 178)); and CDR3: amino acids 118-127 of SEQ ID NO: 42, which also corresponds to SEQ ID NO: 182 (encoded by nucleotides 352-381 of SEQ ID NO: 41 (SEQ ID NO: 179)).

[0189] As another specific example, the antibodies and antigen-binding fragments that are provided may comprise one or more of the following CDR sequences from the 537.2 light chain:

CDR1: amino acids 46-55 of SEQ ID NO: 48, which also corresponds to SEQ ID NO: 198 (encoded by nucleotides 136-165 of SEQ ID NO: 47 (SEQ ID NO: 195)); CDR2: amino acids 71-77 of SEQ ID NO: 48, which also corresponds to SEQ ID NO: 199 (encoded by nucleotides 211-231 of SEQ ID NO: 47 (SEQ ID NO: 196)); and CDR3: amino acids 110-118 of SEQ ID NO: 48, which also corresponds to SEQ ID NO: 200 (encoded by nucleotides 328-354 of SEQ ID NO: 47 (SEQ ID NO: 197)).

[0190] Additional antibodies and antigen-binding fragments of the invention may comprise one or more of the following CDR sequences from the 537.2 heavy chain:

CDR1: amino acids 44-54 of SEQ ID NO: 46, which also corresponds to SEQ ID NO: 192 (encoded by nucleotides 130-162 of SEQ ID NO: 45 (SEQ ID NO: 189)); CDR2: amino acids 69-84 of SEQ ID NO: 46, which also corresponds to SEQ ID NO: 193 (encoded by nucleotides 205-252 of SEQ ID NO: 45 (SEQ ID NO: 190)); and CDR3: amino acids 117-127 of SEQ ID NO: 46, which also corresponds to SEQ ID NO: 194 (encoded by nucleotides 349-381 of SEQ ID NO: 45 (SEQ ID NO: 191)).

[0191] As another specific example, the antibodies and antigen-binding fragments that are provided may comprise one or more of the following CDR sequences from the 606.4 light chain:

CDR1: amino acids 44-54 of SEQ ID NO: 52, which also corresponds to SEQ ID NO: 210 (encoded by nucleotides 130-162 of SEQ ID NO: 51 (SEQ ID NO: 207)); CDR2: amino acids 70-76 of SEQ ID NO: 52, which also corresponds to SEQ ID NO: 211 (encoded by nucleotides 208-228 of SEQ ID NO: 51 (SEQ ID NO: 208)); and CDR3: amino acids 109-117 of SEQ ID NO: 52, which also corresponds to SEQ ID NO: 212 (encoded by nucleotides 325-351 of SEQ ID NO: 51 (SEQ ID NO: 209)).

[0192] Additional antibodies and antigen-binding fragments of the invention may comprise one or more of the following CDR sequences from the 606.4 heavy chain:

CDR1: amino acids 45-54 of SEQ ID NO: 50, which also corresponds to SEQ ID NO: 204 (encoded by nucleotides 133-162 of SEQ ID NO: 49 (SEQ ID NO: 201)); CDR2: amino acids 69-85 of SEQ ID NO: 50, which also corresponds to SEQ ID NO: 205 (encoded by nucleotides 205-255 of SEQ ID NO: 49 (SEQ ID NO: 202)); and CDR3: amino acids 118-127 of SEQ ID NO: 50, which also corresponds to SEQ ID NO: 207 (encoded by nucleotides 352-381 of SEQ ID NO: 49 (SEQ ID NO: 203)).

[0193] As another specific example, the antibodies and antigen-binding fragments that are provided may comprise one or more of the following CDR sequences from the 620.1 light chain:

CDR1: amino acids 44-60 of SEQ ID NO: 56, which also corresponds to SEQ ID NO: 222 (encoded by nucleotides 130-180 of SEQ ID NO: 55 (SEQ ID NO: 219)); CDR2: amino acids 76-82 of SEQ ID NO: 56, which also corresponds to SEQ ID NO: 223 (encoded by nucleotides 226-246 of SEQ ID NO: 55 (SEQ ID NO: 220)); and CDR3: amino acids 115-123 of SEQ ID NO: 56, which also corresponds to SEQ ID NO: 224 (encoded by nucleotides 343-369 of SEQ ID NO: 55 (SEQ ID NO: 221)).

[0194] Additional antibodies and antigen-binding fragments of the invention may comprise one or more of the following CDR sequences from the 620.1 heavy chain:

CDR1: amino acids 58-67 of SEQ ID NO: 54, which also corresponds to SEQ ID NO: 216 (encoded by nucleotides 172-201 of SEQ ID NO: 53 (SEQ ID NO: 213)); CDR2: amino acids 82-98 of SEQ ID NO: 54, which also corresponds to SEQ ID NO: 217 (encoded by nucleotides 244-294 of SEQ ID NO: 53 (SEQ ID NO: 214)); and CDR3: amino acids 131-142 of SEQ ID NO: 54, which also corresponds to SEQ ID NO: 218 (encoded by nucleotides 391-426 of SEQ ID NO: 53 (SEQ ID NO: 215)).

[0195] As another specific example, the antibodies and antigen-binding fragments that are provided may comprise one or more of the following CDR sequences from the 856.6 light chain:

CDR1: amino acids 28-37 of SEQ ID NO: 60, which also corresponds to SEQ ID NO: 234 (encoded by nucleotides 82-111 of SEQ ID NO: 59 (SEQ ID NO: 231)); CDR2: amino acids 53-59 of SEQ ID NO: 60, which also corresponds to SEQ ID NO: 235 (encoded by nucleotides 157-177 of SEQ ID NO: 59 (SEQ ID NO: 232)); and CDR3: amino acids 92-100 of SEQ ID NO: 60, which also corresponds to SEQ ID NO: 236 (encoded by nucleotides 274-300 of SEQ ID NO: 59 (SEQ ID NO: 233)).

[0196] Additional antibodies and antigen-binding fragments of the invention may comprise one or more of the following CDR sequences from the 856.6 heavy chain:

CDR1: amino acids 45-54 of SEQ ID NO: 58, which also corresponds to SEQ ID NO: 228 (encoded by nucleotides 133-162 of SEQ ID NO: 57 (SEQ ID NO: 225)); CDR2: amino acids 69-85 of SEQ ID NO: 58, which also corresponds to SEQ ID NO: 229 (encoded by nucleotides 205-255 of SEQ ID NO: 57 (SEQ ID NO: 226)); and CDR3: amino acids 118-129 of SEQ ID NO: 58, which also corresponds to SEQ ID NO: 230 (encoded by nucleotides 352-387 of SEQ ID NO: 57 (SEQ ID NO: 227)).

[0197] As another specific example, the antibodies and antigen-binding fragments that are provided may comprise one or more of the following CDR sequences from the 923.3 light chain:

CDR1: amino acids 44-54 of SEQ ID NO: 64, which also corresponds to SEQ ID NO: 246 (encoded by nucleotides 130-162 of SEQ ID NO: 63 (SEQ ID NO: 243)); CDR2: amino acids 70-76 of SEQ ID NO: 64, which also corresponds to SEQ ID NO: 247 (encoded by nucleotides 208-228 of SEQ ID NO: 63 (SEQ ID NO: 244)); and CDR3: amino acids 109-117 of SEQ ID NO: 64, which also corresponds to SEQ ID NO: 248 (encoded by nucleotides 325-351 of SEQ ID NO: 63 (SEQ ID NO: 245)).

[0198] Additional antibodies and antigen-binding fragments of the invention may comprise one or more of the following CDR sequences from the 923.3 heavy chain:

CDR1: amino acids 45-54 of SEQ ID NO: 62, which also corresponds to SEQ ID NO: 240 (encoded by nucleotides 133-162 of SEQ ID NO: 61 (SEQ ID NO: 237)); CDR2: amino acids 69-85 of SEQ ID NO: 62, which also corresponds to SEQ ID NO: 241 (encoded by nucleotides 205-255 of SEQ ID NO: 61 (SEQ ID NO: 238)); and CDR3: amino acids 118-128 of SEQ ID NO: 62, which also corresponds to SEQ ID NO: 242 (encoded by nucleotides 352-384 of SEQ ID NO: 61 (SEQ ID NO: 239)).

[0199] As another specific example, the antibodies and antigen-binding fragments that are provided may comprise one or more of the following CDR sequences from the 931.1 light chain:

CDR1: amino acids 44-54 of SEQ ID NO: 68, which also corresponds to SEQ ID NO: 258 (encoded by nucleotides 130-162 of SEQ ID NO: 67 (SEQ ID NO: 255)); CDR2: amino acids 70-76 of SEQ ID NO: 68, which also corresponds to SEQ ID NO: 259 (encoded by nucleotides 208-228 of SEQ ID NO: 67 (SEQ ID NO: 256)); and CDR3: amino acids 109-117 of SEQ ID NO: 68, which also corresponds to SEQ ID NO: 260 (encoded by nucleotides 325-351 of SEQ ID NO: 67 (SEQ ID NO: 257)).

[0200] Additional antibodies and antigen-binding fragments of the invention may comprise one or more of the following CDR sequences from the 931.1 heavy chain:

CDR1: amino acids 45-54 of SEQ ID NO: 66, which also corresponds to SEQ ID NO: 252 (encoded by nucleotides 133-162 of SEQ ID NO: 65 (SEQ ID NO: 249)); CDR2: amino acids 69-85 of SEQ ID NO: 66, which also corresponds to SEQ ID NO: 253 (encoded by nucleotides 205-255 of SEQ ID NO: 65 (SEQ ID NO: 250)); and CDR3: amino acids 118-127 of SEQ ID NO: 66, which also corresponds to SEQ ID NO: 254 (encoded by nucleotides 352-381 of SEQ ID NO: 65 (SEQ ID NO: 251)).

[0201] Additional antibodies and antigen-binding fragments of the invention may comprise one or more of the following CDR sequences from the 993.9 heavy chain:

CDR1: amino acids 44-54 of SEQ ID NO: 70, which also corresponds to SEQ ID NO: 264 (encoded by nucleotides 130-162 of SEQ ID NO: 69 (SEQ ID NO: 261)); CDR2: amino acids 69-74 of SEQ ID NO: 70, which also corresponds to SEQ ID NO: 265 (encoded by nucleotides 205-222 of SEQ ID NO: 69 (SEQ ID NO: 262)); and CDR3: amino acids 117-127 of SEQ ID NO: 70, which also corresponds to SEQ ID NO: 266 (encoded by nucleotides 349-381 of SEQ ID NO: 69 (SEQ ID NO: 263)).

[0202] Additional antibodies and antigen-binding fragments of the invention may comprise one or more of the following CDR sequences from the 995.5 heavy chain:

CDR1: amino acids 45-55 of SEQ ID NO: 72, which also corresponds to SEQ ID NO: 270 (encoded by nucleotides 133-165 of SEQ ID NO: 71 (SEQ ID NO: 267)); CDR2: amino acids 70-85 of SEQ ID NO: 72, which also corresponds to SEQ ID NO: 271 (encoded by nucleotides 208-255 of SEQ ID NO: 71 (SEQ ID NO: 268)); and CDR3: amino acids 118-128 of SEQ ID NO: 72, which also corresponds to SEQ ID NO: 272 (encoded by nucleotides 352-384 of SEQ ID NO: 71 (SEQ ID NO: 269)).

[0203] As another specific example, the antibodies and antigen-binding fragments that are provided may comprise one or more of the following CDR sequences from the 1115.3 light chain:

CDR1: amino acids 44-54 of SEQ ID NO: 76, which also corresponds to SEQ ID NO: 282 (encoded by nucleotides 133-162 of SEQ ID NO: 75 (SEQ ID NO: 279)); CDR2: amino acids 70-76 of SEQ ID NO: 76, which also corresponds to SEQ ID NO: 283 (encoded by nucleotides 208-228 of SEQ ID NO: 75 (SEQ ID NO: 280)); and CDR3: amino acids 109-117 of SEQ ID NO: 76, which also corresponds to SEQ ID NO: 284 (encoded by nucleotides 325-351 of SEQ ID NO: 75 (SEQ ID NO: 281)).

[0204] Additional antibodies and antigen-binding fragments of the invention may comprise one or more of the following CDR sequences from the 1115.3 heavy chain:

CDR1: amino acids 45-54 of SEQ ID NO: 74, which also corresponds to SEQ ID NO: 276 (encoded by nucleotides 133-162 of SEQ ID NO: 73 (SEQ ID NO: 273)); CDR2: amino acids 69-85 of SEQ ID NO: 74, which also corresponds to SEQ ID NO: 277 (encoded by nucleotides 205-255 of SEQ ID NO: 73 (SEQ ID NO: 274)); and CDR3: amino acids 118-128 of SEQ ID NO: 74, which also corresponds to SEQ ID NO: 278 (encoded by nucleotides 352-384 of SEQ ID NO: 73 (SEQ ID NO: 275)).

[0205] As another specific example, the antibodies and antigen-binding fragments that are provided may comprise one or more of the following CDR sequences from the 1213.2 light chain:

CDR1: amino acids 44-54 of SEQ ID NO: 80, which also corresponds to SEQ ID NO: 294 (encoded by nucleotides 130-162 of SEQ ID NO: 79 (SEQ ID NO: 291)); CDR2: amino acids 70-76 of SEQ ID NO: 80, which also corresponds to SEQ ID NO: 295 (encoded by nucleotides 208-228 of SEQ ID NO: 79 (SEQ ID NO: 292)); and CDR3: amino acids 109-117 of SEQ ID NO: 80, which also corresponds to SEQ ID NO: 296 (encoded by nucleotides 323-351 of SEQ ID NO: 79 (SEQ ID NO: 293)).

[0206] Additional antibodies and antigen-binding fragments of the invention may comprise one or more of the following CDR sequences from the 1213.2 heavy chain:

CDR1: amino acids 45-54 of SEQ ID NO: 78, which also corresponds to SEQ ID NO: 288 (encoded by nucleotides 133-162 of SEQ ID NO: 77 (SEQ ID NO: 285)); CDR2: amino acids 69-85 of SEQ ID NO: 78, which also corresponds to SEQ ID NO: 289 (encoded by nucleotides 205-255 of SEQ ID NO: 77 (SEQ ID NO: 286)); and CDR3: amino acids 118-127 of SEQ ID NO: 78, which also corresponds to SEQ ID NO: 290 (encoded by nucleotides 352-381 of SEQ ID NO: 771 (SEQ ID NO: 287)).

[0207] As another specific example, the antibodies and antigen-binding fragments that are provided may comprise one or more of the following CDR sequences from the 1253.12 light chain:

CDR1: amino acids 46-55 of SEQ ID NO: 84, which also corresponds to SEQ ID NO: 306 (encoded by nucleotides 136-165 of SEQ ID NO: 83 (SEQ ID NO: 303)); CDR2: amino acids 71-77 of SEQ ID NO: 84, which also corresponds to SEQ ID NO: 307 (encoded by nucleotides 211-231 of SEQ ID NO: 83 (SEQ ID NO: 304)); and CDR3: amino acids 110-118 of SEQ ID NO: 84, which also corresponds to SEQ ID NO: 308 (encoded by nucleotides 328-354 of SEQ ID NO: 83 (SEQ ID NO: 305)).

[0208] Additional antibodies and antigen-binding fragments of the invention may comprise one or more of the following CDR sequences from the 1253.12 heavy chain:

CDR1: amino acids 61-70 of SEQ ID NO: 82, which also corresponds to SEQ ID NO: 300 (encoded by nucleotides 181-210 of SEQ ID NO: 81 (SEQ ID NO: 297)); CDR2: amino acids 85-101 of SEQ ID NO: 82, which also corresponds to SEQ ID NO: 301 (encoded by nucleotides 253-303 of SEQ ID NO: 81 (SEQ ID NO: 298)); and CDR3: amino acids 134-142 of SEQ ID NO: 82, which also corresponds to SEQ ID NO: 302 (encoded by nucleotides 400-426 of SEQ ID NO: 81 (SEQ ID NO: 299)).

[0209] As another specific example, the antibodies and antigen-binding fragments that are provided may comprise one or more of the following CDR sequences from the 1281.1 light chain:

CDR1: amino acids 44-59 of SEQ ID NO: 88, which also corresponds to SEQ ID NO: 318 (encoded by nucleotides 130-177 of SEQ ID NO: 87 (SEQ ID NO: 315)); CDR2: amino acids 75-81 of SEQ ID NO: 88, which also corresponds to SEQ ID NO: 319 (encoded by nucleotides 225-243 of SEQ ID NO: 87 (SEQ ID NO: 316)); and CDR3: amino acids 104-122 of SEQ ID NO: 88, which also corresponds to SEQ ID NO: 320 (encoded by nucleotides 310-366 of SEQ ID NO: 87 (SEQ ID NO: 317)).

[0210] Additional antibodies and antigen-binding fragments of the invention may comprise one or more of the following CDR sequences from the 1281.1 heavy chain:

CDR1: amino acids 45-54 of SEQ ID NO: 86, which also corresponds to SEQ ID NO: 312 (encoded by nucleotides 133-162 of SEQ ID NO: 85 (SEQ ID NO: 309)); CDR2: amino acids 69-85 of SEQ ID NO: 86, which also corresponds to SEQ ID NO: 313 (encoded by nucleotides 205-255 of SEQ ID NO: 85 (SEQ ID NO: 310)); and CDR3: amino acids 118-133 of SEQ ID NO: 86, which also corresponds to SEQ ID NO: 314 (encoded by nucleotides 352-399 of SEQ ID NO: 85 (SEQ ID NO: 311)).

[0211] As another specific example, the antibodies and antigen-binding fragments that are provided may comprise one or more of the following CDR sequences from the 1393.11 light chain:

CDR1: amino acids 46-55 of SEQ ID NO: 92, which also corresponds to SEQ ID NO: 330 (encoded by nucleotides 136-165 of SEQ ID NO: 91 (SEQ ID NO: 327)); CDR2: amino acids 71-77 of SEQ ID NO: 92, which also corresponds to SEQ ID NO: 331 (encoded by nucleotides 211-231 of SEQ ID NO: 91 (SEQ ID NO: 328)); and CDR3: amino acids 100-118 of SEQ ID NO: 92, which also corresponds to SEQ ID NO: 332 (encoded by nucleotides 298-354 of SEQ ID NO: 91 (SEQ ID NO: 329)).

[0212] Additional antibodies and antigen-binding fragments of the invention may comprise one or more of the following CDR sequences from the 1293.11 heavy chain:

CDR1: amino acids 61-70 of SEQ ID NO: 90, which also corresponds to SEQ ID NO: 324 (encoded by nucleotides 181-210 of SEQ ID NO: 89 (SEQ ID NO: 321)); CDR2: amino acids 85-101 of SEQ ID NO: 90, which also corresponds to SEQ ID NO: 325 (encoded by nucleotides 253-303 of SEQ ID NO: 89 (SEQ ID NO: 322)); and CDR3: amino acids 134-142 of SEQ ID NO: 90, which also corresponds to SEQ ID NO: 326 (encoded by nucleotides 400-426 of SEQ ID NO: 89 (SEQ ID NO: 323)).

[0213] As another specific example, the antibodies and antigen-binding fragments that are provided may comprise one or more of the following CDR sequences from the 1433.8 light chain:

CDR1: amino acids 21-36 of SEQ ID NO: 96, which also corresponds to SEQ ID NO: 342 (encoded by nucleotides 61-108 of SEQ ID NO: 95 (SEQ ID NO: 339)); CDR2: amino acids 52-58 of SEQ ID NO: 96, which also corresponds to SEQ ID NO: 343 (encoded by nucleotides 154-174 of SEQ ID NO: 95 (SEQ ID NO: 340)); and CDR3: amino acids 91-99 of SEQ ID NO: 96, which also corresponds to SEQ ID NO: 344 (encoded by nucleotides 371-397 of SEQ ID NO: 95 (SEQ ID NO: 341)).

[0214] Additional antibodies and antigen-binding fragments of the invention may comprise one or more of the following CDR sequences from the 1433.8 heavy chain:

CDR1: amino acids 45-54 of SEQ ID NO: 94, which also corresponds to SEQ ID NO: 336 (encoded by nucleotides 133-162 of SEQ ID NO: 93 (SEQ ID NO: 333)); CDR2: amino acids 69-85 of SEQ ID NO: 94, which also corresponds to SEQ ID NO: 337 (encoded by nucleotides 205-255 of SEQ ID NO: 93 (SEQ ID NO: 334)); and CDR3: amino acids 118-133 of SEQ ID NO: 94, which also corresponds to SEQ ID NO: 338 (encoded by nucleotides 352-399 of SEQ ID NO: 93 (SEQ ID NO: 335)).

[0215] As another specific example, the antibodies and antigen-binding fragments that are provided may comprise one or more of the following CDR sequences from the 1470.2 light chain:

CDR1: amino acids 44-54 of SEQ ID NO: 100, which also corresponds to SEQ ID NO: 354 (encoded by nucleotides 130-162 of SEQ ID NO: 99 (SEQ ID NO: 351)); CDR2: amino acids 70-76 of SEQ ID NO: 100, which also corresponds to SEQ ID NO: 355 (encoded by nucleotides 208-228 of SEQ ID NO: 99 (SEQ ID NO: 352)); and CDR3: amino acids 109-117 of SEQ ID NO: 100, which also corresponds to SEQ ID NO: 356 (encoded by nucleotides 325-351 of SEQ ID NO: 99 (SEQ ID NO: 353)).

[0216] Additional antibodies and antigen-binding fragments of the invention may comprise one or more of the following CDR sequences from the 1470.2 heavy chain:

CDR1: amino acids 45-54 of SEQ ID NO: 98, which also corresponds to SEQ ID NO: 348 (encoded by nucleotides 133-162 of SEQ ID NO: 97 (SEQ ID NO: 345)); CDR2: amino acids 69-85 of SEQ ID NO: 98, which also corresponds to SEQ ID NO: 349 (encoded by nucleotides 205-255 of SEQ ID NO: 97 (SEQ ID NO: 346)); and CDR3: amino acids 118-127 of SEQ ID NO: 98, which also corresponds to SEQ ID NO: 350 (encoded by nucleotides 352-381 of SEQ ID NO: 97 (SEQ ID NO: 347)).

[0217] As another specific example, the antibodies and antigen-binding fragments that are provided may comprise one or more of the following CDR sequences from the 1903.1 light chain:

CDR1: amino acids 48-58 of SEQ ID NO: 104, which also corresponds to SEQ ID NO: 366 (encoded by nucleotides 142-174 of SEQ ID NO: 103 (SEQ ID NO: 363)); CDR2: amino acids 74-80 of SEQ ID NO: 104, which also corresponds to SEQ ID NO: 367 (encoded by nucleotides 222-240 of SEQ ID NO: 103 (SEQ ID NO: 364)); and CDR3: amino acids 113-121 of SEQ ID NO: 104, which also corresponds to SEQ ID NO: 368 (encoded by nucleotides 337-363 of SEQ ID NO: 103 (SEQ ID NO: 365)).

[0218] Additional antibodies and antigen-binding fragments of the invention may comprise one or more of the following CDR sequences from the 1903.1 heavy chain:

CDR1: amino acids 45-54 of SEQ ID NO: 102, which also corresponds to SEQ ID NO: 360 (encoded by nucleotides 133-162 of SEQ ID NO: 101 (SEQ ID NO: 357)); CDR2: amino acids 69-85 of SEQ ID NO: 102, which also corresponds to SEQ ID NO: 361 (encoded by nucleotides 205-255 of SEQ ID NO: 101 (SEQ ID NO: 358)); and CDR3: amino acids 118-127 of SEQ ID NO: 102, which also corresponds to SEQ ID NO: 362 (encoded by nucleotides 352-381 of SEQ ID NO: 101 (SEQ ID NO: 359)).

[0219] Certain antibodies that are disclosed herein comprise one or more amino acid sequences that comprise one or more CDRs that begin at least one amino acid before (N-terminal to) the beginning amino acid of the CDRs as summarized in Table 2. Yet other antibodies that are disclosed herein comprise one or more amino acid sequences that comprise one or more CDRs that begin at least two, at least three, or at least four amino acids before (N-terminal to) the beginning amino acid of the CDRs as summarized in Table 2. Certain other antibodies that are disclosed herein comprise one or more amino acid sequences that comprise one or more CDRs that end at least one amino acid after (C-terminal to) the last amino acid of the CDRs as summarized in Table 2. Yet other antibodies that are disclosed herein comprise one or more amino acid sequences that comprise one or more CDRs that end at least two, at least three, or at least four amino acids after (C-terminal to) the last amino acid of the CDRs as summarized in Table 2. Other antibodies disclosed herein comprise one or more amino acid sequences that comprise a combination of one or more CDRs with one, two, three or four amino acid differences at the start and/or stop of the CDRs as summarized in Table 2.

[0220] Polypeptides comprising one or more of the light or heavy chain CDRs may be produced by using a suitable vector to express the polypeptides in a suitable host cell as described in greater detail below.

[0221] The heavy and light chain variable regions and the CDRs that are disclosed in Tables 1 and 2 can be used to prepare any of the various types of antigen-binding fragments that are known in the art including, but not limited to, domain antibodies, Fab fragments, Fab' fragments, F(ab').sub.2 fragments, Fv fragments, single-chain antibodies, and scFvs.

D. Antibodies and Binding Epitopes

[0222] When an antibody is said to bind an epitope within specified residues, such as LRP6, for example, what is meant is that the antibody binds with high affinity to a polypeptide consisting of the specified residues (e.g., a specified segment of LRP6). Such an antibody does not necessarily contact every residue within LRP6. Nor does every single amino acid substitution or deletion within LRP6 necessarily significantly affect binding affinity. Epitope specificity of an antibody can be determined in a variety of ways. One approach, for example, involves testing a collection of overlapping peptides of about 15 amino acids spanning the sequence of LRP6 and differing in increments of a small number of amino acids (e.g., 3 to 30 amino acids). The peptides are immobilized in separate wells of a microtiter dish. Immobilization can be effected by biotinylating one terminus of the peptides. Optionally, different samples of the same peptide can be biotinylated at the N or C terminus and immobilized in separate wells for purposes of comparison. This is useful for identifying end-specific antibodies. Optionally, additional peptides can be included terminating at a particular amino acid of interest. This approach is useful for identifying end-specific antibodies to internal fragments of LRP6. An antibody or antigen-binding fragment is screened for binding to each of the various peptides. The epitope is defined as occurring with a segment of amino acids that is common to all peptides to which the antibody shows high affinity binding. Details regarding a specific approach for defining an epitope are set forth in Example 3.

[0223] Antibodies and antigen-binding fragments thereof that bind to an epitope that is located in the carboxy-terminal portion of the first propeller domain of LRP6 (e.g., SEQ ID NO: 13, 16 or 371) or the second propeller domain (e.g., SEQ ID NO: 27; see FIG. 3) are also provided. Exemplary antibodies capable of binding to the aforementioned epitope are the monoclonal antibodies 77.2, 135.16, 213.7, 240.8, 413.1, 421.1, 498.3, 537.2, 606.4, 620.1, 856.6, 923.3, 931.1, 993.9, 995.5, 1115.3, 1213.2, 1253.12, 1281.1, 1293.11, 1433.8, 1470.2, or 1903.1, each of which comprise a light chain and a heavy chain.

[0224] In one aspect of the invention, peptides comprising or consisting of amino acids 43-324 of SEQ ID NO: 2 (e.g., SEQ ID NO: 13 or 16) are provided. Other peptides comprise or consist of amino acids 43-627 of SEQ ID NO: 2 (e.g., SEQ ID NO: 15), or amino acids 263-283 of SEQ ID NO: 2 (e.g. SEQ ID NO: 317), or amino acids 352-627 of SEQ ID NO: 2 (e.g., SEQ ID NO: 370) are provided. Such peptides are shorter than the full-length protein sequence of a native LRP6 (e.g., the peptides may include one or more of the forgoing regions and be 8, 9, 10, 11, 12, 13, 14, 15, 20, 21, 22, 23, 24, 25, 30, 40, 50, 75, 100, 150, or 200 amino acids in length). These peptides may be fused to another peptide to increase immunogenicity and thus be in the form of a fusion protein.

E. Monoclonal Antibodies

[0225] The antibodies that are provided include monoclonal antibodies that bind to LRP6. Monoclonal antibodies may be produced using any technique known in the art, e.g., by immortalizing spleen cells harvested from a transgenic or non-transgenic animal after completion of the immunization schedule. The spleen cells can be immortalized using any technique known in the art, e.g., by fusing them with myeloma cells to produce hybridomas. Myeloma cells for use in hybridoma-producing fusion procedures preferably are non-antibody-producing, have high fusion efficiency, and enzyme deficiencies that render them incapable of growing in certain selective media which support the growth of only the desired fused cells (hybridomas). Examples of suitable cell lines for use in mouse fusions include Sp-20, P3-X63/Ag8, P3-X63-Ag8.653, NS1/1.Ag41, Sp210-Ag14, FO, NSO/U, MPC-11, MPC11-X45-GTG 1.7 and 5194/5XXO Bul; examples of cell lines used in rat fusions include R210.RCY3, Y3-Ag1.2.3, IR983F and 4B210. Other cell lines useful for cell fusions are U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6.

[0226] In some instances, a hybridoma cell line is produced by immunizing an animal (e.g., a transgenic animal having human immunoglobulin sequences) with an LRP6 immunogen; harvesting spleen cells from the immunized animal; fusing the harvested spleen cells to a myeloma cell line, thereby generating hybridoma cells; establishing hybridoma cell lines from the hybridoma cells, and identifying a hybridoma cell line that produces an antibody that binds a LRP6 polypeptide. Such hybridoma cell lines, and anti-LRP6 monoclonal antibodies produced by them, are encompassed by the present invention.

[0227] Monoclonal antibodies secreted by a hybridoma cell line can be purified using any technique known in the art. Hybridomas or mAbs may be further screened to identify mAbs with particular properties, such as blocking LRP6 activity, enhancing LRP6 activity, enhancing Wnt activity or antagonizing Dkk1 activity.

F. Chimeric and Humanized Antibodies

[0228] Chimeric and humanized antibodies based upon the foregoing sequences are also provided. Monoclonal antibodies for use as therapeutic agents may be modified in various ways prior to use. One example is a "chimeric" antibody, which is an antibody composed of protein segments from different antibodies that are covalently joined to produce functional immunoglobulin light or heavy chains or antigen-binding fragments thereof. Generally, a portion of the heavy chain and/or light chain is identical with, or homologous to, a corresponding sequence in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is/are identical or homologous to a corresponding sequence in antibodies derived from another species or belonging to another antibody class or subclass. For methods relating to chimeric antibodies, see, for example, U.S. Pat. No. 4,816,567; and Morrison et al. Proc. Natl. Acad. Sci. USA 81:6851-6855 (1985), which are hereby incorporated by reference. CDR grafting is described, for example, in U.S. Pat. Nos. 6,180,370, 5,693,762, 5,693,761, 5,585,089, and 5,530,101, which are all hereby incorporated by reference for all purposes.

[0229] Generally, the goal of making a chimeric antibody is to create a chimera in which the number of amino acids from the intended patent species is maximized. One example is the "CDR-grafted" antibody, in which the antibody comprises one or more complementarity determining regions (CDRs) from a particular species or belonging to a particular antibody class or subclass, while the remainder of the antibody chain(s) is/are identical with or homologous to a corresponding sequence in antibodies derived from another species or belonging to another antibody class or subclass. For use in humans, the V region or selected CDRs from a rodent antibody often are grafted into a human antibody, replacing the naturally-occurring V regions or CDRs of the human antibody.

[0230] One useful type of chimeric antibody is a "humanized" antibody. Generally, a humanized antibody is produced from a monoclonal antibody raised initially in a non-human animal. Certain amino acid residues in this monoclonal antibody, typically from non-antigen recognizing portions of the antibody, are modified to be homologous to corresponding residues in a human antibody or corresponding isotype. Preferably, anti-LRP6 humanized antibodies contain minimal sequence derived from non-human immunoglobulin sequences. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity. See, for example, U.S. Pat. Nos. 5,225,539; 5,585,089; 5,693,761; 5,693,762; 5,859,205. In some instances, framework residues of the human immunoglobulin are replaced by corresponding non-human residues (see, for example, U.S. Pat. Nos. 5,585,089; 5,693,761; 5,693,762). Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance (e.g., to obtain desired affinity). In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details see Jones et al., Nature 331:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992); Verhoeyen et al., Science 239:1534-36 (1988)).

[0231] In one aspect of the invention, the CDRs of the light and heavy chain variable regions of the antibodies provided herein (see Table 2) are grafted to framework regions (FRs) from antibodies from the same, or a different, phylogenetic species. For example, the CDRs of the light and heavy chain variable regions of the 77.2, 135.16, 213.7, 240.8, 413.1, 421.1, 498.3, 537.2, 606.4, 620.1, 856.6, 923.3, 931.1, 993.9, 995.5, 1115.3, 1213.2, 1253.12, 1281.1, 1293.11, 1433.8, 1470.2, or 1903.1 antibody can be grafted to consensus human FRs. To create consensus human FRs, FRs from several human heavy chain or light chain amino acid sequences may be aligned to identify a consensus amino acid sequence. In other embodiments, the FRs of the 77.2, 135.16, 213.7, 240.8, 413.1, 421.1, 498.3, 537.2, 606.4, 620.1, 856.6, 923.3, 931.1, 993.9, 995.5, 1115.3, 1213.2, 1253.12, 1281.1, 1293.11, 1433.8, 1470.2, or 1903.1 antibody heavy or light chain are replaced with the FRs from a different heavy chain or light chain. In one aspect of the invention, rare amino acids in the FRs of the heavy and light chains of anti-LRP6 antibody are not replaced, while the rest of the FR amino acids are replaced. A "rare amino acid" is a specific amino acid that is in a position in which this particular amino acid is not usually found in an FR. Alternatively, the grafted variable regions from the 77.2, 135.16, 213.7, 240.8, 413.1, 421.1, 498.3, 537.2, 606.4, 620.1, 856.6, 923.3, 931.1, 993.9, 995.5, 1115.3, 1213.2, 1253.12, 1281.1, 1293.11, 1433.8, 1470.2, or 1903.1 antibody may be used with a constant region that is different from the constant region of 77.2, 135.16, 213.7, 240.8, 413.1, 421.1, 498.3, 537.2, 606.4, 620.1, 856.6, 923.3, 931.1, 993.9, 995.5, 1115.3, 1213.2, 1253.12, 1281.1, 1293.11, 1433.8, 1470.2, or 1903.1. In another aspect of this embodiment, the CDRs of the light and heavy chain variable regions of the 77.2, 135.16, 213.7, 240.8, 413.1, 421.1, 498.3, 537.2, 606.4, 620.1, 856.6, 923.3, 931.1, 993.9, 995.5, 1115.3, 1213.2, 1253.12, 1281.1, 1293.11, 1433.8, 1470.2, or 1903.1 antibody can be used. In other embodiments of the invention, the grafted variable regions are part of a single chain Fv antibody.

[0232] In certain embodiments, constant regions from species other than human can be used along with the human variable region(s) to produce hybrid antibodies.

[0233] Also encompassed are xenogeneic or modified anti-LRP6 antibodies produced in a non-human mammalian host, more particularly a transgenic mouse, characterized by inactivated endogenous immunoglobulin (Ig) loci. In such transgenic animals, competent endogenous genes for the expression of light and heavy subunits of host immunoglobulins are rendered non-functional and substituted with the analogous human immunoglobulin loci. These transgenic animals produce human antibodies in the substantial absence of light or heavy host immunoglobulin subunits. See, for example, U.S. Pat. No. 5,939,598.

[0234] Antibody fragments that retain the ability to recognize the antigen of interest, will also find use herein. A number of antibody fragments are known in the art which comprise antigen-binding sites capable of exhibiting immunological binding properties of an intact antibody molecule. For example, functional antibody fragments can be produced by cleaving a constant region, not responsible for antigen binding, from the antibody molecule, using e.g., pepsin, to produce F(ab').sub.2 fragments. These fragments can contain two antigen binding sites, but lack a portion of the constant region from each of the heavy chains. Similarly, if desired, Fab fragments, comprising a single antigen binding site, can be produced, e.g., by digestion of polyclonal or monoclonal antibodies with papain. Functional fragments, including only the variable regions of the heavy and light chains, can also be produced, using standard techniques such as recombinant production or preferential proteolytic cleavage of immunoglobulin molecules. These fragments are known as Fv. See, e.g., Inbar et al., Proc. Nat. Acad. Sci. USA 69:2659-2662 (1972); Hochman et al., Biochem. 15:2706-2710 (1976); and Ehrlich et al., Biochem. 19:4091-4096 (1980).

[0235] A phage-display system can be used to expand antibody molecule populations in vitro. Saiki, et al., Nature 324:163 (1986); Scharf et al., Science 233:1076 (1986); U.S. Pat. Nos. 4,683,195 and 4,683,202; Yang et al., J Mol Biol. 254:392 (1995); Barbas, III et al., Methods: Comp. Meth Enzymol. 8:94 (1995); Barbas, III et al., Proc Natl Acad Sci USA 88:7978 (1991).

[0236] Once generated, the phage display library can be used to improve the immunological binding affinity of the Fab molecules using known techniques. See, e.g., Figini et al., J. Mol. Biol. 239:68 (1994). The coding sequences for the heavy and light chain portions of the Fab molecules selected from the phage display library can be isolated or synthesized, and cloned into any suitable vector or replicon for expression. Any suitable expression system can be used, including those described above.

[0237] Single chain antibodies are also within the scope of the present invention. A single-chain Fv ("sFv" or "scFv") polypeptide is a covalently linked V.sub.H-V.sub.L heterodimer which is expressed from a gene fusion including V.sub.H- and V.sub.L-encoding genes linked by a peptide-encoding linker. Huston et al., Proc. Nat. Acad. Sci. USA 85:5879-5883 (1988). A number of methods have been described to discern and develop chemical structures (linkers) for converting the naturally aggregated, but chemically separated, light and heavy polypeptide chains from an antibody V region into an scFv molecule which will fold into a three-dimensional structure substantially similar to the structure of an antigen-binding site. See, e.g., U.S. Pat. Nos. 5,091,513, 5,132,405 and 4,946,778. The scFv molecules may be produced using methods described in the art. See, e.g., Huston et al., Proc. Nat. Acad. Sci. USA 85:5879-5883 (1988); U.S. Pat. Nos. 5,091,513, 5,132,405 and 4,946,778. Design criteria include determining the appropriate length to span the distance between the C-terminus of one chain and the N-terminus of the other, wherein the linker is generally formed from small hydrophilic amino acid residues that do not tend to coil or form secondary structures. Such methods have been described in the art and are well known. See, e.g., U.S. Pat. Nos. 5,091,513, 5,132,405 and 4,946,778. Suitable linkers generally comprise polypeptide chains of alternating sets of glycine and serine residues, and may include glutamic acid and lysine residues inserted to enhance solubility.

[0238] "Mini-antibodies" or "minibodies" are also within the scope of the present invention. Minibodies are scFv polypeptide chains that include oligomerization domains at their C-termini, separated from the sFv by a hinge region. Pack et al., Biochem. 31:1579-1584 (1992). The oligomerization domain comprises self-associating .alpha.-helices, e.g., leucine zippers, that can be further stabilized by additional disulfide bonds. The oligomerization domain is designed to be compatible with vectorial folding across a membrane, a process thought to facilitate in vivo folding of the polypeptide into a functional binding protein. Generally, minibodies are produced using recombinant methods well known in the art. See, e.g., Pack et al., Biochem. 31:1579-1584 (1992); Cumber et al., J. Immunology 149B:120-126 (1992).

G. Fully Human Antibodies

[0239] Fully human antibodies are also provided. Methods are available for making fully human antibodies specific for a given antigen without exposing human beings to the antigen ("fully human antibodies"). One means for implementing the production of fully human antibodies is the "humanization" of the mouse humoral immune system. Introduction of human immunoglobulin (Ig) loci into mice in which the endogenous Ig genes have been inactivated is one means of producing fully human monoclonal antibodies (mAbs) in mouse, an animal that can be immunized with any desirable antigen. Using fully human antibodies can minimize the immunogenic and allergic responses that can sometimes be caused by administering mouse or mouse-derivatized mAbs to humans as therapeutic agents.

[0240] In one embodiment, human antibodies may be produced in a non-human transgenic animal, e.g., a transgenic mouse capable of producing multiple isotypes of human antibodies to LRP6 (e.g., IgG, IgA, and/or IgE) by undergoing V-D-J recombination and isotype switching. Accordingly, aspects of the invention include not only antibodies, antibody fragments, and pharmaceutical compositions thereof, but also non-human transgenic animals, B-cells, host cells, and hybridomas which produce anti-LRP6 monoclonal antibodies. Methods of using the anti-LRP6 antibodies or antigen-binding fragments to detect a cell expressing LRP6, either in vivo or in vitro, are also encompassed by the invention. Further, the present invention encompasses pharmaceutical preparations containing the anti-LRP6 antibodies, and methods of treating physiological disorders, e.g., bone diseases and other disorders modulated by Wnt signaling, including but not limited to, gastrointestinal diseases such as inflammatory bowel disease, ulcerative colitis and radiation- or chemotherapy-induced mucositis, and wound healing, by administering the anti-LRP6 antibodies or antigen-binding fragments provided herein.

[0241] Fully human antibodies can be produced by immunizing transgenic animals (usually mice) that are capable of producing a repertoire of human antibodies in the absence of endogenous immunoglobulin production. Antigens for this purpose typically have six or more contiguous amino acids, and optionally are conjugated to a carrier, such as a hapten. See, for example, Jakobovits et al., Proc. Natl. Acad. Sci. USA 90:2551-2555 (1993); Jakobovits et al., Nature 362:255-258 (1993); Bruggermann et al., Year in Immunol. 7:33 (1993). In one example of such a method, transgenic animals are produced by incapacitating the endogenous mouse immunoglobulin loci encoding the mouse heavy and light immunoglobulin chains therein, and inserting into the mouse genome large fragments of human genome DNA containing loci that encode human heavy and light chain proteins. Partially modified animals, which have less than the full complement of human immunoglobulin loci, are then cross-bred to obtain an animal having all of the desired immune system modifications. When administered an immunogen, these transgenic animals produce antibodies that are immunospecific for the immunogen but have human rather than murine amino acid sequences, including the variable regions. For further details of such methods, see, for example, International Patent Application Publication Nos. WO 96/33735 and WO 94/02602, which are hereby incorporated by reference in their entirety. Additional methods relating to transgenic mice for making human antibodies are described in U.S. Pat. Nos. 5,545,807; 6,713,610; 6,673,986; 6,162,963; 6,300,129; 6,255,458; 5,877,397; 5,874,299 and 5,545,806; in International Patent Application Publication Nos. WO 91/10741 and WO 90/04036; and in European Patent Nos. EP 546073B1 and EP 546073A1, all of which are hereby incorporated by reference in their entirety for all purposes.

[0242] The transgenic mice described above, referred to herein as "HuMAb" mice, contain a human immunoglobulin gene minilocus that encodes unrearranged human heavy (.mu., and .gamma.) and .kappa. light chain immunoglobulin sequences, together with targeted mutations that inactivate the endogenous .mu. and .kappa. chain loci (Lonberg et al., Nature 368:856-859 (1994)). Accordingly, the mice exhibit reduced expression of mouse IgM or .kappa. chains and in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to generate high affinity human IgG .kappa. monoclonal antibodies (Lonberg et al., supra; Lonberg and Huszar, Intern. Ref Immunol. 13:65-93 (1995); Harding and Lonberg, Ann. N.Y. Acad. Sci. 764:536-546 (1995)). The preparation of HuMAb mice is described in detail in Taylor et al., Nucl. Acids Res. 20:6287-6295 (1992); Chen et al., Int. Immunol. 5:647-656 (1993); Tuaillon et al., J. Immunol. 152:2912-2920 (1994); Lonberg et al., supra; Lonberg, Handbook of Exp. Pharmacol. 113:49-101 (1994); Taylor et al., Int. Immunol. 6:579-591 (1994); Lonberg and Huszar, Intern. Ref Immunol. 13:65-93 (1995); Harding and Lonberg, Ann. N.Y. Acad. Sci. 764:536-546 (1995); Fishwild et al., Nat. Biotechnol. 14:845-851 (1996); the foregoing references are herein incorporated by reference in their entirety for all purposes. See further, U.S. Pat. Nos. 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,877,397; 5,661,016; 5,814,318; 5,874,299; 5,770,429; and 5,545,807; as well as International Patent Application Publication Nos. WO 93/1227; WO 92/22646; and WO 92/03918, the disclosures of all of which are hereby incorporated by reference in their entirety for all purposes. Technologies utilized for producing human antibodies in these transgenic mice are disclosed also in WO 98/24893, and Mendez et al., Nat. Genetics 15:146-156 (1997), which are herein incorporated by reference. For example, the HCo7 and HCo12 transgenic mice strains can be used to generate human anti-LRP6 antibodies.

[0243] Using hybridoma technology, antigen-specific human mAbs with the desired specificity can be produced and selected from the transgenic mice such as those described above. Such antibodies may be cloned and expressed using a suitable vector and host cell, or the antibodies can be harvested from cultured hybridoma cells.

[0244] Fully human antibodies can also be derived from phage-display libraries (as disclosed in Hoogenboom et al., J. Mol. Biol. 227:381 (1991); and Marks et al., J. Mol. Biol. 222:581 (1991)). Phage-display techniques 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 International Patent Application Publication No. WO 99/10494 (herein incorporated by reference), which describes the isolation of high affinity and functional agonistic antibodies for MPL.sup.- and msk.sup.- receptors using such an approach.

H. Bispecific or Bifunctional Antibodies

[0245] The antibodies that are provided also include bispecific and bifunctional antibodies that include one or more CDRs or one or more variable regions as described above. A bispecific or bifunctional antibody in some instances is an artificial hybrid antibody having two different heavy/light chain pairs and two different binding sites. Bispecific antibodies may be produced by a variety of methods including, but not limited to, fusion of hybridomas or linking of Fab' fragments. See, e.g., Songsivilai and Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelny et al., J. Immunol. 148:1547-1553 (1992).

I. Various Other Forms

[0246] Some of the antibodies or antigen-binding fragments that are provided are variant forms of the antibodies and fragments disclosed above (e.g., those having the sequences listed in Tables 1 and 2). For instance, some of the antibodies or antigen-binding fragments are ones having one or more conservative amino acid substitutions in one or more of the heavy or light chains, variable regions or CDRs listed in Tables 1 and 2.

[0247] Naturally-occurring amino acids may be divided into classes based on common side chain properties: [0248] 1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; [0249] 2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; [0250] 3) acidic: Asp, Glu; [0251] 4) basic: His, Lys, Arg; [0252] 5) residues that influence chain orientation: Gly, Pro; and [0253] 6) aromatic: Trp, Tyr, Phe.

[0254] Conservative amino acid substitutions may involve exchange of a member of one of these classes with another member of the same class. Conservative amino acid substitutions may encompass non-naturally occurring amino acid residues, which are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include peptidomimetics and other reversed or inverted forms of amino acid moieties.

[0255] Non-conservative substitutions may involve the exchange of a member of one of the classes for a member from another class. Such substituted residues may be introduced into regions of the antibody that are homologous with human antibodies, or into the non-homologous regions of the molecule.

[0256] In making such changes, according to certain embodiments, the hydropathic profile of a protein is calculated by assigning each amino acid a numerical value ("hydropathy index") and then repetitively averaging these values along the peptide chain. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics. They are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cysteine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0257] The importance of the hydropathic profile in conferring interactive biological function on a protein is understood in the art (see, for example, Kyte et al., J. Mol. Biol. 157:105-131 (1982)). It is known that certain amino acids may be substituted for other amino acid shaving a similar hydropathic index or score and still retain a similar biological activity. In making changes based upon the hydropathic index, in certain embodiments, the substitution of amino acids whose hydropathic indices are within .+-.2 is included. In some aspects of the invention, those which are within .+-.1 are included, and in other aspects of the invention, those within .+-.0.5 are included).

[0258] It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity, particularly where the biologically functional protein or peptide thereby created is intended for use in immunological embodiments, as in the present case. In certain embodiments, the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigen-binding, that is, with a biological property of the protein.

[0259] The following hydrophilicity values have been assigned to these amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0.+-.1); glutamate (+3.0.+-.1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5.+-.1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); and tryptophan (-3.4). In making changes based upon similar hydrophilicity values, in certain embodiments, the substitution of amino acids whose hydrophilicity values are within .+-.2 are included, in other embodiments, whose which are within .+-.1 are included, and in still other embodiments, those within .+-.0.5 are included. In some instances, one may also identify epitopes from primary amino acid sequences on the basis of hydrophilicity. These regions are also referred to as "epitopic core regions."

[0260] Exemplary conservative amino acid substitutions are set forth in Table 3.

TABLE-US-00003 TABLE 3 Original Residues Exemplary Substitutions Ala Val, Leu, Ile Arg Lys, Gln, Asn Asn Gln Asp Glu Cys Ser, Ala Gln Asn Glu Asp Gly Pro, Ala His Asn, Gln, Lys, Arg Ile Leu, Val, Met, Ala, Phe, Norleucine Leu Norleucine, Ile, Val, Met, Ala, Phe Lys Arg, Gln, Asn, 1,4 diamine-butryic acid Met Leu, Phe, Ile Phe Leu, Val, Ile, Ala, Tyr Pro Ala Ser Thr, Ala, Cys Thr Ser Trp Tyr, Phe Tyr Trp, Phe, Thr, Ser Val Ile, Met, Leu, Phe, Ala, Norleucine

[0261] A skilled artisan will be able to determine suitable variants of polypeptides as set forth herein using well-known techniques. One skilled in the art may identify suitable areas of the molecule that may be changed without destroying activity by targeting regions not believed to be important for activity. The skilled artisan also will be able to identify residues and portions of the molecules that are conserved among similar polypeptides. In further embodiments, even areas that may be important for biological activity or for structure may be subject to conservative amino acid substitutions without destroying the biological activity or without adversely affecting the polypeptide structure.

[0262] Additionally, one skilled in the art can review structure-function studies identifying residues in similar polypeptides that are important for activity or structure. In view of such a comparison, one can predict the importance of amino acid residues in a protein that correspond to amino acid residues important for activity or structure in similar proteins. One skilled in the art may opt for chemically similar amino acid substitutions for such predicted important amino acid residues.

[0263] One skilled in the art can also analyze the three-dimensional structure and amino acid sequence in relation to that structure in similar polypeptides. In view of such information, one skilled in the art may predict the alignment of amino acid residues of an antibody with respect to its three-dimensional structure. One skilled in the art may choose not to make radical changes to amino acid residues predicted to be on the surface of the protein, since such residues may be involved in important interactions with other molecules. Moreover, one skilled in the art may generate test variants containing a single amino acid substitution at each desired amino acid residue. These variants can then be screened using assays for LRP6 binding, LRP6 activating activity, Wnt activating activity or Dkk1 antagonistic activity (see Examples below) thus yielding information gathered from such routine experiments, one skilled in the art can readily determine the amino acid positions where further substitutions should be avoided either alone or in combination with other mutations.

[0264] A number of scientific publications have been devoted to the prediction of secondary structure. See, Moult, Curr. Op. Biotech 7:422-427 (1996); Chou et al., Biochemistry 13:222-245 (1974); Chou et al., Biochemistry 13:211-222 (1974); Chou et al., Adv. Enzymol. Relat. Areas Mol. Biol. 47:45-148 (1978); Chou et al., Ann. Rev. Biochem. 47:251-276 (1979); and Chou et al., Biophys J. 26:367-384 (1979). Moreover, computer programs are currently available to assist with predicting secondary structure. One method of predicting secondary structure is based upon homology modeling. For example, two polypeptides or proteins that have a sequence identity of greater than 30% or similarity of greater than 40% often have similar structural topologies. The growth of the protein structural database (PDB) has provided enhanced predictability of secondary structure, including the potential number of folds within a polypeptide's or protein's structure. See, Holm et al., Nucl. Acids Res. 27:244-247 (1999). It has been suggested (Brenner et al., Curr. Op. Struct. Biol. 7:369-376 (1997)) that there are a limited number of folds in a given polypeptide or protein and that once a critical number of structures have been resolved, structural prediction will become dramatically more accurate.

[0265] Additional methods of predicting secondary structure include "threading" (Jones, Curr. Opin. Struct. Biol. 7:377-87 (1997); Sippl et al., Structure 4:15-19 (1996)), "profile analysis" (Bowie et al., Science 253:164-170 (1991); Gribskov et al., Proc. Natl. Acad. Sci. USA 84:4355-4358 (1987)), and "evolutionary linkage" (See, Holm, 1999, supra; and Brenner, 1997, supra).

[0266] In some embodiments of the invention, amino acid substitutions are made that: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter ligand or antigen binding affinities, and/or (5) confer or modify other physicochemical or functional properties on such polypeptides. For example, single or multiple amino acid substitutions (in certain embodiments, conservative amino acid substitutions) may be made in the naturally-occurring sequence. Substitutions can be made in that portion of the antibody that lies outside the domain(s) forming intermolecular contacts. In such embodiments, conservative amino acid substitutions can be used that do not substantially change the structural characteristics of the parent sequence (e.g., one or more replacement amino acids that do not disrupt the secondary structure that characterizes the parent or native antibody). Examples of art-recognized polypeptide secondary and tertiary structures are described in Proteins, Structures and Molecular Principles (Creighton, Ed.), 1984, W.H. New York: Freeman and Company; Introduction to Protein Structure (Brandon and Tooze, eds.), 1991 New York: Garland Publishing; and Thornton et al., Nature 354:105 (1991), each of which is incorporated herein by reference in its entirety for all purposes.

[0267] The invention also encompasses glycosylation variants of the anti-LRP6 antibodies wherein the number and/or type of glycosylation site(s) has been altered compared to the amino acid sequences of the parent polypeptide. In certain embodiments, antibody protein variants comprise a greater or a lesser number of N-linked glycosylation sites than the native antibody. An N-linked glycosylation site is characterized by the sequence: Asn-X-Ser or Asn-X-Thr, wherein the amino acid residue designated as X may be any amino acid residue except proline. The substitution of amino acid residues to create this sequence provides a potential new site for the addition of an N-linked carbohydrate chain. Alternatively, substitutions that eliminate or alter this sequence will prevent addition of an N-linked carbohydrate chain present in the native polypeptide. For example, the glycosylation can be reduced by the deletion of an Asn or by substituting the Asn with a different amino acid. In other embodiments, one or more new N-linked glycosylation sites are created. Antibodies typically have an N-linked glycosylation site in the Fc region.

[0268] Additional preferred antibody variants include cysteine variants wherein one or more cysteine residues in the parent or native amino acid sequence are deleted from or substituted with another amino acid (e.g., serine). Cysteine variants are useful, inter alia, when antibodies must be refolded into a biologically active conformation. Cysteine variants may have fewer cysteine residues than the native antibody, and typically have an even number to minimize interactions resulting from unpaired cysteines.

[0269] The heavy and light chains, variable region domains and CDRs that are disclosed can be used to prepare polypeptides that contain an antigen-binding fragment that can specifically bind to a LRP6 molecule. For example, one or more of the CDRs listed in Table 2 can be incorporated into a molecule (e.g., a polypeptide) covalently or noncovalently to make an immunoadhesion. An immunoadhesion 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 CDR(s) enable the immunoadhesion to bind specifically to a particular antigen of interest (e.g., an LRP6 polypeptide or epitope thereof).

[0270] Mimetics (e.g., "peptide mimetics" or "peptidomimetics") based upon the variable region domains and CDRs that are described herein are also provided. These analogs can be peptides, non-peptides or combinations of peptide and non-peptide regions. Fauchere, Adv. Drug Res. 15:29 (1986); Veber and Freidiner, TINS p. 392 (1985); and Evans et al., J. Med. Chem. 30:1229 (1987), which are incorporated herein by reference in their entirety for any purpose. Peptide mimetics that are structurally similar to therapeutically useful peptides may be used to produce a similar therapeutic or prophylactic effect. Such compounds are often developed with the aid of computerized molecular modeling. Generally, peptidomimetics are proteins that are structurally similar to an antibody displaying a desired biological activity, such as the ability to bind LRP6, but have one or more peptide linkages optionally replaced by a linkage selected from: --CH.sub.2NH--, --CH.sub.2S--, --CH.sub.2--CH.sub.2--, --CH.dbd.CH-- (cis and trans), --COCH.sub.2--, --CH(OH)CH.sub.2--, and --CH.sub.2SO-- by methods well known in the art. Systematic substitution of one or more amino acids of a consensus sequence with a D-amino acid of the same type (e.g., D-lysine in place of L-lysine) may be used in certain embodiments of the invention to generate more stable proteins. In addition, constrained peptides comprising a consensus sequence or a substantially identical consensus sequence variation may be generated by methods known in the art (Rizo and Gierasch, Ann. Rev. Biochem. 61:387 (1992), incorporated herein by reference), for example, by adding internal cysteine residues capable of forming intramolecular disulfide bridges which cyclize the peptide.

[0271] Derivatives of the antibodies and antigen binding fragments that are described herein are also provided. The derivatized antibody or fragment may comprise any molecule or substance that imparts a desired property to the antibody or fragment, such as increased half-life in a particular use. The derivatized antibody can comprise, for example, a detectable (or labeling) moiety (e.g., a radioactive, colorimetric, antigenic or enzymatic molecule, a detectable bead [such as a magnetic or electrodense (e.g., gold) bead], or a molecule that binds to another molecule (e.g., biotin or streptavidin), a therapeutic or diagnostic moiety (e.g., a radioactive, cytotoxic, or pharmaceutically active moiety), or a molecule that increases the suitability of the antibody for a particular use (e.g., administration to a subject, such as a human subject, or other in vivo or in vitro uses). Examples of molecules that can be used to derivatize an antibody include albumin (e.g., human serum albumin) and polyethylene glycol (PEG). Albumin-linked and PEGylated derivatives of antibodies can be prepared using techniques well known in the art. In one embodiment, the antibody is conjugated or otherwise linked to transthyretin (TTR) or a TTR variant. The TTR or TTR variant can be chemically modified with, for example, a chemical selected from the group consisting of dextran, poly(n-vinyl pyrrolidone), polyethylene glycols, propropylene glycol homopolymers, polypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols and polyvinyl alcohols.

[0272] Other derivatives include covalent or aggregative conjugates of anti-LRP6 antibodies, or antigen-binding fragments thereof, with other proteins or polypeptides, such as by expression of recombinant fusion proteins comprising heterologous polypeptides fused to the N-terminus or C-terminus of an anti-LRP6 antibody polypeptide. For example, the conjugated peptide may be a heterologous signal (or leader) polypeptide, e.g., the yeast alpha-factor leader, or a peptide such as an epitope tag (e.g., V5-His). Anti-LRP6 antibody-containing fusion proteins can comprise peptides added to facilitate purification or identification of the anti-LRP6 antibody (e.g., poly-His). An anti-LRP6 antibody polypeptide also can be linked to the FLAG.RTM. (Sigma-Aldrich, St. Louis, Mo.) peptide as described in Hopp et al., Bio/Technology 6:1204 (1988), and U.S. Pat. No. 5,011,912. The FLAG.RTM. peptide is highly antigenic and provides an epitope reversibly bound by a specific monoclonal antibody enabling reversibly rapid assay and facile purification of expressed recombinant protein. Reagents useful for preparing fusion proteins in which the FLAG.RTM. peptide is fused to a given polypeptide are commercially available (Sigma, St. Louis, Mo., USA).

[0273] Oligomers that contain one or more anti-LRP6 antibody polypeptide may be employed as LRP6 agonists or antagonists. Oligomers may be in the form of covalently-linked or non-covalently linked dimers, trimers, or higher oligomers. Oligomers comprising two or more anti-LRP6 antibody polypeptides are contemplated for use, with one example being a homodimer. Other oligomers include heterodimers, homotrimers, heterotrimers, homotetramers, heterotetramers, etc.

[0274] One embodiment is directed to oligomers comprising multiple anti-LRP6 antibody polypeptides joined via covalent or non-covalent interactions between peptide moieties fused to the anti-LRP6 antibody polypeptides. Such peptides may be peptide linkers (spacers), or peptides that have the property of promoting oligomerization. Leucine zippers and certain polypeptides derived from antibodies are among the peptides that can promote oligomerization of anti-LRP6 antibody polypeptides attached thereto, as described in more detail below.

[0275] In particular embodiments, the oligomers comprise from two to four anti-LRP6 polypeptides. The anti-LRP6 antibody moieties of the oligomer may be in any of the forms described above, e.g., variants or fragments. Preferably, the oligomers comprise anti-LRP6 antibody polypeptides that have LRP6 binding activity.

[0276] In one embodiment, an oligomer is prepared using polypeptides derived from immunoglobulins. Preparation of fusion proteins comprising certain heterologous polypeptides fused to various portions of antibody-derived polypeptides (including the Fc domain) has been described, e.g., by Ashkenazi et al., Proc. Natl. Acad. Sci. USA 88:10535 (1991); Byrn et al., Nature 344:677 (1990); and Hollenbaugh et al., 1992 "Construction of Immunoglobulin Fusion Proteins," in Current Protocols in Immunology," Suppl 4, pages 10.19.1-10.19.11.

[0277] One embodiment of the present invention is directed to a dimer comprising two fusion proteins created by fusing a LRP6 binding fragment of an anti-LRP6 antibody to the Fc region of an antibody. The dimer can be made by, for example, inserting a gene fusion encoding the fusion protein into an appropriate expression vector, expressing the gene fusion in host cells transformed with the recombinant expression vector, and allowing the expressed fusion protein to assemble much like antibody molecules, whereupon interchain disulfide bonds form between the Fc moieties to yield the dimer.

[0278] The term "Fc polypeptide" as used herein includes native and mutein forms of polypeptides derived from the Fc region of an antibody. Truncated forms of such polypeptides containing the hinge region that promotes dimerization also are included. Fusion proteins comprising Fc moieties (and oligomers formed therefrom) offer the advantage of facile purification by affinity chromatography over Protein A or Protein G columns.

[0279] One suitable Fc polypeptide, described in International Patent Application Publication No. WO 93/10151 and U.S. Pat. Nos. 5,426,048 and 5,262,522 (each of which is herein incorporated by reference), is a single chain polypeptide extending from the N-terminal hinge region to the native C-terminus of the Fc region of a human IgG.sub.1 antibody. Another useful Fc polypeptide is the Fc mutein described in U.S. Pat. No. 5,457,035 and in Baum et al., EMBO J. 13:3992-4001 (1994). The amino acid sequence of this mutein is identical to that of the native Fc sequence presented in WO 93/10151, except that amino acid 19 has been changed from Leu to Glu, and amino acid 22 has been changed from Gly to Ala. The mutein exhibits reduced affinity for Fc receptors.

[0280] In other embodiments, the variable portion of the heavy and/or light chains of an anti-LRP6 antibody such as disclosed herein may be substituted for the variable portion of an antibody heavy and/or light chain.

[0281] Alternatively, the oligomer is a fusion protein comprising multiple anti-LRP6 antibody polypeptides, with or without peptide linkers (spacer peptides). Among the suitable peptide linkers are those described in U.S. Pat. Nos. 4,751,180 and 4,935,233.

[0282] Another method for preparing oligomeric anti-LRP6 antibody derivatives involves use of a leucine zipper. Leucine zipper domains are peptides that promote oligomerization of the proteins in which they are found. Leucine zippers were originally identified in several DNA-binding proteins (Landschulz et al., Science 240:1759 (1988)), and have since been found in a variety of different proteins. Among the known leucine zippers are naturally occurring peptides and derivatives thereof that dimerize or trimerize. Examples of leucine zipper domains suitable for producing soluble oligomeric proteins are described in International Patent Application Publication No. WO 94/10308, and the leucine zipper derived from lung surfactant protein D (SPD) described in Hoppe et al., FEBS Lett. 344:191 (1994), hereby incorporated by reference. The use of a modified leucine zipper that allows for stable trimerization of a heterologous protein fused thereto is described in Fanslow et al., Semin. Immunol. 6:267-78 (1994). In one approach, recombinant fusion proteins comprising an anti-LRP6 antibody fragment or derivative fused to a leucine zipper peptide are expressed in suitable host cells, and the soluble anti-LRP6 antibody fragments or derivatives that form are recovered from the culture supernatant.

[0283] Some antibodies that are provided have a binding affinity (k.sub.a) for LRP6 of at least 10.sup.4 or 10.sup.5 M.sup.-1 sec.sup.-1 measured, for instance, as described in the examples below. Other antibodies have a k.sub.a of at least 10.sup.6, 10.sup.7, 10.sup.8 or 10.sup.9M.sup.-2 sec.sup.-1. Certain antibodies that are provided have a low disassociation rate. Some antibodies, for instance, have a k.sub.off of 1.times.10.sup.-4 s.sup.-1, 1.times.10.sup.-5 s.sup.-1 or lower. In another embodiment, the K.sub.off is the same as an antibody having combinations of variable region domains according to the formula V.sub.LxV.sub.Hy, wherein x=the number of the light chain variable region and y=the number of the heavy chain variable region as listed in Table 1, wherein x and y are each 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23.

[0284] In another aspect, the present invention provides an anti-LRP6 antibody or antigen-binding fragment having a half-life of at least one day in vitro or in vivo (e.g., when administered to a human subject). In one embodiment, the antibody or antigen-binding fragment has a half-life of at least three days. In another embodiment, the antibody or antigen-binding fragment has a half-life of four days or longer. In another embodiment, the antibody or antigen-binding fragment has a half life of eight days or longer. In another embodiment, the antibody or antigen-binding fragment is derivatized or modified such that it has a longer half-life as compared to the underivatized or unmodified antibody. In another embodiment, the antibody contains point mutations to increase serum half life, such as described in International Patent Application Publication No. WO 00/09560, which is herein incorporated by reference.

[0285] J. Immunoconjugates

[0286] The invention also pertains to immunoconjugates, or antibody-drug conjugates (ADC), comprising an antibody or antigen-binding fragment thereof conjugated to a cytotoxic agent such as a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate). In one embodiment of the invention, an anti-LRP6 antibody may be conjugated to various therapeutic substances in order to target the LRP6 cell surface antigen. Examples of conjugated agents include, but are not limited to, metal chelate complexes, drugs, toxins and other effector molecules, such as cytokines, lymphokines, chemokines, immunomodulators, radiosensitizers, asparaginase, carboranes and radioactive halogens. Additionally, enzymes useful for activating a prodrug or increasing the target-specific toxicity of a drug can be conjugated to the antibodies. Such substances are described in further detail below.

[0287] The use of antibody-drug conjugates for the local delivery of cytotoxic or cytostatic agents, i.e. drugs to kill or inhibit tumor cells in the treatment of cancer (Syrigos and Epenetos, Anticancer Res. 19:605-614 (1999); Niculescu-Duvaz and Springer, Adv. Drg. Del. Rev. 26:151-172 (1997); U.S. Pat. No. 4,975,278) theoretically allows targeted delivery of the drug moiety to tumors, and intracellular accumulation therein, where systematic administration of these unconjugated drug agents may result in unacceptable levels of toxicity to normal cells as well as the tumor cells sought to be eliminated (Baldwin et al., Lancet 1:603-5 (1986); Thorpe, (1985) "Antibody Carriers of Cytotoxic Agents in Cancer Therapy: A Review," In: Monoclonal Antibodies '84: Biological and Clinical Applications, A. Pincera et al., (eds.) pp. 475-506). Maximal efficacy with minimal toxicity is sought thereby. Both polyclonal antibodies and monoclonal antibodies have been reported as useful in these strategies (Rowland et al., Cancer Immunol. Immunother. 21:183-87 (1986)). Drugs used in these methods include danuomycin, doxorubicin, methotrexate and vindesine (Rowland et al., (1986) supra). Toxins used in antibody-toxin conjugates include bacterial toxins such as diphtheria toxin, plant toxins such as ricin, small molecule toxins such as geldamanycin (Mandler et al., J. Nat. Cancer Inst. 92:1573-1581 (2000); Mandler et al., Bioorganic Med. Chem. Lett. 10:1025-1028 (2000); Mandler et al., Bioconjugate Chem. 13:786-791 (2002)), maytansinoids (European Patent No. EP 1391213; Liu et al., Proc. Natl. Acad. Sci. USA 93:8618-8623 (1996)), and calicheamicin (Lode et al., Cancer Res. 58:2928 (1998); Rinman et al., Cancer Res. 53:3336-3342 (1993)). The toxins may effect their cytotoxin and cytostatic effects by mechanisms including tubulin binding, DNA binding, or topoisomerase inhibition. Some cytotoxic drugs tend to be inactive or less active when conjugated to large antibodies or protein receptor ligands.

[0288] The antibodies provided herein may be used in combination with various chemotherapeutic agents, toxins and regimens. The agents and/or toxins can either be administered before, after or concurrently with the antibodies of the invention. Alternatively, if appropriate, the agents and toxins can be conjugated to the antibodies of the invention to target the agent directly to tumor cells.

[0289] A variety of radionuclides are available for the production of radioconjugated antibodies. Goodwin and Meares, Cancer Supplement 80:2675-2680 (1997) have described the use of yttrium-90-labeled monoclonal antibodies in various strategies to maximize the dose to tumor while limiting normal tissue toxicity. Other known cytotoxic radionuclides include, but are not limited to phosphorus-32, copper-67, arsenic-77, rhodium-105, palladium-109, silver-111, tin-121, iodine-125 or 131, holmium-166, lutetium-177, rhenium-186 or 188, iridium-194, gold-199, astatium-211, yttrium-90, samarium-153, or bismuth-212, all of which can be used to label antibodies directed against the LRP6 cell surface antigen for the treatment of cancer. When the conjugate is used for detection, it may comprise a radioactive atom for scintigraphic studies, for example technetium-99m or iodine-123, or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging (MRI)), such as iodine-123, iodine-131, iodine-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

[0290] The anti-LRP6 antibodies or antigen-binding fragments thereof can be conjugated to radionuclides using an indirect labeling or indirect labeling approach. The anti-LRP6 antibodies may be labeled by an "indirect labeling" or "indirect labeling approach" wherein a chelating agent is covalently attached to an antibody and at least one radionuclide is inserted into the chelating agent. See, for example, the chelating agents and radionuclides described in Srivastava and Mease, Int. J. Rad. Appl. Instrum. B. 18:589-603 (1991). Alternatively, the anti-LRP6 antibody may be labeled using "direct labeling" or a "direct labeling approach", where a label, such as a radionuclide is covalently attached directly to an antibody (typically via an amino acid residue). For example, the peptide may be biosynthesized or may be synthesized by chemical amino acid synthesis using amino acid precursors involving, for example, fluorine-19 in place of hydrogen. Labels such as technetium-99m, iodine-123, rhenium-186, rhenium-188, and indium-111 can be attached via a cysteine residue in the peptide. Yttrium-90 can be attached via a lysine residue. The iodogen method (Franker et al., Biochem. Biophys. Res. Commun. 80:49-57 (1978)) can be used to incorporate iodine-123. "Monoclonal Antibodies in Immunoscintigraphy" (Chantal, CRC Press, 1989, which is herein incorporated by reference in its entirety) describes other methods in detail.

[0291] Conjugates of an anti-LRP6 antibody and a cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bos(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238:1098 (1987). .sup.14C-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See, International Patent Application Publication No. WO 94/11026.

[0292] Further, the invention provides an embodiment wherein the anti-LRP6 antibody or antigen-binding fragment thereof is linked to an enzyme that converts a prodrug into a cytotoxic drug.

[0293] The enzymes cleave the non-toxic "prodrug" into the toxic "drug", which leads to tumor cell death. Suitable prodrug enzymes include thymidine kinase (TK), xanthine-guanine phosphoribosyltransferase (GPT) gene from E. coli or E. coli cytosine deaminase (CD), or hypoxanthine phosphoribosyl transferase (HPRT). Additional representative examples of enzymes and associated prodrug molecules include alkaline phosphatase and various toxic phosphorylated compounds such as phenolmustard phosphate, doxorubicin phosphate, mitomycin phosphate and etoposide phosphate; .beta.-galactosidase and N-[4-(.beta.-D-galactopyranosyl)benzyloxycarbonyl]-daunorubicin; azoreductase and azobenzene mustards; .beta.-glucosidase and amygdalin; .beta.-glucuronidase and phenolmustard-glucuronide and epirubicin-glucuronide; carboxypeptidase A and methotrexate-alanine; cytochrome P450 and cyclophosphamide or ifosfamide; DT diaphorase and 5-(aziridine-1-yl)-2,4,dinitrobenzamide (CB1954) (Cobb et al., Biochem. Pharmacol 18:1519 (1969), Knox et al., Cancer Metastasis Rev. 12:195 (1993)); .beta.-glutamyl transferase and .beta.-glutamyl p-phenylenediamine mustard; nitroreductase and CB1954 or derivatives of 4-nitrobenzyloxycarbonyl; glucose oxidase and glucose; xanthine oxidase and hypoxanthine; and plasmin and peptidyl-p-phenylenediamine-mustard.

[0294] Conjugates of an antibody and one or more small molecule toxins, such as calcheamicin, maytansinoids, a trichothecene, and CC1065, and the derivatives of these toxins that have toxin activity, are also contemplated herein.

[0295] The present invention further contemplates an immunoconjugate formed between an antibody and a compound with nucleolytic activity (e.g., a ribonuclease or a DNA endonuclease such as deoxyribonuclease; DNase).

[0296] Additionally, the anti-LRP6 antibodies can be attached to various labels in order to screen biological samples such as blood, tissues and/or tumors for the presence or absence of the proteins, as an indication of disease, as described further below.

K. Preparation of Antibody Drug Conjugates

[0297] In the antibody drug conjugates (ADC) of the invention, an antibody (Ab) is conjugated to one or more drug moieties (D), e.g., about 1 to about 20 drug moieties per antibody, through a linker (L). The ADC of Formula I (see below) may be prepared by several routes, employing organic chemistry reactions, conditions, and reagents known to those skilled in the art, including: (1) reaction of a nucleophilic group of an antibody with a bivalent linker reagent, to form Ab-L, via a covalent bond, followed by reaction with a drug moiety D; and (2) reaction of a nucleophilic group of a drug moiety with a bivalent linker reagent, to form D-L, via a covalent bond, followed by reaction with the nucleophilic group of an antibody.

Ab-(L-D).sub.P (I)

[0298] Nucleophilic groups on antibodies include, but are not limited to: (i) N-terminal amine groups, (ii) side chain amine groups, e.g., lysine, (iii) side chain thiol groups, e.g., cysteine, and (iv) sugar hydroxyl or amino groups where the antibody is glycosylated. Amine, thiol, and hydroxyl groups are nucleophilic and capable of reacting to form covalent bonds with electrophilic groups on linker moieties and linker reagents including: (i) active esters such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl and benzyl halides such as haloacetamides; (iii) aldehydes, ketones, carboxyl, and maleimide groups. Certain antibodies have reducible interchain disulfides, i.e., cysteine bridges. Antibodies may be made reactive for conjugation with linker reagents by treatment with a reducing agent such as DTT (dithiolthreitol). Each cysteine bridge will thus form, theoretically, two reactive thiol nucleophiles. Additional nucleophilic groups can be introduced into antibodies through the reaction of lysines with 2-iminothiolane (Traut's reagent) resulting in conversion of an amine into a thiol.

[0299] Antibody drug conjugates may also be produced by modification of the antibody to introduce electrophilic moieties, which can react with nucleophilic substituents on the linker reagent or drug. The sugars of glycosylated antibodies may be oxidized, e.g., with periodate oxidizing reagents, to form aldehyde or ketone groups which may react with the amine group of linker reagents or drug moieties. The resulting imine Schiff base groups may form a stable linkage, or may be reduced, e.g. by borohydride reagents to form stable amine linkages. In one embodiment, reaction of the carbohydrate portion of a glycosylated antibody with either galactose oxidase or sodium metaperiodate may yield carbonyl (aldehyde and ketone) groups in the protein that can react with appropriate groups on the drug (Hermanson, Bioconjugate Techniques). In another embodiment, proteins containing N-terminal serine or threonine residues can react with sodium meta-periodate, resulting in production of an aldehyde in place of the first amino acid (Geoghegan and Stroh, Bioconjugate Chem. 3:138-146 (1992); U.S. Pat. No. 5,362,852). Such aldehyde can be reacted with a drug moiety or linker nucleophile.

[0300] Likewise, nucleophilic groups on a drug moiety include, but are not limited to: amine, thiol, hydroxyl, hydrazide, oxime, hydrazine, thiosemicarbazone, hydrazine, carboxylate, and arylhydrazide groups capable of reacting to form covalent bonds with electrophilic groups on linker moieties and linker reagents including: (i) active esters such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl and benzyl halides such as haloacetamides; (iii) aldehydes, ketones, carboxyl, and maleimide groups.

[0301] Alternatively, a fusion protein comprising the antibody and cytotoxic agent may be made, e.g., by recombinant techniques or peptide synthesis. The length of DNA may comprise respective regions encoding the two portions of the conjugate either adjacent one another or separated by a region encoding a linker peptide which does not destroy the desired properties of the conjugate.

[0302] In yet another embodiment, the antibody may be conjugated to a "receptor" (such as streptavidin) for utilization in tumor or cancer cell pre-targeting" wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a "ligand" (e.g., avidin) which is conjugated to a cytotoxic agent (e.g., a radionucleotide).

IV. LRP6 Nucleic Acids

A. Nucleic Acids

[0303] Polynucleotide sequences encoding the anti-LRP6 antibodies and immunoreactive fragments thereof, described above, are readily obtained using standard techniques, well known in the art, such as those techniques described above with respect to the recombinant production of the LRP6 cell surface receptor.

[0304] Nucleic acids that encode one or both chains of an anti-LRP6 antibody, or a fragment, derivative, mutein, or variant thereof, polynucleotides sufficient for use as hybridization probes, PCR primers or sequencing primers for identifying, analyzing, mutating or amplifying a polynucleotide encoding a polypeptide, anti-sense nucleic acids for inhibiting expression of a polynucleotide, and complementary sequences of the foregoing are also provided. The nucleic acids can be any length. They can be, for example, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 175, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1500, 3000, 5000 or more nucleotides in length, and/or can comprise one or more additional sequences, for example, regulatory sequences, and/or be a part of a larger nucleic acid, for example, a vector. The nucleic acids can be single-stranded or double-stranded and can comprise RNA and/or DNA nucleotides, and artificial variants thereof (e.g., peptide nucleic acids).

[0305] Nucleic acids that encode the epitope to which certain of the antibodies provided herein are also provided. Thus, nucleic acids that encode SEQ ID NO: 16, 370 and 371 are included as are those that encode SEQ ID NO: 13 and 15. Nucleic acids encoding fusion proteins that include these peptides are also provided.

[0306] DNA encoding anti-LRP6 antibody polypeptides (e.g., heavy or light chain, variable domain only, or full-length) may be isolated from B cells of mice that have been immunized with LRP6 or an immunogenic fragment thereof. The DNA may be isolated by conventional procedures such as polymerase chain reaction (PCR). Phage display is another example of a known technique whereby derivatives of antibodies may be prepared. In one approach, polypeptides that are components of an antibody of interest are expressed in any suitable recombinant expression system, and the expressed polypeptides are allowed to assemble to form antibody molecules.

[0307] Exemplary nucleic acids that encode the light and heavy chains, variable regions and CDRs of the antibodies and antigen-binding fragments are provided in Tables 1 and 2 above. Due to the degeneracy of the genetic code, each of the polypeptide sequences listed in Tables 1 and 2 is also encoded by a large number of other nucleic acid sequences besides those listed in Tables 1 and 2. The present invention provides each degenerate nucleotide sequence encoding each anti-LRP6 antibody or antigen-binding fragment thereof.

[0308] Nucleic acid molecules encoding anti-LRP6 antibodies or antigen-binding fragments thereof are provided. In one embodiment, the nucleic acid molecule encodes a heavy and/or light chain of an anti-LRP6 immunoglobulin. In a preferred embodiment, a single nucleic acid molecule encodes a heavy chain of an anti-LRP6 immunoglobulin and another nucleic acid molecule encodes the light chain of an anti-LRP6 immunoglobulin. In a more preferred embodiment, the encoded immunoglobulin is a human immunoglobulin, preferably a human IgG. The encoded light chain may be a .lamda. chain or a .kappa. chain.

[0309] The invention provides nucleic acid molecules comprising a nucleic acid sequence that encodes the amino acid sequence of the variable region of the light chain (V.sub.L) of 77.2, 135.16, 213.7, 240.8, 413.1, 421.1, 498.3, 537.2, 606.4, 620.1, 856.6, 923.3, 931.1, 993.9, 995.5, 1115.3, 1213.2, 1253.12, 1281.1, 1293.11, 1433.8, 1470.2, or 1903.1. The invention also provides nucleic acid molecules comprising a nucleic acid sequence that encodes the amino acid sequence of one or more of the CDRs of any one of the light chains of 77.2, 135.16, 213.7, 240.8, 413.1, 421.1, 498.3, 537.2, 606.4, 620.1, 856.6, 923.3, 931.1, 993.9, 995.5, 1115.3, 1213.2, 1253.12, 1281.1, 1293.11, 1433.8, 1470.2, or 1903.1. In a preferred embodiment, the nucleic acid molecule comprises a nucleic acid sequence that encodes the amino acid sequence of all of the CDRs of any one of the light chains of 77.2, 135.16, 213.7, 240.8, 413.1, 421.1, 498.3, 537.2, 606.4, 620.1, 856.6, 923.3, 931.1, 993.9, 995.5, 1115.3, 1213.2, 1253.12, 1281.1, 1293.11, 1433.8, 1470.2, or 1903.1. In another embodiment, the nucleic acid molecule comprises a nucleic acid sequence that encodes the amino acid sequence of one of SEQ ID NO: 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 76, 80, 84, 88, 92, 96, 100, or 104, or comprises a nucleic acid sequence of one of SEQ ID NO: 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 75, 79, 83, 87, 91, 95, 99, or 103. In another preferred embodiment, the nucleic acid molecule comprises a nucleic acid sequence that encodes the amino acid sequence of one or more of the CDRs of any one of SEQ ID NO: 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 76, 80, 84, 88, 92, 96, 100, or 104, or comprises a nucleic acid sequence of one or more of the CDRs of any one of SEQ ID NO: 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 75, 79, 83, 87, 91, 95, 99, or 103. In a more preferred embodiment, the nucleic acid molecule comprises a nucleic acid sequence that encodes the amino acid sequence of all of the CDRs of any one of SEQ ID NO: 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 76, 80, 84, 88, 92, 96, 100, or 104, or comprises a nucleic acid sequence of all the CDRs of any one of SEQ ID NO: 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 75, 79, 83, 87, 91, 95, 99, or 103.

[0310] The invention also provides nucleic acid molecules that encode an amino acid sequence of a V.sub.L that has an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a V.sub.L described above, particularly to a V.sub.L that comprises an amino acid sequence of one of SEQ ID NO: 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 76, 80, 84, 88, 92, 96, 100, or 104. The invention also provides a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a nucleic acid sequence of one of SEQ ID NO: 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 75, 79, 83, 87, 91, 95, 99, or 103. In another embodiment, the invention provides a nucleic acid molecule encoding a V.sub.L that hybridizes under stringent conditions to a nucleic acid molecule encoding a V.sub.L as described above, particularly a nucleic acid molecule that comprises a nucleic acid sequence encoding an amino acid sequence of SEQ ID NO: 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 76, 80, 84, 88, 92, 96, 100, or 104. The invention also provides a nucleic acid sequence encoding a V.sub.L that hybridizes under stringent conditions to a nucleic acid molecule comprising a nucleic acid sequence of one of SEQ ID NO: 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 75, 79, 83, 87, 91, 95, 99, or 103.

[0311] The invention also provides a nucleic acid molecule encoding the variable region of the heavy chain (V.sub.H) of 77.2, 135.16, 213.7, 240.8, 413.1, 421.1, 498.3, 537.2, 606.4, 620.1, 856.6, 923.3, 931.1, 993.9, 995.5, 1115.3, 1213.2, 1253.12, 1281.1, 1293.11, 1433.8, 1470.2, or 1903.1. In one embodiment, the nucleic acid molecule comprises a nucleic acid sequence that encodes the amino acid sequence of the V.sub.H of 77.2, 135.16, 213.7, 240.8, 413.1, 421.1, 498.3, 537.2, 606.4, 620.1, 856.6, 923.3, 931.1, 993.9, 995.5, 1115.3, 1213.2, 1253.12, 1281.1, 1293.11, 1433.8, 1470.2, or 1903.1. In another embodiment, the nucleic acid molecule comprises a nucleic acid sequence that encodes the amino acid sequence of one or more of the CDRs of the heavy chain of 77.2, 135.16, 213.7, 240.8, 413.1, 421.1, 498.3, 537.2, 606.4, 620.1, 856.6, 923.3, 931.1, 993.9, 995.5, 1115.3, 1213.2, 1253.12, 1281.1, 1293.11, 1433.8, 1470.2, or 1903.1. In a preferred embodiment, the nucleic acid molecule comprises a nucleic acid sequence that encodes the amino acid sequences of all of the CDRs of the heavy chain of 77.2, 135.16, 213.7, 240.8, 413.1, 421.1, 498.3, 537.2, 606.4, 620.1, 856.6, 923.3, 931.1, 993.9, 995.5, 1115.3, 1213.2, 1253.12, 1281.1, 1293.11, 1433.8, 1470.2, or 1903.1. In another preferred embodiment, the nucleic acid molecule comprises a nucleic acid sequence that encodes the amino acid sequence of one of SEQ ID NO: 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 72, 74, 78, 82, 86, 90, 94, 98, 102, or that comprises a nucleic acid sequence of one of SEQ ID NO: 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 71, 73, 77, 81, 85, 89, 93, 97, 101. In another preferred embodiment, the nucleic acid molecule comprises a nucleic acid sequence that encodes the amino acid sequence of one or more of the CDRs of any one of SEQ ID NO: 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 72, 74, 78, 82, 86, 90, 94, 98, 102, or comprises a nucleic acid sequence of one or more of the CDRs of any one of SEQ ID NO: 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 71, 73, 77, 81, 85, 89, 93, 97, 101. In a preferred embodiment, the nucleic acid molecule comprises a nucleic acid sequence that encodes the amino acid sequences of all of the CDRs of any one of SEQ ID NO: 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 72, 74, 78, 82, 86, 90, 94, 98, 102, or comprises a nucleic acid sequence of all of the CDRs for any one of SEQ ID NO: 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 71, 73, 77, 81, 85, 89, 93, 97, 101.

[0312] In another embodiment, the nucleic acid molecule encodes an amino acid sequence of a V.sub.H that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of the amino acid sequences encoding a V.sub.H as described immediately above, particularly to a V.sub.H that comprises an amino acid sequence of one of SEQ ID NO: 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 72, 74, 78, 82, 86, 90, 94, 98, 102. In another embodiment, the nucleic acid molecule encoding a V.sub.H is one that hybridizes under stringent conditions to a nucleic acid sequence encoding a V.sub.H as described above, particularly to a V.sub.H that comprises an amino acid sequence of one of SEQ ID NO: 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 72, 74, 78, 82, 86, 90, 94, 98, 102. The invention also provides a nucleic acid sequence encoding a V.sub.H that hybridizes under stringent conditions to a nucleic acid molecule comprising a nucleic acid sequence of one of SEQ ID NO: 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 71, 73, 77, 81, 85, 89, 93, 97, 101.

[0313] The nucleic acid molecule encoding either or both of the entire heavy and light chains of an anti-LRP6 antibody or the variable regions thereof may be obtained from any source that produces an anti-LRP6 antibody. Methods of isolating mRNA encoding an antibody are well-known in the art. See e.g., Sambrook et al., supra. The mRNA may be used to produce cDNA for use in the polymerase chain reaction (PCR) or cDNA cloning of antibody genes. In one embodiment of the invention, the nucleic acid molecules may be obtained from a hybridoma that expresses an anti-LRP6 antibody as described above, preferably a hybridoma that has as one of its fusion partners a transgenic animal cell that expresses human immunoglobulin genes, such as a XENOMOUSE.RTM. (Amgen, Thousand Oaks, Calif., USA), non-human mouse transgenic animal, or a non-human, non-mouse transgenic animal. In another embodiment, the hybridoma is derived from a non-human, non-transgenic animal, which may be used, e.g., for humanized antibodies.

[0314] A nucleic acid molecule encoding the entire heavy chain of an anti-LRP6 antibody may be constructed by fusing a nucleic acid molecule encoding the variable domain of a heavy chain or an antigen-binding domain thereof with a constant domain of a heavy chain. Similarly, a nucleic acid molecule encoding the light chain of an anti-LRP6 antibody may be constructed by fusing a nucleic acid molecule encoding the variable domain of a light chain or an antigen-binding fragment thereof with a constant domain of a light chain. The nucleic acid molecules encoding the V.sub.H and V.sub.L chain may be converted to full-length antibody genes by inserting them into expression vectors already encoding heavy chain constant and light chain constant regions, respectively, such that the V.sub.H segment is operatively linked to the heavy chain constant region (C.sub.H) segment(s) within the vector and the V.sub.L segment is operatively linked to the light chain constant region (C.sub.L) segment within the vector. Alternatively, the nucleic acid molecules encoding the V.sub.H or V.sub.L chains are converted into full-length antibody genes by linking, e.g., ligating, the nucleic acid molecule encoding a V.sub.H chain to a nucleic acid molecule encoding a C.sub.H chain using standard molecule biological techniques. The same may be achieved using nucleic acid molecules encoding V.sub.L and C.sub.L chains. The sequences of human heavy and light chain constant region genes are known in the art. See, e.g., Kabat et al., 1991, supra. Nucleic acid molecules encoding the full-length heavy and/or light chains may then be expressed from a cell into which they have been introduced and the anti-LRP6 antibody isolated.

[0315] In another embodiment, a nucleic acid molecule encoding either the heavy chain of an anti-LRP6 antibody or an antigen-binding fragment thereof or the light chain of an anti-LRP6 antibody or an antigen-binding fragment thereof may be isolated from a non-human, non-mouse animal that expresses human immunoglobulin genes and has been immunized with a LRP6 antigen. In another embodiment, the nucleic acid molecule may be isolated from an anti-LRP6 antibody producing cell derived from a non-transgenic animal or from a human patient who produces anti-LRP6 antibodies. Methods of isolating mRNA from the anti-LRP6 antibody-producing cells may be isolated by standard techniques, cloned and/or amplified using PCR and library construction techniques, and screened using standard protocols to obtain nucleic acid molecules encoding anti-LRP6 heavy and light chains.

[0316] The nucleic acid molecules may be used to recombinantly express large quantities of anti-LRP6 antibodies, as described below. The nucleic acid molecules may also be used to produce chimeric antibodies, single chain antibodies, immunoadhesins, diabodies, mutated antibodies and antibody derivatives. If the nucleic acid molecules are derived from a non-human, non-transgenic animal, the nucleic acid molecules may be used for antibody humanization.

[0317] The invention further provides nucleic acids that hybridize to other nucleic acids (e.g., nucleic acids comprising a nucleotide sequence listed in Tables 1-2) under particular hybridization conditions. Methods for hybridizing nucleic acids are well-known in the art. See, e.g., Current Protocols in Molecular Biology, John Wiley and Sons, N.Y. (1989), 6.3.1-6.3.6. As defined herein, a moderately stringent hybridization condition uses a prewashing solution containing 5.times. sodium chloride/sodium citrate (SSC), 0.5% SDS, 1.0 mM EDTA (pH 8.0), hybridization buffer of about 50% formamide, 6.times.SSC, and a hybridization temperature of 55.degree. C. (or other similar hybridization solutions, such as one containing about 50% formamide, with a hybridization temperature of 42.degree. C.), and washing conditions of 60.degree. C. in 0.5.times.SSC, 0.1% SDS. A stringent hybridization condition hybridizes in 6.times.SSC at 45.degree. C., followed by one or more washes in 0.1.times.SSC, 0.2% SDS at 68.degree. C. Furthermore, one of skill in the art can manipulate the hybridization and/or washing conditions to increase or decrease the stringency of hybridization such that nucleic acids comprising nucleotide sequence that are at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to each other typically remain hybridized to each other.

[0318] The basic parameters affecting the choice of hybridization conditions and guidance for devising suitable conditions are set forth by, for example, Sambrook, Fritsch, and Maniatis (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., chapters 9 and 11 (1989); Current Protocols in Molecular Biology, Ausubel et al., eds., John Wiley and Sons, Inc., sections 2.10 and 6.3-6.4 (1995), both of which are herein incorporated by reference in their entirety for all purposes) and can be readily determined by those having ordinary skill in the art based on, for example, the length and/or base composition of the DNA.

[0319] Changes can be introduced by mutation into a nucleic acid, thereby leading to changes in the amino acid sequence of a polypeptide (e.g., an anti-LRP6 antibody or antibody derivative) that it encodes. Mutations can be introduced using any technique known in the art. In one embodiment, one or more particular amino acid residues are changed using, for example, a site-directed mutagenesis protocol. In another embodiment, one or more randomly selected residues is changed using, for example, a random mutagenesis protocol. However it is made, a mutant polypeptide can be expressed and screened for a desired property.

[0320] Mutations can be introduced into a nucleic acid without significantly altering the biological activity of a polypeptide that it encodes. For example, one can make nucleotide substitutions leading to amino acid substitutions at non-essential amino acid residues. Alternatively, one or more mutations can be introduced into a nucleic acid that selectively change the biological activity of a polypeptide that it encodes. For example, the mutation can quantitatively or qualitatively change the biological activity. Examples of quantitative changes include increasing, reducing or eliminating the activity. Examples of qualitative changes include changing the antigen specificity of an antibody.

[0321] In another aspect, the present invention provides nucleic acid molecules that are suitable for use as primers or hybridization probes for the detection of the nucleic acid sequences provided herein. A nucleic acid molecule may comprise only a portion of a nucleic acid sequence encoding a full-length polypeptide of an anti-LRP6 antibody, for example, a fragment that can be used as a probe or primer or a fragment encoding an active portion (e.g., an LRP6 binding portion) of the polypeptide.

[0322] In another embodiment, the nucleic acid molecules may be used as probes or PCR primers for specific antibody sequences. For instance, a nucleic acid molecule probe may be used in diagnostic methods or a nucleic acid molecule PCR primer may be used to amplify regions of DNA that could be used, inter alia, to isolate nucleic acid sequences for use in producing variable domains of anti-LRP6 antibodies. In a preferred embodiment, the nucleic acid molecules are oligonucleotides. In a more preferred embodiment, the oligonucleotides are from highly variable regions of the heavy and light chains of the antibody of interest. In an even more preferred embodiment, the oligonucleotides encode all or part of one or more of the CDRs.

[0323] Probes based on the sequence of a nucleic acid provided herein can be used to detect the nucleic acid or similar nucleic acids, for example, transcripts encoding a polypeptide of an anti-LRP6 antibody. The probe can comprise a label group, e.g., a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor. Such probes can be used to identify a cell that expresses the polypeptide.

B. Vectors

[0324] The invention provides vectors comprising the nucleic acid molecules that encode the heavy chain or the antigen-binding portion thereof. Also provided are vectors comprising the nucleic acid molecules that encode the light chain or antigen-binding portion thereof. In addition, vectors comprising nucleic acid molecules encoding fusion proteins, modified antibodies, antibody fragments, and probes thereof are provided herein.

[0325] Also provided are vectors comprising a nucleic acid encoding a polypeptide of an anti-LRP6 antibody or a portion thereof (e.g., a fragment containing one or more CDRs or one or more variable region domains). Examples of vectors include, but are not limited to, plasmids, viral vectors, non-episomal mammalian vectors and expression vectors, for example, recombinant expression vectors. The recombinant expression vectors may comprise a nucleic acid in a form suitable for expression of the nucleic acid in a host cell. The recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operably linked to the nucleic acid sequence to be expressed. Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cells (e.g., Simian Virus 40 (SV40) early gene enhancer, Rous sarcoma virus (RSV) promoter and cytomegalovirus (CMV) promoter), those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences, see Voss et al., Trends Biochem. Sci. 11:287 (1986); Maniatis et al., Science 236:1237 (1986), incorporated by reference herein in their entireties), and those that direct inducible expression of a nucleotide sequence in response to particular treatment or condition (e.g., the metallothionin promoter in mammalian cells and the tet-responsive and/or streptomycin responsive promoter in both prokaryotic and eukaryotic systems. It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. The expression vectors may be introduced into host cells to thereby produce proteins or peptides, including fusion protein or peptides, encoded by nucleic acids as described herein.

[0326] To express the antibodies, or antigen-binding fragments thereof, DNAs encoding partial or full-length light and heavy chains, obtained as described above, are inserted into expression vectors such that the genes area operatively linked to transcriptional and translational control sequences. Expression vectors include plasmids, retroviruses, cosmids, YACs, EBV-derived episomes, and the like. The antibody gene is ligated into a vector such that transcriptional and translational control sequences within the vector serve their intended function of regulating the transcription and translation of the antibody gene. The expression vector and expression control sequences are chosen to be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene can be inserted into separate vectors. In a preferred embodiment, both genes are inserted into the same expression vector. The antibody genes are inserted into the expression vector by standard methods (e.g., ligation of complementary restriction sites on the antibody gene fragment and vector, or blunt end ligation if no restriction sites are present).

[0327] A convenient vector is one that encodes a functionally complete human C.sub.H or C.sub.L immunoglobulin sequence, with appropriate restriction sites engineered so that any V.sub.H or V.sub.L sequence can be easily inserted and expressed, as described above. In such vectors, splicing usually occurs between the splice donor site in the inserted J region and the splice acceptor site preceding the human C region, and also at the splice regions that occur within the human C.sub.H exons. Polyadenylation and transcription termination occur at native chromosomal sites downstream of the coding regions. The recombinant expression vector can also encode a signal peptide that facilitates secretion of the antibody chain from a host cell. The antibody chain gene may be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (e.g., a signal peptide from a non-immunoglobulin protein).

[0328] In addition to the antibody chain genes, the recombinant expression vectors carry regulatory sequences that control the expression of the antibody chain genes in a host cell. It will be appreciated by those skilled in the art that the design of the expression vector, including the selection of regulatory sequences may depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. Preferred regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from retroviral LTRs, CMV (such as the CMV promoter/enhancer), SV40 (such as the SV40 promoter/enhancer), adenovirus (e.g., the adenovirus major late promoter (AdMLP)), polyoma and strong mammalian promoters such as native immunoglobulin and actin promoters. For further description of viral regulatory elements, and sequences thereof, see e.g., U.S. Pat. Nos. 5,168,062 4,510,245, and 4,968,615.

[0329] In addition to the antibody chain genes and regulatory sequences, the recombinant expression vectors may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see e.g., U.S. Pat. Nos. 4,399,216, 4,634,665, and 5,179,017). For example, typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin or methotrexate, on a host cell into which the vector has been introduced. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr.sup.- host cells with methotrexate selection/amplification) and the neomycin gene (for G418 selection).

C. Host Cells

[0330] In another aspect, the present invention provides host cells into which a recombinant expression vector has been introduced. A host cell can be any prokaryotic cell (for example, E. coli) or eukaryotic cell (for example, yeast (for example, Pichia pastoris), insect, or mammalian cells (e.g., CHO cells)). Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (e.g., for resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Preferred selectable markers include those which confer resistance to drugs, such as G418, hygromycin and methotrexate. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die), among other methods.

V. Preparation of Antibodies

[0331] As explained above, the LRP6 antigen is used to produce antibodies for therapeutic, diagnostic and purification purposes. These antibodies may be polyclonal or monoclonal antibody preparations, monospecific antisera, human antibodies, or may be hybrid or chimeric antibodies, such as humanized antibodies, altered antibodies, F(ab').sub.2 fragments, Fab fragments, Fv fragments, single-domain antibodies, dimeric or trimeric antibody fragment constructs, minibodies, or functional fragments thereof which bind to the antigen in question. Antibodies are produced using techniques well known to those of skill in the art and disclosed in, for example, U.S. Pat. Nos. 4,011,308; 4,722,890; 4,016,043; 3,876,504; 3,770,380; and 4,372,745.

[0332] For example, the LRP6 antigens can be used to produce LRP6-specific polyclonal and monoclonal antibodies for use in diagnostic and detection assays, for purification and for use as therapeutics. LRP6-specific polyclonal and monoclonal antibodies bind with high affinity to LRP6 antigens. The non-human antibodies that are provided can be, for example, derived from any antibody-producing animal, such as mouse, rat, rabbit, goat, donkey, or non-human primate (such as monkey (e.g., cynomologous or rhesus monkey) or ape (e.g., chimpanzee)). Serum from the immunized animal is collected and the antibodies are purified from the plasma by, for example, precipitation with ammonium sulfate, followed by chromatography, preferably affinity chromatography. Techniques for producing and processing polyclonal antisera are known in the art.

[0333] Non-human antibodies can be used, for instance, in in vitro cell culture and cell-culture based applications, or any other application where an immune response to the antibody does not occur or is insignificant, can be prevented, is not a concern, or is desired. In certain embodiments of the invention, the antibodies may be produced by immunizing with full-length LRP6 (i.e., SEQ ID NO: 2) or with the extracellular domain (i.e. SEQ ID NO: 3). Alternatively, the certain non-human antibodies may be raised by immunizing with amino acids 43-324 of SEQ ID NO: 2 (i.e., SEQ ID NO: 13 or 16), amino acids 263-283 of SEQ ID NO: 2 (i.e. SEQ ID NO: 271), or amino acids 43-627 of SEQ ID NO: 2 (i.e., SEQ ID NO: 15) which are segments of human LRP6 that form part of the epitope to which certain antibodies provided herein bind. In yet further embodiments, anti-LRP6 antibodies may be raised by immunizing non-human animals with amino acids 43-324 of SEQ ID NO: 2 (i.e., SEQ ID NO: 13 or 16), or amino acids 43-627 of SEQ ID NO: 2 (i.e., SEQ ID NO: 15). The antibodies may be polyclonal, monoclonal, or may be synthesized in host cells by expressing recombinant DNA.

[0334] Fully human antibodies may be prepared as described above by immunizing transgenic animals containing human immunoglobulin loci or by selecting a phage display library that is expressing a repertoire of human antibodies.

[0335] Mouse and/or rabbit monoclonal antibodies directed against epitopes present in the LRP6 antigen can also be readily produced. In order to produce such monoclonal antibodies, the mammal of interest, such as a rabbit or mouse, is immunized, such as by mixing or emulsifying the antigen in saline, preferably in an adjuvant such as Freund's complete adjuvant (FCA), and injecting the mixture or emulsion parenterally (generally subcutaneously or intramuscularly). The animal is generally boosted 2-6 weeks later with one or more injections of the antigen in saline, preferably using Freund's incomplete adjuvant (FIA).

[0336] The anti-LRP6 monoclonal antibodies (mAbs) can be produced by a variety of techniques, including conventional monoclonal antibody methodology, e.g., the standard somatic cell hybridization technique of Kohler and Milstein, Nature 256:495 (1975), herein incorporated by reference in its entirety for all purposes. Alternatively, other techniques for producing monoclonal antibodies can be employed, for example, the viral or oncogenic transformation of B-lymphocytes. One suitable animal system for preparing hybridomas is the murine system, which is a very well-established procedure. Immunization protocols and techniques for isolation of immunized splenocytes for fusion are known in the art. For such procedures, B cells from immunized mice are fused with a suitable immortalized fusion partner, such as a murine myeloma cell line. If desired, rats or other mammals can be immunized instead of mice and B cells from such animals can be fused with the murine myeloma cell line to form hybridomas. Alternatively, a myeloma cell line from a source other than mouse may be used. Fusion procedures for making hybridomas are also well-known.

[0337] Antibodies may also be generated by in vitro immunization, using methods known in the art. See, e.g., James et al., J. Immunol. Meth. 100:5-40 (1987). Polyclonal antisera are then obtained from the immunized animal. However, rather than bleeding the animal to extract serum, the spleen (and optionally several large lymph nodes) is removed and dissociated into single cells. If desired, the spleen cells (splenocytes) may be screened (after removal of nonspecifically adherent cells) by applying a cell suspension to a plate or well coated with the antigen. B-cells, expressing membrane-bound immunoglobulin specific for the antigen, will bind to the plate, and are not rinsed away with the rest of the suspension. Resulting B-cells, or all dissociated splenocytes, are then induced to fuse with cells from an immortalized cell line (also termed a "fusion partner"), to form hybridomas. Typically, the fusion partner includes a property that allows selection of the resulting hybridomas using specific media. For example, fusion partners can be hypoxanthine/aminopterin/thymidine (HAT)-sensitive.

[0338] If rabbit-rabbit hybridomas are desired, the immortalized cell line will be from a rabbit. Such rabbit-derived fusion partners are known in the art and include, for example, cells of lymphoid origin, such as cells from a rabbit plasmacytoma as described in Spieker-Polet et al., Proc. Natl. Acad. Sci. USA 92:9348-9352 (1995) and U.S. Pat. No. 5,675,063, or the TP-3 fusion partner described in U.S. Pat. No. 4,859,595, incorporated herein by reference in their entireties. If a rabbit-mouse hybridoma or a rat-mouse or mouse-mouse hybridoma, or the like, is desired, the mouse fusion partner will be derived from an immortalized cell line from a mouse, such as a cell of lymphoid origin, typically from a mouse myeloma cell line. A number of such cell lines are known in the art and are available from ATCC (American Type Culture Collection, Manassas, Va., USA).

[0339] Fusion is accomplished using techniques well known in the art. Chemicals that promote fusion are commonly referred to as fusogens. These agents are extremely hydrophilic and facilitate membrane contact. One particularly preferred method of cell fusion uses polyethylene glycol (PEG). Another method of cell fusion is electrofusion. In this method, cells are exposed to a predetermined electrical discharge that alters the cell membrane potential. Additional methods for cell fusion include bridged-fusion methods. In this method, the antigen is biotinylated and the fusion partner is avidinylated. When the cells are added together, an antigen-reactive B cell-antigen-biotin-avidin-fusion partner bridge is formed. This permits the specific fusion of an antigen-reactive cell with an immortalizing cell. The method may additionally employ chemical or electrical means to facilitate cell fusion.

[0340] Following fusion, the cells are cultured in a selective medium (e.g., HAT medium). In order to enhance antibody secretion, an agent that has secretory stimulating effects can optionally be used, such as IL-6. See, e.g., Liguori et al., Hybridoma 20:189-198 (2001). The resulting hybridomas can be plated by limiting dilution, and are assayed for the production of antibodies which bind specifically to the immunizing antigen (and which do not bind to unrelated antigens). The selected monoclonal antibody-secreting hybridomas are then cultured either in vitro (e.g., in tissue culture bottles or hollow fiber reactors), or in vivo (e.g., as ascites in mice). For example, hybridomas producing LRP6-specific antibodies can be identified using RIA or ELISA and isolated by cloning in semi-solid agar or by limiting dilution. Clones producing the desired antibodies can be isolated by another round of screening.

[0341] An alternative technique for generating the anti-LRP6 monoclonal antibodies is the selected lymphocyte antibody method (SLAM). This method involves identifying a single lymphocyte that is producing an antibody with the desired specificity or function within a large population of lymphoid cells. The genetic information that encodes the specificity of the antibody (i.e., the immunoglobulin V.sub.H and V.sub.L DNA) is then rescued and cloned. See, e.g., Babcook et al., Proc. Natl. Acad. Sci. USA 93:7843-7848 (1996), for a description of this method.

[0342] For further descriptions of rabbit monoclonal antibodies and methods of making the same from rabbit-rabbit and rabbit-mouse fusions, see, e.g., U.S. Pat. No. 5,675,063 (rabbit-rabbit); U.S. Pat. No. 4,859,595 (rabbit-rabbit); U.S. Pat. No. 5,472,868 (rabbit-mouse); and U.S. Pat. No. 4,977,081 (rabbit-mouse).

[0343] The single-chain antibodies that are provided may be formed by linking heavy and light chain variable domain (Fv region) fragments (see, e.g., Table 1) via an amino acid bridge (short peptide linker), resulting in a single polypeptide chain. Such single-chain Fvs (scFvs) may be prepared by fusing DNA encoding a peptide linker between DNAs encoding the two variable domain polypeptides (V.sub.L and V.sub.H). The resulting polypeptides can fold back on themselves to form antigen-binding monomers, or they can form multimers (e.g., dimers, trimers, or tetramers), depending on the length of a flexible linker between the two variable domains (Kortt et al., Prot. Eng. 10:423 (1997); Kort et al., Biomol. Eng. 18:95-108 (2001)). By combining different V.sub.L and V.sub.H-comprising polypeptides, one can form multimeric scFvs that bind to different epitopes (Kriangkum et al., Biomol. Eng. 18:31-40 (2001)). Techniques developed for the production of single-chain antibodies include those described in U.S. Pat. No. 4,946,778; Bird, Science 242:423 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879 (1988); Ward et al., Nature 334:544 (1989); de Graff et al., Methods Mol. Biol. 178:379-87 (2002)). Single-chain antibodies derived from antibodies provided herein include, but are not limited to scFvs comprising the variable domain combinations designated by the formula "V.sub.LxV.sub.Hy," wherein "x" is the number of the light chain variable region and "y" corresponds to the number of the heavy chain variable region as listed in Table 1. In general, x and y are each 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23.

[0344] Antibodies provided herein that are of one subclass can be changed to antibodies of a different subclass using subclass switching methods. Thus, IgG antibodies may be derived from an IgM antibody, for example, and vice versa. Such techniques allow the preparation of new antibodies that possess the antigen-binding properties of a given antibody (the parent antibody), but also exhibit biological properties associated with an antibody isotype or subclass different from that of the parent antibody. Recombinant DNA techniques may be employed. Cloned DNA encoding particular antibody polypeptides may be employed in such procedures, e.g., DNA encoding the constant domain of an antibody of the desired isotype. See, e.g., Lantto et al., Methods Mol. Biol. 178:303-16 (2002).

[0345] Accordingly, the antibodies that are provided include those comprising, for example, the following variable domain combinations designated by the formula "V.sub.LxV.sub.Hy," (see definition above) having a desired isotype (for example, IgA, IgG.sub.1, IgG.sub.2, IgG.sub.3, IgG.sub.4, IgE, and IgD) as well as Fab or F(ab').sub.2 fragments thereof. Moreover, if an IgG.sub.4 is desired, it may also be desired to introduce a point mutation (e.g. CPSCP.fwdarw.CPPCP) in the hinge region as described in Bloom et al., Protein Sci. 6:407 (1997), incorporated by reference herein) to alleviate a tendency to form intra-H chain disulfide bonds that can lead to heterogeneity in the IgG.sub.4 antibodies.

[0346] Moreover, techniques for deriving antibodies having different properties (i.e., varying affinities for the antigen to which they bind) are also known. One such technique, referred to as chain shuffling, involves displaying immunoglobulin variable domain gene repertoires on the surface of filamentous bacteriophage, often referred to as phage display. Chain shuffling has been used to prepare high affinity antibodies to the hapten 2-phenyloxazol-5-one, as described by Marks et al., BioTechnology 10:770 (1992).

[0347] Conservative modifications may be made to the heavy and light chains (and corresponding modifications to the encoding nucleic acids) to produce an anti-LRP6 antibody having functional and biochemical characteristics. Methods for achieving such modifications are described above.

[0348] Antibodies and functional fragments thereof may be further modified in various ways. For example, if they are to be used for therapeutic purposes, they may be conjugated with polyethylene glycol (PEGylated) to prolong the serum half-life or to enhance protein delivery. Alternatively, the V region of the subject antibodies or fragments thereof may be fused with the Fc region of a different antibody molecule. The Fc region used for this purpose may be modified so that it does not bind complement, thus reducing the likelihood of inducing cell lysis in the patient when the fusion protein is used as a therapeutic agent. In addition, the subject antibodies or functional fragments thereof may be conjugated with human serum albumin to enhance the serum half-life of the antibody of fragment thereof. Another useful fusion is transthyretin (TTR). TTR has the capacity to form a tetramer, thus an antibody-TTR fusion protein can form a multivalent antibody which may increase its binding avidity.

[0349] Alternatively, substantial modifications in the functional and/or biochemical characteristics of the antibodies and fragments described herein may be achieved by creating substitutions in the amino acid sequence of the heavy and light chains that differ significantly in their effect on maintaining (a) the structure of the molecular backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulkiness of the side chain. A "conservative amino acid substitution" may involve a substitution of a native amino acid residue with a normative residue that has little or no effect on the polarity or charge of the amino acid residue at that position. Furthermore, any native residue in the polypeptide may also be substituted with alanine, as has been previously described for alanine scanning mutagenesis.

[0350] Amino acid substitutions (whether conservative or non-conservative) of the subject antibodies can be implemented by those skilled in the art by applying routine techniques. Amino acid substitutions can be used to identify important residues of the antibodies provided herein, or to increase or decrease the affinity of these antibodies for human LRP6 or for modifying the binding affinity of other anti-LRP6 antibodies described herein.

Vi. Expression of Anti-LRP6 Antibodies

[0351] The anti-LRP6 antibodies and antigen-binding fragments can be prepared by any of a number of conventional techniques. For example, anti-LRP6 antibodies may be produced by recombinant expression systems, using any technique known in the art. See, for example, Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Kennet et al. (eds.) Plenum Press, N.Y. (1980); Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1988).

[0352] The antibodies may be expressed in hybridoma cell lines or in cell lines other than hybridomas. Expression constructs encoding the antibodies may be used to transform a mammalian, insect, or microbial host cell. Transformation may be performed using any known method for introducing polynucleotides into a host cell, including, for example packaging the polynucleotide in a virus or bacteriophage and transducing a host cell with the construct by transfection procedures known in the art, as exemplified by U.S. Pat. Nos. 4,399,216, 4,912,040, 4,740,461, and 4,959,455 (which patents are hereby incorporated herein by reference for any purpose). The optimal transformation procedure used will depend upon which type of host cell is being transformed. Methods for introduction of heterologous polynucleotides into mammalian cells are well known in the art and include, but are not limited to, dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, mixing nucleic acid with positively-charged lipids, and direct microinjection of the DNA into nuclei.

[0353] Recombinant expression constructs typically comprise a nucleic acid molecule encoding a polypeptide comprising one or more of the following: a heavy chain constant region (e.g., C.sub.H1, C.sub.H2 and/or C.sub.H3); a heavy chain variable region; a light chain constant region; a light chain variable region; one or more CDRs of the light or heavy chain of the anti-LRP6 antibody. These nucleic acid sequences are inserted into an appropriate expression vector using standard ligation techniques. In one embodiment, the 77.2, 135.16, 213.7, 240.8, 413.1, 421.1, 498.3, 537.2, 606.4, 620.1, 856.6, 923.3, 931.1, 993.3, 995.5, 1115.3, 1213.2, 1253.12, 1281.1, 1293.11, 1433.8, 1470.2, or 1903.1 heavy or light chain constant region is appended to the C-terminus of the LRP6-specific heavy or light chain variable region and is ligated into an expression vector. The vector is typically selected to be functional in the particular host cell employed (i.e., the vector is compatible with the host cell machinery, permitting amplification and/or expression of the gene can occur). In some embodiments, vectors are used that employ protein-fragment complementation assays using protein reporters, such as dihydrofolate reductase (see, for example, U.S. Pat. No. 6,270,964, which is hereby incorporated by reference). Suitable expression vectors can be purchased, for example, from Invitrogen Life Technologies or BD Biosciences. Other useful vectors for cloning and expressing the anti-LRP6 antibodies and fragments include those described in Bianchi and McGrew, Biotech. Biotechnol. Bioeng. 84:439-44 (2003), herein incorporated by reference. Additional suitable expression vectors are discussed, for example, in Methods Enzymol, vol. 185 (D. V. Goeddel, ed.), 1990, New York: Academic Press, herein incorporated by reference.

[0354] Typically, expression vectors used in any of the host cells contain sequences for plasmid or virus maintenance and for cloning and expression of exogenous nucleotide sequences. Such sequences, collectively referred to as "flanking sequences" typically include one or more of the following operatively linked nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcriptional termination sequence, a complete intron sequence containing a donor and acceptor splice site, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for inserting the nucleic acid encoding the polypeptide to be expressed, and a selectable marker element.

[0355] Optionally, the vector may contain a "tag"-encoding sequence, that is, an oligonucleotide molecule located at the 5' or 3' end of the coding sequence, the oligonucleotide sequence encoding polyHis (such as hexaHis), or another "tag" for which commercially available antibodies exist, such as V5-His, FLAG.RTM., HA (hemaglutinin from influenza virus), or myc. The tag is typically fused to the antibody protein upon expression, and can serve as a means for affinity purification of the antibody from the host cell. Affinity purification can be accomplished, for example, by column chromatography using antibodies against the tag as an affinity matrix. Optionally, the tag can subsequently be removed from the purified antibody polypeptide by various means such as using certain peptidases for cleavage.

[0356] Flanking sequences in the expression vector may be homologous (i.e., from the same species and/or strain as the host cell), heterologous (i.e., from a species other than the host cell species or strain), hybrid (i.e., a combination of flanking sequences from more than one source), synthetic or native. As such, the source of a flanking sequence may be any prokaryotic or eukaryotic organism, any vertebrate or invertebrate organism, or any plant, provided that the flanking sequence is functional in, and can be activated by, the host cell machinery.

[0357] Flanking sequences useful in the vectors provided herein may be obtained by any of several methods well known in the art. Typically, flanking sequences useful herein will have been previously identified by mapping and/or by restriction endonuclease digestion and can thus be isolated from the proper tissue source using the appropriate restriction endonucleases. In some cases, the full nucleotide sequence of a flanking sequence may be known. Here, the flanking sequence may be synthesized using the methods described herein for nucleic acid synthesis or cloning.

[0358] Where all or only a portion of the flanking sequence is known, it may be obtained using PCR and/or by screening a genomic library with a suitable oligonucleotide and/or flanking sequence fragment from the same or another species. Where the flanking sequence is not known, a fragment of DNA containing a flanking sequence may be isolated from a larger piece of DNA that may contain, for example, a coding sequence or even another gene or genes. Isolation may be accomplished by restriction endonuclease digestion to produce the proper DNA fragment followed by isolation using agarose gel purification, QIAGEN.RTM. column chromatography (Qiagen, Chatsworth, Calif., USA), or other methods known to the skilled artisan. The selection of suitable enzymes to accomplish this purpose will be readily apparent to those skilled in the art.

[0359] An origin of replication is typically a part of prokaryotic expression vectors, particularly those purchased commercially, and the origin aids in the amplification of the vector in a host cell. If the vector of choice does not contain an origin of replication site, one may be chemically synthesized based on a known sequence, and ligated into the vector. For example, the origin of replication from the plasmid pBR322 (New England Biolabs, Beverly, Mass., USA.) is suitable for most gram-negative bacteria and various origins of replication (e.g., SV40, polyoma, adenovirus, vesicular stomatitis virus (VSV), or papilloma viruses such as HPV or BPV) are useful for cloning vectors in mammalian cells. Generally, a mammalian origin of replication is not needed for mammalian expression vectors (for example, the SV40 origin is often used only because it contains the early promoter).

[0360] The expression and cloning vectors of the present invention will typically contain a promoter that is recognized by the host organism and operably linked to nucleic acid encoding an anti-LRP6 antibody or antigen-binding fragment thereof. Promoters are untranscribed sequences located upstream (i.e., 5') to the start codon of a structural gene (generally within about 100 to 1000 bp) that control transcription of the structural gene. Promoters are conventionally grouped into one of two classes: inducible promoters and constitutive promoters. Inducible promoters initiate increased levels of transcription from DNA under their control in response to some change in culture conditions, such as the presence or absence of a nutrient or a change in temperature. Constitutive promoters, on the other hand, initiate continuous gene product production; that is, there is little or no experimental control over gene expression. A large number of promoters, recognized by a variety of potential host cells, are well known. A suitable promoter is operably linked to the DNA encoding an anti-LRP6 antibody by removing the promoter from the source DNA by restriction enzyme digestion or amplifying the promoter by polymerase chain reaction and inserting the desired promoter sequence into the vector.

[0361] Suitable promoters for use with yeast hosts are also well known in the art. Yeast enhancers are advantageously used with yeast promoters. Suitable promoters for use with mammalian host cells are well known and include, but are not limited to, those obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retroviruses, hepatitis-B virus and most preferably SV40. Other suitable mammalian promoters include heterologous mammalian promoters, for example, heat-shock promoters and the actin promoter.

[0362] Particular promoters useful in the practice of the recombinant expression vectors of the invention include, but are not limited to: the SV40 early promoter region (Bemoist and Chambon, Nature 290:304-10 (1981)); the CMV promoter; the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto, et al., Cell 22:787-97 (1980)); the herpes thymidine kinase promoter (Wagner et al., Proc. Natl. Acad. Sci. U.S.A. 78:1444-45 (1981)); the regulatory sequences of the metallothionine gene (Brinster et al., Nature 296:39-42 (1982)); prokaryotic expression vectors such as the beta-lactamase promoter (VIIIa-Kamaroff et al., Proc. Natl. Acad. Sci. U.S.A., 75:3727-31 (1978)); or the tac promoter (DeBoer et al., Proc. Natl. Acad. Sci. U.S.A. 80:21-25 (1983)). Also available for use are the following animal transcriptional control regions, which exhibit tissue specificity and have been utilized in transgenic animals: the elastase I gene control region that is active in pancreatic acinar cells (Swift et al., Cell 38:63946 (1984); Ornitz et al., Cold Spring Harbor Symp. Quant. Biol. 50:399409 (1986); MacDonald, Hepatology 7:425-515 (1987)); the insulin gene control region that is active in pancreatic beta cells (Hanahan, Nature 315:115-22 (1985)); the mouse mammary tumor virus control region that is active in testicular, breast, lymphoid and mast cells (Leder et al., Cell 45:485-95 (1986)); the albumin gene control region that is active in liver (Pinkert et al., Genes Devel. 1:268-76 (1987)); the alpha-feto-protein gene control region that is active in liver (Krumlauf et al., Mol. Cell. Biol. 5:1639-48 (1985); Hammer et al., Science 235:53-58 (1987)); the alpha 1-antitrypsin gene control region that is active in the liver (Kelsey et al., Genes Devel. 1:161-71 (1987)); the beta-globin gene control region that is active in myeloid cells (Mogram et al., Nature 315:338-40 (1985); Kollias et al., Cell 46:89-94 (1986)); the myelin basic protein gene control region that is active in oligodendrocyte cells in the brain (Readhead et al., Cell 48:703-12 (1987)); the myosin light chain-2 gene control region that is active in skeletal muscle (Sani, Nature 314:283-86 (1985)); the gonadotropic releasing hormone gene control region that is active in the hypothalamus (Mason et al., Science 234:1372-78 (1986)); and most particularly the immunoglobulin gene control region that is active in lymphoid cells (Grosschedl et al., Cell 38:647-58 (1984); Adames et al., Nature 318 533-38 (1985); Alexander et al., Mol. Cell Biol. 7:1436-44 (1987)).

[0363] An enhancer sequence may be inserted into the vector to increase the transcription in higher eukaryotes of a nucleic acid encoding an anti-LRP6 antibody or antigen-binding fragment thereof. Enhancers are cis-acting elements of DNA, usually about 10-300 bp in length, that act on promoters to increase transcription. Enhancers are relatively orientation and position independent. They have been found 5' and 3' to the transcription unit. Several enhancer sequences available from mammalian genes are known (e.g., globin, elastase, albumin, alpha-feto-protein and insulin). An enhancer sequence from a virus also can be used. The SV40 enhancer, the cytomegalovirus early promoter enhancer, the polyoma enhancer, and adenovirus enhancers are exemplary enhancing elements for the activation of eukaryotic promoters. While an enhancer may be spliced into the vector at a position 5' or 3' to a nucleic acid molecule, it is typically placed at a site 5' to the promoter.

[0364] In expression vectors, a transcription termination sequence is typically located 3' of the end of a polypeptide-coding region and serves to terminate transcription. A transcription termination sequence used for expression in prokaryotic cells typically is a G-C rich fragment followed by a poly-T sequence. While the sequence is easily cloned from a library or even purchased commercially as part of a vector, it can also be readily synthesized using methods for nucleic acid synthesis such as those described herein.

[0365] A selectable marker gene element encodes a protein necessary for the survival and growth of a host cell grown in a selective culture medium. Typical selection marker genes used in expression vectors encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, tetracycline, or kanamycin for prokaryotic host cells; (b) complement auxotrophic deficiencies of the cell; or (c) supply critical nutrients not available from complex media. Examples of selectable markers include the kanamycin resistance gene, the ampicillin resistance gene and the tetracycline resistance gene. A bacterial neomycin resistance gene can also be used for selection in both prokaryotic and eukaryotic host cells.

[0366] Other selection genes can be used to amplify the gene that will be expressed. Amplification is a process whereby genes that cannot in single copy be expressed at high enough levels to permit survival and growth of cells under certain selection conditions are reiterated in tandem within the chromosomes of successive generations of recombinant cells. Examples of suitable amplifiable selectable markers for mammalian cells include dihydrofolate reductase (DHFR) and promoterless thymidine kinase. In the use of these markers mammalian cell transformants are placed under selection pressure wherein only the transformants are uniquely adapted to survive by virtue of the selection gene present in the vector. Selection pressure is imposed by culturing the transformed cells under conditions in which the concentration of selection agent in the medium is successively increased, thereby permitting survival of only those cells in which the selection gene has been amplified. Under these circumstances, DNA adjacent to the selection gene, such as DNA encoding an anti-LRP6 antibody, is co-amplified with the selection gene. As a result, increased quantities of anti-LRP6 antibody polypeptides are synthesized from the amplified DNA.

[0367] A ribosome-binding site is usually necessary for translation initiation of mRNA and is characterized by a Shine-Dalgarno sequence (prokaryotes) or a Kozak sequence (eukaryotes). The element is typically located 3' to the promoter and 5' to the coding sequence of the polypeptide to be expressed.

[0368] In some cases, for example where glycosylation is desired in a eukaryotic host cell expression system, various presequences can be manipulated to improve glycosylation or yield. For example, the peptidase cleavage site of a particular signal peptide can be altered, or pro-sequences added, which also may affect glycosylation. The final protein product may have in the -1 position (relative to the first amino acid of the mature protein) one or more additional amino acids incident to expression which may not have been totally removed. For example, the final protein product may have one or two amino acid residues found in the peptidase cleavage site attached to the amino-terminus. Alternatively, use of some enzyme cleavage sites may result in a slightly truncated yet active form of the desired polypeptide if the enzyme cuts at such area within the mature polypeptide.

[0369] Where a commercially available expression vector lacks some of the desired flanking sequences as described above, the vector can be modified by individually ligating these sequences into the vector. After the vector has been chosen and modified as desired, a nucleic acid molecule encoding an anti-LRP6 antibody or antigen-binding fragment thereof is inserted into the proper site of the vector.

[0370] The completed vector containing sequences encoding the anti-LRP6 antibody or antigen-binding region thereof is inserted into a suitable host cell for amplification and/or polypeptide expression. The transformation of an expression vector for an anti-LRP6 antibody or antigen-binding fragment thereof into a selected host cell may be accomplished by well-known methods including methods such as transfection, infection, calcium chloride, electroporation, microinjection, lipofection, DEAE-dextran method, or other known techniques. The method selected will in part be a function of the type of host cell to be used. These methods and other suitable methods are well known to the skilled artisan.

[0371] The transformed host cell, when cultured under appropriate conditions, synthesizes an anti-LRP6 antibody or antigen-binding fragment thereof that can subsequently be collected from the culture medium (if the host cell secretes it into the medium) or directly from the host cell producing it (if it is not secreted). The selection of an appropriate host cell will depend upon various factors, such as desired expression levels, polypeptide modifications that are desirable or necessary for activity (such as glycosylation or phosphorylation) and ease of folding into a biologically active molecule.

[0372] Mammalian cell lines available as hosts for expression are well known in the art and include, but are not limited to, many immortalized cell lines available from the American Type Culture Collection (ATCC), such as Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) cells, HEK293 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), and a number of other cell lines. In certain embodiments, the best cell line for expressing a particular DNA construct may be selected by testing various cell lines to determine which ones have the highest levels of expression levels and produce antibodies that bind LRP6.

VII. Pharmaceutical Compositions

A. Exemplary Formulations

[0373] In certain embodiments, the invention also provides compositions comprising the subject anti-LRP6 antibodies or antigen-binding fragments thereof together with one or more of the following: a pharmaceutically acceptable diluent; a carrier; a solubilizer; an emulsifier; a preservative; and/or an adjuvant. Such compositions may contain an effective amount of the anti-LRP6 antibody or antigen-binding fragment thereof that are provided herein in the preparation of a pharmaceutical composition of medicament is also included. Such compositions can be used in the treatment of a variety of diseases such as listed below.

[0374] The anti-LRP6 antibodies and antigen-binding fragments thereof may be formulated into therapeutic compositions in a variety of dosage forms such as, but not limited to, liquid solutions or suspensions, tablets, pills, powders, suppositories, polymeric microcapsules or microvesicles, liposomes, and injectable or infusible solutions. The preferred form depends upon the mode of administration and the particular disease or disorder targeted. The compositions also preferably include pharmaceutically acceptable vehicles, carriers or adjuvants, well known in the art.

[0375] A "pharmaceutically acceptable" vehicle, carrier or adjuvant is a non-toxic agent that can be tolerated by a recipient patient. Representative non-limiting examples of such agents include human serum albumin, ion exchangers, alumina, lecithin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, and salts or electrolytes such as protamine sulfate. Suitable vehicles are, for example, water, saline, phosphate-buffered saline, dextrose, glycerol, ethanol, or the like, and combinations thereof. Other suitable agents are well-known to those in the art. See, for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 19th edition, 1995. Actual methods of preparing such compositions are also known, or will be apparent, to those skilled in the art. See, e.g., Remington's Pharmaceutical Sciences, 1995, supra.

[0376] Acceptable formulation components for pharmaceutical preparations are nontoxic to recipients at the dosages and concentrations employed. In addition to the antibodies and antigen-binding regions that are provided, compositions according to the invention may contain components for modifying, maintaining or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition. Suitable materials for formulating pharmaceutical compositions include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen-sulfite); buffers (such as acetate, borate, bicarbonate, Tris-HCl, citrates, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); coloring, flavoring and diluting agents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancing agents (such as sucrose or sorbitol); tonicity enhancing agents (such as alkali metal halides, preferably sodium or potassium chloride, mannitol sorbitol); delivery vehicles; diluents; excipients and/or pharmaceutical adjuvants. (see Remington's Pharmaceutical Sciences, 1995, supra), hereby incorporated by reference in its entirety for all purposes.

[0377] The primary vehicle or carrier in a pharmaceutical composition may be either aqueous or non-aqueous in nature. Suitable vehicles or carriers for such compositions include water for injection, physiological saline solution or artificial cerebrospinal fluid, possibly supplemented with other materials common in compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. Compositions comprising anti-LRP6 antibodies or antigen-binding fragments thereof may be prepared for storage by mixing the selected composition having the desired degree of purity with optional formulation agents in the form of a lyophilized cake or an aqueous solution. Further the anti-LRP6 antibodies or antigen-binding fragments thereof may be formulated as a lyophilizate using appropriate excipients such as sucrose.

[0378] Formulation components are present in concentrations that are acceptable to the site of administration. Buffers are advantageously used to maintain the composition at physiological pH or at a slightly lower pH, typically within a pH range of from about 4.0 to about 8.5, or alternatively, between about 5.0 to 8.0. Pharmaceutical compositions can comprise TRIS buffer of about pH 6.5-8.5, or acetate buffer of about pH 4.0-5.5, which may further include sorbitol or a suitable substitute therefore.

[0379] The pharmaceutical composition to be used for in vivo administration typically is sterile. Sterilization may be accomplished by filtration through sterile filtration membranes. If the composition is lyophilized, sterilization may be conducted either prior to or following lyophilization and reconstitution. The composition for parenteral administration may be stored in lyophilized form or in a solution. In certain embodiments, parenteral compositions are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle, or a sterile pre-filled syringe ready to use for injection.

[0380] Additional pharmaceutical methods may be employed to control the duration of action of an antibody in a therapeutic application. Control release preparations can be prepared through the use of polymers to complex or adsorb the antibody. For example, biocompatible polymers include matrices of poly(ethylene-co-vinyl acetate) and matrices of a polyanhydride copolymer of a stearic acid dimer and sebacic acid. Sherwood et al., Bio/Technology 10:1446 (1992). The rate of release of an antibody from such a matrix depends upon the molecular weight of the protein, the amount of antibody within the matrix, and the size of dispersed particles. Saltzman et al., Biophys. J. 55:163 (1989); Sherwood et al., supra. Other solid dosage forms are described in Remington's Pharmaceutical Sciences, 1995, supra.

[0381] The above compositions can be administered using conventional modes of delivery including, but not limited to, intravenous, intraperitoneal, oral, intralymphatic, subcutaneous administration, intraarterial, intramuscular, intrapleural, intrathecal, and by perfusion through a regional catheter. Local administration to a tumor in question, will also find use with the present invention. Eye drops can be used for intraocular administration. When administering the compositions by injection, the administration may be by continuous infusion or by single or multiple boluses. Intravenous injection provides a useful mode of administration due to the thoroughness of the circulation in rapidly distributing antibodies. For parenteral administration, the antibodies may be administered in a pyrogen-free, parenterally acceptable aqueous solution comprising the desired anti-LRP6 antibodies or antigen-binding fragments thereof in a pharmaceutically acceptable vehicle. A particularly suitable vehicle for parenteral injection is sterile distilled water in which the anti-LRP6 antibodies or antigen-binding fragments thereof are formulated as a sterile, isotonic solution, properly preserved.

[0382] Once the pharmaceutical composition has been formulated, it may be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder. Such formulations may be stored either in a ready-to-use form or in a form (e.g., lyophilized) that is reconstituted prior to administration.

[0383] The components used to formulate the pharmaceutical compositions are preferably of high purity and are substantially free of potentially harmful contaminants (e.g., at least National Food (NF) grade, generally at least analytical grade, and more typically at least pharmaceutical grade). Moreover, compositions intended for in vivo use are usually sterile. To the extent that a given compound must be synthesized prior to use, the resulting product is typically substantially free of any potentially toxic agents, particularly any endotoxins, which may be present during the synthesis or purification process. Compositions for parental administration are also sterile, substantially isotonic and made under GMP conditions.

[0384] The present invention provides kits for producing multi-dose or single-dose administration units. For example, kits according to the invention may each contain both a first container having a dried protein and a second container having an aqueous diluent, including for example single and multi-chambered pre-filled syringes (e.g., liquid syringes, lyosyringes or needle-free syringes).

[0385] The subject compositions comprising anti-LRP6 antibodies or antigen-binding fragments thereof also may be used ex vivo. In such instances, cells, tissues or organs that have been removed from the patient are exposed to or cultured with the anti-LRP6 antibody or antigen-binding fragment thereof. The cultured cells may then be implanted back into the patient or a different patient or used for other purposes.

[0386] In certain embodiments, anti-LRP6 antibodies or antigen-binding fragments thereof can be delivered by implanting certain cells that have been genetically engineered, using methods such as those described herein, to express and secrete the polypeptide. Such cells may be animal or human cells, and may be autologous, heterologous, or xenogenic, or may be immortalized. In order to decrease the chance of an immunological response, the cells may be encapsulated to avoid infiltration of surrounding tissues. Encapsulation materials are typically biocompatible, semi-permeable polymeric enclosures or membranes that allow the release of the protein product(s) but prevent the destruction of the cells by the patient's immune system or by other detrimental factors from the surrounding tissues.

[0387] Pharmaceutical compositions for use in accordance with the present invention thus may be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. These pharmaceutical compositions may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes. Proper formulation is dependent upon the route of administration chosen. When a therapeutically effective amount of an anti-LRP6 antibody, protein or other active ingredient provided herein is administered orally, the antibody, protein or other active ingredient will be in the form of a tablet, capsule, powder, solution or elixir. When administered in tablet form, the pharmaceutical composition may additionally contain a solid carrier such as a gelatin or an adjuvant. The tablet, capsule, and powder contain from about 5 to 95% antibody, protein or other active ingredient, and preferably from about 25 to 90% antibody, protein or other active ingredient. When administered in liquid form, a liquid carrier such as water, petroleum, oils of animal or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic oils may be added. The liquid form of the pharmaceutical composition may further contain physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol. When administered in liquid form, the pharmaceutical composition contains from about 0.5 to 90% by weight of antibody, protein or other active ingredient, and preferably from about 1 to 50% antibody, protein or other active ingredient.

[0388] When a therapeutically effective amount of an antibody, protein or other active ingredient provided herein is administered by intravenous, cutaneous or subcutaneous injection, the antibody, protein or other active ingredient will be in the form of a pyrogen-free, parenterally acceptable aqueous solution. The preparation of such parenterally acceptable antibody, protein or other active ingredient solutions, having due regard to pH, isotonicity, stability, and the like, is within the skill in the art. A preferred pharmaceutical composition for intravenous, cutaneous, or subcutaneous injection should contain, in addition to the antibody, protein or other active ingredient, an isotonic vehicle such as Sodium Chloride Injection, Ringers Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringers Injection, or other vehicle as known in the art. The pharmaceutical composition may also contain stabilizers, preservatives, buffers, antioxidants, or other additives known to those of skill in the art. For injection, the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

[0389] For oral administration, the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. Pharmaceutical preparations for oral use can be obtained solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

[0390] Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for such administration. For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

[0391] For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch. The compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

[0392] Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0393] The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides. In addition to the formulations described previously, the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

[0394] A pharmaceutical carrier for hydrophobic compounds is a co-solvent system comprising benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase. The co-solvent system may be the VPD co-solvent system. VPD is a solution of 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant polysorbate 80, and 65% w/v polyethylene glycol 300, made up to volume in absolute ethanol. The VPD co-solvent system (VPD:5W) consists of VPD diluted 1:1 with a 5% dextrose in water solution. This co-solvent system dissolves hydrophobic compounds well, and itself produces low toxicity upon systemic administration. Naturally, the proportions of a co-solvent system may be varied considerably without destroying its solubility and toxicity characteristics. Furthermore, the identity of the co-solvent components may be varied: for example, other low-toxicity nonpolar surfactants may be used instead of polysorbate 80; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g. polyvinyl pyrrolidone; and other sugars or polysaccharides may substitute for dextrose. Alternatively, other delivery systems for hydrophobic pharmaceutical compounds may be employed. Liposomes and emulsions are well known examples of delivery vehicles or carriers for hydrophobic drugs. Certain organic solvents such as dimethylsulfoxide also may be employed, although usually at the cost of greater toxicity. Additionally, the compounds may be delivered using a sustained-release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent. Various types of sustained-release materials have been established and are well known by those skilled in the art. Sustained-release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days. Depending on the chemical nature and the biological stability of the therapeutic reagent, additional strategies for antibody, protein or other active ingredient stabilization may be employed.

[0395] The pharmaceutical compositions also may comprise suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols. Many of the active ingredients provided herein may be provided as salts with pharmaceutically compatible counter ions. Such pharmaceutically acceptable base addition salts are those salts which retain the biological effectiveness and properties of the free acids and which are obtained by reaction with inorganic or organic bases such as sodium hydroxide, magnesium hydroxide, ammonia, trialkylamine, dialkylamine, monoalkylamine, dibasic amino acids, sodium acetate, potassium benzoate, triethanol amine and the like.

[0396] The pharmaceutical composition may be in the form of a complex of the antibody, protein(s) or other active ingredient along with protein or peptide antigens. The protein and/or peptide antigen will deliver a stimulatory signal to both B and T lymphocytes. B lymphocytes will respond to antigen through their surface immunoglobulin receptor. T lymphocytes will respond to antigen through the T cell receptor (TCR) following presentation of the antigen by MHC proteins. MHC and structurally related proteins including those encoded by class I and class II MHC genes on host cells will serve to present the peptide antigen(s) to T lymphocytes. The antigen components could also be supplied as purified MHC-peptide complexes alone or with co-stimulatory molecules that can directly signal T cells. Alternatively antibodies able to bind surface immunoglobulin and other molecules on B cells as well as antibodies able to bind the TCR and other molecules on T cells can be combined with the pharmaceutical composition of the invention.

[0397] The pharmaceutical composition may be in the form of a liposome in which the anti-LRP6 antibody is combined, in addition to other pharmaceutically acceptable carriers, with amphipathic agents such as lipids which exist in aggregated form as micelles, insoluble monolayers, liquid crystals, or lamellar layers in aqueous solution. Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithins, phospholipids, saponin, bile acids, and the like. Preparation of such liposomal formulations is within the level of skill in the art, as disclosed, for example, in U.S. Pat. Nos. 4,235,871; 4,501,728; 4,837,028; and 4,737,323, all of which are incorporated herein by reference.

[0398] The amount of antibody, protein or other active ingredient in the pharmaceutical composition will depend upon the nature and severity of the condition being treated, and on the nature of prior treatments which the patient has undergone. Ultimately, the attending physician will decide the amount of antibody, protein or other active ingredient with which to treat each individual patient. Initially, the attending physician will administer low doses of antibody, protein or other active ingredient and observe the patient's response. Larger doses of antibody, protein or other active ingredient may be administered until the optimal therapeutic effect is obtained for the patient, and at that point the dosage is not increased further. It is contemplated that the various pharmaceutical compositions used to practice the method of the present invention should contain about 0.01 .mu.g to about 100 mg (preferably about 0.1 .mu.g to about 10 mg, more preferably about 0.1 .mu.g to about 1 mg) of antibody, protein or other active ingredient per kg body weight. For compositions of the present invention which are useful for bone, cartilage, tendon or ligament regeneration, the therapeutic method includes administering the composition topically, systematically (i.e., via intravenous, intraperitoneal, intramuscular, or oral administration), or locally as an implant or device. When administered, the therapeutic composition for use in this invention is, of course, in a pyrogen-free, physiologically acceptable form. Further, the composition may desirably be encapsulated or injected in a viscous form for delivery to the site of bone, cartilage or tissue damage. Topical administration may be suitable for wound healing and tissue repair. Therapeutically useful agents other than an antibody, protein or other active ingredient which may also optionally be included in the composition as described above, may alternatively or additionally, be administered simultaneously or sequentially with the composition in the methods provided herein. Preferably for bone and/or cartilage formation, the composition would include a matrix capable of delivering the protein-containing or other active ingredient-containing composition to the site of bone and/or cartilage damage, providing a structure for the developing bone and cartilage and optimally capable of being resorbed into the body. Such matrices may be formed of materials presently in use for other implanted medical applications.

[0399] The choice of matrix material is based on biocompatibility, biodegradability, mechanical properties, cosmetic appearance and interface properties. The particular application of the compositions will define the appropriate formulation. Potential matrices for the compositions may be biodegradable and chemically defined calcium sulfate, tricalcium phosphate, hydroxyapatite, polylactic acid, polyglycolic acid and polyanhydrides. Other potential materials are biodegradable and biologically well-defined, such as bone or dermal collagen. Further matrices are comprised of pure proteins or extracellular matrix components. Other potential matrices are nonbiodegradable and chemically defined, such as sintered hydroxyapatite, bioglass, aluminates, or other ceramics. Matrices may be comprised of combinations of any of the above mentioned types of material, such as polylactic acid and hydroxyapatite or collagen and tricalcium phosphate. The bioceramics may be altered in composition, such as in calcium-aluminate-phosphate and processing to alter pore size, particle size, particle shape, and biodegradability. Presently preferred is a 50:50 (mole weight) copolymer of lactic acid and glycolic acid in the form of porous particles having diameters ranging from 150 to 800 microns. In some applications, it will be useful to utilize a sequestering agent, such as carboxymethyl cellulose or autologous blood clot, to prevent the protein compositions from disassociating from the matrix.

[0400] A preferred family of sequestering agents is cellulosic materials such as alkylcelluloses (including hydroxyalkylcelluloses), including methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl-methylcellulose, and carboxymethylcellulose, the most preferred being cationic salts of carboxymethylcellulose (CMC). Other preferred sequestering agents include hyaluronic acid, sodium alginate, poly(ethylene glycol), polyoxyethylene oxide, carboxyvinyl polymer and poly(vinyl alcohol). The amount of sequestering agent useful herein is 0.5-20 weight percent, preferably 1-10 weight percent based on total formulation weight, which represents the amount necessary to prevent desorption of the antibody or protein from the polymer matrix and to provide appropriate handling of the composition, yet not so much that the progenitor cells are prevented from infiltrating the matrix, thereby providing the antibody or protein the opportunity to assist the osteogenic activity of the progenitor cells. In further compositions, the antibodies, proteins or other active ingredient may be combined with other agents beneficial to the treatment of the bone and/or cartilage defect, wound, or tissue in question. These agents include various growth factors such as epidermal growth factor (EGF), platelet derived growth factor (PDGF), transforming growth factors (TGF-.alpha. and TGF-.beta.), and insulin-like growth factor (IGF).

[0401] The therapeutic compositions are also presently valuable for veterinary applications. Particularly domestic animals and thoroughbred horses, in addition to humans, are desired patients for such treatment with antibodies, proteins or other active ingredient of the present invention. The dosage regimen of an antibody- or protein-containing pharmaceutical composition to be used in tissue regeneration will be determined by the attending physician considering various factors which modify the action of the proteins, e.g., amount of tissue weight desired to be formed, the site of damage, the condition of the damaged tissue, the size of a wound, type of damaged tissue (e.g., bone), the patient's age, sex, and diet, the severity of any infection, time of administration and other clinical factors. The dosage may vary with the type of matrix used in the reconstitution and with inclusion of other proteins in the pharmaceutical composition. For example, the addition of other known growth factors, such as IGF I (insulin like growth factor I), to the final composition, may also effect the dosage. Progress can be monitored by periodic assessment of tissue/bone growth and/or repair, for example, X-rays, histomorphometric determinations and tetracycline labeling.

B. Dosage

[0402] For purposes of therapy, antibodies are administered to a patient in a therapeutically effective amount. A "therapeutically effective amount" is one that is physiologically significant. An agent is physiologically significant if its presence results in a detectable change in the physiology or disease or disorder state of a recipient. A "prophylactically effective amount" refers to an amount that is effective to prevent, hinder or retard the onset of a disease state or symptom.

[0403] Therapeutically effective doses will be easily determined by one of skill in the art and will depend on the severity and course of the disease, the patient's health and response to treatment, the patient's age, weight, height, sex, previous medical history and the judgment of the treating physician. Typically, it is desirable to provide the recipient with a dosage of antibody component or immunoconjugate which is in the range of from about 1 pg/kg to 10 mg/kg (amount of agent/body weight of patient), although a lower or higher dosage also may be administered as circumstances dictate. In preferred embodiments, anti-LRP6 antibodies are administered at low protein doses, such as 20 mg to 2 g protein per dose, given once, or repeatedly, parenterally. Alternatively, antibodies are administered in doses of 20 to 1000 mg protein per dose, or 20 to 500 mg protein per dose, or 20 to 100 mg protein per dose.

[0404] Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve its intended purpose. More specifically, a therapeutically effective amount means an amount effective to prevent development of or to alleviate the existing symptoms of the subject being treated. Determination of the effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein. For any compound used in the methods of the invention, the therapeutically effective dose can be estimated initially from appropriate in vitro assays. For example, a dose can be formulated in animal models to achieve a circulating concentration range that can be used to more accurately determine useful doses in humans. For example, a dose can be formulated in animal models to achieve a circulating concentration range that includes the IC.sub.50 as determined in cell culture (i.e., the concentration of the test compound which achieves a half-maximal inhibition of the protein's biological activity). Such information can be used to more accurately determine useful doses in humans.

[0405] A therapeutically effective dose refers to that amount of the compound that results in amelioration of symptoms or a prolongation of survival in a patient. Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD.sub.50 (the dose lethal to 50% of the population) and the ED.sub.50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between LD.sub.50 and ED.sub.50. Compounds which exhibit high therapeutic indices are preferred. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED.sub.50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. See, e.g., Fingl et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p. 1. Dosage amount and interval may be adjusted individually to provide plasma levels of the active moiety which are sufficient to maintain the desired effects, or minimal effective concentration (MEC). The MEC will vary for each compound but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. However, HPLC assays or bioassays can be used to determine plasma concentrations.

[0406] Dosage intervals can also be determined using MEC value. Compounds should be administered using a regimen which maintains plasma levels above the MEC for 10-90% of the time, preferably between 30-90% and most preferably between 50-90%. In cases of local administration or selective uptake, the effective local concentration of the drug may not be related to plasma concentration.

[0407] The amount of composition administered will, of course, be dependent on the subject being treated, on the subject's age and weight, the severity of the affliction, the manner of administration and the judgment of the prescribing physician.

C. Routes of Administration

[0408] Suitable routes of administration of anti-LRP6 antibodies for the treatment of bone diseases and disorders may, for example, include oral, rectal, transmucosal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections. Administration of anti-LRP6 antibodies or other active ingredient used in the pharmaceutical composition or to practice the methods of the present invention can be carried out in a variety of conventional ways, such as oral ingestion, inhalation, topical application or cutaneous, subcutaneous, intraperitoneal (IP), parenteral or intravenous injection.

[0409] Alternatively, one may administer the compound in a local rather than systemic manner, for example, via injection of the compound directly into the tissue, often in a depot or sustained release formulation.

[0410] The compounds provided herein are administered by any route that delivers an effective dosage to the desired site of action. The determination of a suitable route of administration and an effective dosage for a particular indication is within the level of skill in the art. Preferably for bone disorders, one administers the anti-LRP6 antibodies systemically. Suitable dosage ranges for the anti-LRP6 antibodies can be extrapolated from these dosages or from similar studies in appropriate animal models. Dosages can then be adjusted as necessary by the clinician to provide maximal therapeutic benefit. Alternatively, bone disorders and diseases may be treated via local administration of the anti-LRP6 antibodies.

VIII. Diagnostic Assays

[0411] Antibodies of the present invention can be used in vivo, i.e., injected into subjects, for diagnostic or therapeutic uses. The use of antibodies for in vivo diagnosis is well known in the art. Sumerdon et al., Nucl. Med. Biol 17:247-254 (1990) have described an optimized antibody-chelator for the radioimmunoscintographic imaging of carcinoembryonic antigen (CEA)-expressing tumors using Indium-111 as the label. Griffin et al., J Clin Onc 9:631-640 (1991) have described the use of this agent in detecting tumors in patients suspected of having recurrent colorectal cancer. The use of similar agents with paramagnetic ions as labels for magnetic resonance imaging is known in the art (R. B. Lauffer, Magnetic Resonance in Medicine 22:339-342 (1991). Thus, antibodies directed against the LRP6 antigen can be injected into subjects suspected of having a disease or disorder in which LRP6, Wnt or Dkk1 is implicated for the purpose of diagnosing or staging the disease status of the patient. The label used will depend on the imaging modality chosen. Radioactive labels such as Indium-111, Technetium-99m, or Iodine-131 can be used for planar scans or single photon emission computed tomography (SPECT). Positron emitting labels such as Fluorine-19 can also be used for positron emission tomography (PET). For MRI, paramagnetic ions such as Gadolinium (III) or Manganese (II) can be used. Localization of the label within the patient allows determination of the presence and/or spread of the disease.

[0412] The antibodies generated against LRP6 can also be used in standard in vitro immunoassays, to screen biological samples such as blood, tissues and/or tumors for the presence or absence of LRP6. Thus, the anti-LRP6 antibodies produced as described above, can be used in diagnostic assays. The anti-LRP6 antibodies can be used as either the capture component and/or the detection component in the assays, as described further below. Thus, the presence of LRP6 antigen can be determined by the presence of LRP6 antigens and/or anti-LRP6 antibodies.

[0413] For example, the presence of LRP6 cell surface receptors can be detected using standard electrophoretic and immunodiagnostic techniques, including immunoassays such as competition, direct reaction, or sandwich type assays. Such assays include, but are not limited to, Western blots; agglutination tests; enzyme-labeled and mediated immunoassays, such as enzyme-linked immunosorbent assays ("ELISAs"); biotin/avidin type assays; radioimmunoassays; immunoelectrophoresis; immunoprecipitation, etc. The reactions generally include revealing labels such as fluorescent, chemiluminescent, radioactive, or enzymatic labels or dye molecules, or other methods for detecting the formation of a complex between the antigens and the antibodies described above.

[0414] Assays can also be conducted in solution, such that the antigens and antibodies thereto form complexes under precipitating conditions. The precipitated complexes can then be separated from the test sample, for example, by centrifugation. The reaction mixture can be analyzed to determine the presence or absence of antibody-antigen complexes using any of a number of standard methods, such as those immunodiagnostic methods described above. The antigens and antibodies can be provided in kits, with suitable instructions and other necessary reagents, in order to conduct immunoassays as described above. The kit can also contain, depending on the particular immunoassay used, suitable labels and other packaged reagents and materials (i.e. wash buffers and the like). Standard immunoassays, such as those described above, can be conducted using these kits.

IX. Therapeutic Uses

[0415] The present invention provides antibodies or antigen-binding fragments thereof that bind to LRP6 epitopes that are useful for the treatment of human diseases and pathological conditions. Anti-LRP6 antibodies may be used in combination with other therapeutic agents to enhance their therapeutic effects or decrease potential side effects.

[0416] Supplemental active compounds can also be incorporated into the compositions. In certain embodiments, an anti-LRP6 antibody of antigen-binding fragment can be co-formulated with one or more additional therapeutic agents, such as a chemotherapeutic agent, an antineoplastic agent, or an anti-tumor agent. These agents include without limitation, antibodies that bind other targets (e.g., antibodies that bind one or more growth factors, cytokines, or cell surface receptors), LRP6 binding proteins, antineoplastic agents, chemotherapeutic agents, anti-tumor agents, antisense oligonucleotides against LRP6, LRP6 peptide analogs, and/or one or more chemical agents that inhibit LRP6 production or activity, which are known in the art.

[0417] In another aspect, the anti-LRP6 antibody may be co-administered with other therapeutic agents, such as antineoplastic drugs or molecules, to a patient who has a hyperproliferative disorder, such as cancer (for example multiple myeloma or prostate cancer with associated osteolytic lesions) or a tumor. In one aspect, the invention relates to a method for the treatment of the hyperproliferative disorder in a mammal comprising administering to said mammal a therapeutically effective amount of a compound of the invention in combination with an anti-tumor agent selected from the group consisting of, but not limited to, mitotic inhibitors, alkylating agents, anti-metabolites, intercalating agents, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, biological response modifiers, anti-hormones, kinase inhibitors, matrix metalloprotease inhibitors, genetic therapeutics and anti-androgens. In a more preferred embodiment, the antibody may be administered with an antineoplastic agent, such as adriamycin or taxol. In another preferred embodiment, the antibody or combination therapy is administered along with radiotherapy, chemotherapy, photodynamic therapy, surgery or other immunotherapy. In yet another preferred embodiment, the antibody will be administered with another antibody. For example, an anti-LRP6 antibody may be administered with an antibody or other agent that is known to inhibit tumor or cancer cell proliferation, e.g., an antibody or agent that inhibits erbB2 receptor, EGF-R, CD20 or VEGF.

[0418] In yet another aspect, the anti-LRP6 mAbs may be administered with other therapeutic agents, such as anti-inflammatory agents including but not limited to steroids, glucocorticoids, corticosteroids, NSAIDS, including cyclooxygenase inhibitors, aspirin, analgesics including paracetamol (acetaminophen), and capsaicin, to a patient with a bone or joint inflammatory disease, such as rheumatoid arthritis, osteoarthritis, ankylosing spondylosis.

[0419] In yet another aspect, the anti-LRP6 mAbs may be administered with other therapeutic agents, such as agents that treat osteoporosis including but not limited to bisphosphonates, including alendronate (FOSAMAX.RTM. (Merck, Whitehouse Station, N.J.), ibandronate (BONIVA.RTM., Roche, Nutley, N.J.), risedronate (ACTONEL.RTM. (Procter & Gamble Pharmaceuticals, Cincinnati, Ohio); selective estrogen receptor modulators (SERM) including raloxifene (EVISTA.RTM., Eli Lilly, Indianapolis, Ind.); calcitonin, including CALCIMAR.RTM. (Rhone-Poulenc-Rorer, Collegeville, Pa.) and MIACALCIN.RTM. (Novartis, East Hanover, N.J.); parathyroid hormone, including teriparatide; estrogen replacement therapy (ERT), hormone replacement therapy (HRT, estrogen with progestin), testosterone, and calcium with vitamin D, for the treatment of bone disorders characterized by low bone density, such as osteoporosis.

[0420] Co-administration of the anti-LRP6 antibody or antigen-binding fragments thereof with an additional therapeutic agent (combination therapy) encompasses administering a pharmaceutical composition comprising an anti-LRP6 antibody and the additional therapeutic agent and administering two or more separate pharmaceutical compositions, one composition comprising an anti-LRP6 antibody and the other(s) comprising the additional therapeutic agent(s). Further, although co-administration or combination therapy generally means that the antibody and additional therapeutic agents are administered at the same time as one another, it also encompasses instances in which the antibody and additional therapeutic agents are administered at different times. For instance, the antibody may be administered once every three days, while the additional therapeutic agent is administered once daily. Alternatively, the antibody may be administered prior to or subsequent to treatment of the disorder with the additional therapeutic agent. Similarly, administration of the anti-LRP6 antibody may be administered prior to or subsequent to other therapy, such as radiotherapy, chemotherapy, photodynamic therapy, surgery or other immunotherapy

[0421] The antibody and one or more additional therapeutic agents (the combination therapy) may be administered once, twice or at least the period of time until the condition is treated, palliated or cured. Preferably, the combination therapy is administered multiple times. The combination therapy may be administered from three times daily to once every six months. The administering may be on a schedule such as three times daily, twice daily, once daily, once every two days, once every three days, once weekly, once every two weeks, once every month, once every two months, once every three months, once every six months, or may be administered continuously via a minipump. The combination therapy may be administered via an oral, mucosal, buccal, intranasal, inhalable, intravenous, subcutaneous, intramuscular, parenteral, intratumor or topical route.

[0422] In one aspect, the present invention provides reagents and methods useful for treating diseases and conditions characterized by reduced levels of Wnt/LRP6 signaling activity and/or increased levels of Wnt inhibitors, such as the LRP5/6 inhibitor Dkk1. In a particular embodiment, the antibodies and derivatives thereof are used in vivo to enhance LRP6 signaling and/or block Dkk1 activity to treat, prevent or diagnose a variety of bone diseases. These diseases include osteoporosis, osteogenesis imperfecta, Paget's disease of bone, myeloma bone disease including osteolytic lesions associated with multiple myeloma, bone spurs (osteophytes), osteoarthritis, diffuse idiopathic skeletal hyperstosis, plantar fasciitis, spondylosis (including cervical and lumbar), spinal stenosis, craniocynostossi, echondroma, fibrous dysplasia, Klippel-Feil syndrome, osteitis condensans ilii, osteochondritis dissecans, osteomyelitis, osteopetroses (marble bone diseases), renal osteodystrophy, unicameral bone cyst, osteomalacia, hyperostosis, and van Buchem disease.

[0423] The diseases treatable by methods of the present invention preferably occur in mammals. Mammals include, for example, humans and other primates, as well as pet or companion animals such as dogs and cats, laboratory animals such as rats, mice and rabbits, and farm animals such as horses, pigs, sheep and cattle.

[0424] The present invention also provides methods of modulating stem cell growth by administering anti-LRP6 antibodies that promote LRP6 activity and/or inhibit Dkk1 activity. For example, such anti-LRP6 antibodies may serve to stimulate proliferation of intestinal epithelial cells including crypt cells and for regeneration of oral and gastrointestinal tissue, i.e., for the treatment of injuries sustained by the epithelial layer which involve degeneration, death or trauma to epithelial cells. More specifically, an anti-LRP6 antibody that promotes LRP6 activity and/or inhibits Dkk1 activity can be used in the treatment of diseases of the gastrointestinal tract as recited herein. Similarly, such anti-LRP6 antibodies can also be used to promote expansion and/or differentiation of other stem cell populations such as, but not limited to, hematopoietic, neuronal, and embryonic stem cells.

[0425] In one aspect, the present invention provides compositions and methods useful for treating diseases and conditions wherein epithelialization is desired. Anti-LRP6 antibodies that promote LRP6 activity and/or inhibit Dkk1 activity can be used to increase cytoprotection, proliferation or differentiation of epithelial cells of the oral and gastrointestinal tract. Specifically, anti-LRP6 antibodies that promote LRP6 activity and/or inhibit Dkk1 activity can be useful to treat or prevent diseases or conditions that include without limitation, gastrointestinal diseases, mucositis of the gastrointestinal tract, mucositis of the oropharynx, lips and esophagus (oral mucositis), inflammatory bowel disease, short bowel syndrome, gastric and duodenal ulcers, erosions of the gastrointestinal tract including erosive gastritis, esophagitis, esophageal reflux and other conditions including wounds, burns, ophthalmic disorders, and any disorder where stimulation of epithelial cell proliferation or regeneration is desired. Treatment of diseases that result in insufficient production of mucus throughout the oral and gastrointestinal tract is also contemplated.

[0426] Anti-LRP6 antibodies that promote LRP6 activity and/or inhibit Dkk1 activity can also be useful to promote better or faster closure of non-healing wounds, including without limitation, pressure ulcers, ulcers associated with vascular insufficiency, surgical and traumatic wounds and the like. Assays for wound healing activity include, without limitation, those described in Winter, Epidermal Wound Healing, pp. 71-112 (Maibach and Rovee, eds), Year Book Medical Publishers, Inc., Chicago, as modified by Eaglstein and Mertz, J. Invest. Dermatol. 71:382-84 (1978).

[0427] The invention further provides methods for treating wounded tissue comprising administering to a subject in need thereof. The anti-LRP6 antibodies that promote LRP6 activity and/or inhibit Dkk1 activity may be administered alone or in combination with other compositions including but not limited to growth factors, antioxidant vitamins, antibiotics, and cellulosic materials. The present invention provides methods for treating wounds on external surfaces such as the skin and mucous membranes as well as treating internal lesions. For example, the methods and compositions of the present invention may be used to treat wounds associated with surgical incisions and other localized injury to internal tissues.

[0428] In yet another embodiment, anti-LRP6 antibodies that promote LRP6 activity and/or inhibit Dkk1 activity may enhance hematopoietic recovery after chemotherapy or radiation therapy by stimulating the growth or proliferation of hematopoietic stem cells. Such anti-LRP6 antibodies may also be used to stimulate bone marrow transplant engraftment by stimulating hematopoietic stem cell proliferation.

[0429] Anti-LRP6 antibodies that promote LRP6 activity and/or inhibit Dkk1 activity may also be useful in treating disorders wherein epithelial stem cell proliferation is desired, for example in stimulating hair growth.

[0430] Anti-LRP6 antibodies that promote LRP6 activity and/or inhibit Dkk1 activity may also be useful in stimulating the growth, expansion or differentiation of stem cells in vivo or in vitro to expand stem cell populations. Expanded stem cell populations can also be used for cell-based therapies in which stem cells are induced to differentiate into specific cell types required to repair damaged or destroyed cells or tissue. Examples of diseases and disorders that can be treated using stem cell-based therapies include, but are not limited to: organ regeneration or generation; neural diseases and disorders such as Parkinson's disease, Alzheimer's disease, spinal cord injury, stroke, neurodegenerative diseases, multiple sclerosis; burns; heart disease; diabetes; bone and cartilage diseases and disorders including osteoporosis and osteoarthritis; kidney diseases, gastrointestinal diseases and disorders, rheumatoid arthritis, sickle cell disease; and cancer such as multiple myeloma, breast and prostate cancer that have associated osteolytic lesions.

X. Articles of Manufacture

[0431] In another embodiment of the invention, an article of manufacture containing materials useful for the treating diseases or disorders implicating LRP6 is provided. The article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition which is effective for treating the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an anti-LRP6 antibody (e.g., 77.2, 135.16, 213.7, 240.8, 413.1, 421.1, 498.3, 537.2, 606.4, 620.1, 856.6, 923.3, 931.1, 993.9, 995.5, 1115.3, 1213.2, 1253.12, 1281.1, 1293.11, 1433.8, 1470.2, or 1903.1). Alternatively, or additionally, the article of manufacture may further comprise a second container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline (PBS), Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles and syringes.

[0432] Further details of the invention are illustrated by the following non-limiting Examples. The disclosures of all citations in the specification are expressly incorporated herein by reference.

Example 1

Generation and Characterization of Anti-LRP6 Monoclonal Antibodies

A. Generation of Hybridomas

[0433] Recombinant human LRP6 (hLRP6) protein containing the complete extracellular domain was purchased from R&D systems (Minneapolis, Minn.). Using standard protocols (see Kohler and Milstein, Nature 256:495-497 (1975) herein incorporated by reference in its entirety), immunizations of Balb/c mice with the extracellular domain of LRP6 (SEQ ID NO: 3) and subsequent fusions with SP20-Ag14 cells (ATCC) resulted in a total of 170 hybridoma supernatants containing antibodies which bound to LRP6 in an ELISA screen, 64 of which scored positive by FACS analysis on 293 Tcells transiently transfected with hLRP6.

B. ELISA Screen of Hybridoma Supernatants for Binding to LRP6

[0434] Human LRP6-Fc (R&D Systems) was coated at 1 .mu.g/ml in Carbonate-Bicarbonate buffer (Sigma #C-3041) on MaxiSorp 96-well plates (Nunc) and incubated overnight at 4.degree. C. After three washes with 300 .mu.g/well TBST (0.1 M Tris-HCl, 0.15 M NaCl, 0.05% Tween-20), wells were blocked using 300 .mu.g/well 2% BSA (Sigma #A9647) in PBS for one hour at room temperature. Hybridoma supernatants were diluted 1:2 in Iscove's Media (Gibco #31980-030) with 10% FBS (Gibco #20012-027) and 100 .mu.l was added to each well followed by incubation for 2 hours on a plate shaker at room temperature. Three washes with TBST were followed by addition of 100 .mu.l of secondary antibody, goat anti-mouse Ig-HRP (BioRad #170-6516), diluted 1:10,000 in 0.5% BSA/PBS and incubated for one hour on a plate shaker. After five washes with TBST, 100 .mu.l TMB substrate (KPL #50-76-03) were added and color was allowed to develop for 10 min. Plates were read at 450 nm on a SpectraMax plate reader (Molecular Devices, Sunnyvale, Calif.).

C. FACS Screening of Hybridoma Supernatants for Binding to LRP6 Expressing Cells

[0435] Briefly, 293 T cells were transiently transfected with HA-tagged hLRP6 plasmid (SEQ ID NO: 4) using Fugene transfection reagent (Roche), according to manufacturer's instructions. Transfected cells were collected 48 hours post transfection and resuspended at 5.times.10.sup.6 cells/ml in blocking buffer (10% heat-inactivated human serum, BioWhittaker, in PBS) and 100 .mu.l were added to each well of a round-bottom 96-well plate and incubated for 15 min on ice. 100 .mu.l of hybridoma supernatant was added to each well and plates were incubated for an additional 20 min on ice. Cells were centrifuged for 5 min at 1500 rpm, supernatant was removed and cells were washed twice in cold FACS buffer (1% BSA in PBS). The pellet was resuspended in 100 .mu.l blocking buffer containing 0.25 .mu.g of secondary antibody (goat anti-mouse PE-conjugated, BD Pharmingen) and incubated for 15 min on ice. Cells were analyzed for fluorescence in FL-2 using an Automated Microsampler from Cytek hooked up to a FACScalibur system (Becton Dickinson, Franklin Lakes, N.J.).

[0436] Based on isotype and ability to recognize LRP6 expressed on cells by FACS, 23 hits were subcloned, re-screened, selected for scale-up and purified using a protein G column. The monoclonal antibodies isolated and described herein were annotated as follows: 77.2, 135.16, 213.7, 240.8, 413.1, 421.1, 498.3, 537.2, 606.4, 620.1, 856.6, 923.3, 931.1, 993.9, 995.5, 1115.3, 1213.2, 1253.12, 1281.1, 1293.11, 1433.8, 1470.2, and 1903.1. These antibodies were subsequently used in detailed expression analysis and efficacy studies (discussed below).

D. Generation of Anti-LRP6 Chimeric Monoclonal Antibodies

[0437] Chimeric monoclonal antibodies (mAbs) against LRP6 are generated as follows: RNA is isolated from hybridoma fusion cells expressing the anti-LRP6 mAb of interest. Using standard RACE/RT-PCR techniques, the heavy and light variable regions are cloned into two separate expression vectors in fusion with cDNA encoding for human IgG1 constant regions. The resulting plasmids are co-transfected into CHO cells and stable cell lines are selected secreting full-length chimeric mAbs. Conditioned medium of these cell lines are subjected to protein G purification to yield purified chimeric mAbs.

Example 2

Affinity Measurements for Anti-LRP6 Monoclonal Antibodies

[0438] Kinetic rate constants (k.sub.a and k.sub.d) were determined using surface plasmon resonance, and affinities (K.sub.D) were then calculated from the rate constants (k.sub.d/k.sub.a). Surface plasmon resonance was carried out on a BIAcore system (Biacore International AB, Uppsala, Sweden). Each murine anti-LRP6 mAb sample was diluted 100 fold and captured onto an anti-mouse sensor chip surface. The running buffer contained 10 mM HEPES, 150 mM NaCl, 0.005% Tween-20 and 0.1 mg/ml BSA. Following the capturing step, the Fc-antigen was injected at 45 nM as the highest concentration in a 3-fold dilution series. The association and dissociation phases were monitored for 8 and 60 minutes, respectively. The antigen response data were fit into a 1:1 interaction model. The apparent binding constants were reported within each plot (see FIG. 2 and summarized in Table 4. The analysis was carried out in HBS, pH 7.4 buffer at 25.degree. C. (Canziani et al, Anal. Biochem. 352:301-307 (2004)).

TABLE-US-00004 TABLE 4 mAb k.sub.a (M.sup.-1s.sup.-1) k.sub.d (s.sup.-1) K.sub.D 77.2 6.32(2)e4 3.5(2)e-5 550(40) pM 135.16 2.63(1)e4 1.9(1)e-5 730(30) pM 213.7 5.563(9)e4 9.6(1)e-5 1.72(2) nM 240.8 6.11(1)e3 .sup. 4(1)e-5 6.55(1) nM 413.1 4.7(2)e3 8.47(8)e-5 18.0(7) nM 421.1 3.54(3)e4 1.14(4)e-4 3.2(1) nM 498.3 5.05(2)e4 5.0(3)e-5 980(50) pM 606.4 5.32(1)e4 5.7(1)e-5 1.08(2) nM 537.2 3.517(6)e4 3.15(6)e-5 900(20) pM 620.1 4.46(2)e4 7.7(2)e-5 1.73(4) nM 856.6 4.74(2)e4 4.5(2)e-5 950(30) pM 923.3 3.16(1)e4 7.4(1)e-5 2.33(4) nM 931.1 5.63(1)e4 6.3(1)e-5 1.12(2) nM 993.9 2.80(1)e4 5.7(1)e-5 2.05(3) nM 995.5 4.138(9)e4 4.51(9)e-5 1.09(2) nM 1115.3 3.675(9)e4 5.34(9)e-5 1.45(2) nM 1213.2 5.88(1)e4 6.3(1)e-5 1.07(2) nM 1253.12 3.40(3)e4 7.3(3)e-5 2.15(7) nM 1281.1 4.55(2)e4 8.3(2)e-5 1.83(4) nM 1293.11 2.2(3)e3 1.24(1)e-4 57(8) nM 1433.8 4.56(2)e4 7.7(2)e-5 1.68(4) nM 1903.1 3.37(4)e4 9.4(4)e-5 2.79(9) nM

Example 3

Epitope Mapping of Anti-LRP6 Monoclonal Antibodies

[0439] To identify the region of LRP6 that is recognized by LRP6 mAbs, a series of LRP6 propeller domain deletion constructs were made (SEQ ID NO: 6-15) and expressed in 293 cells. Binding of anti-LRP6 mAbs was determined by flow cytometry. LRP6 mAb135.16 bound to full-length LRP6 (SEQ ID NO: 5), LRP6.DELTA.2-4 (SEQ ID NO: 13) and LRP6.DELTA.3-4 (SEQ ID NO: 15); however mAb135.16 did not bind LRP6.DELTA.1-2 (SEQ ID NO: 7). Therefore, the results demonstrated binding of the anti-LRP6 mAb 135.16 to a region containing the first propeller domain (propeller domain 1) defined by SEQ ID NO: 16 or amino acids 43-324 of SEQ ID NO: 2 (FIGS. 3A & B). The other antibodies disclosed herein, were mapped in the same way and results are summarized in Table 5. Amino acid sequence alignments of the heavy and light chain variable domains of the anti-LRP6 mAbs disclosed herein are shown in FIGS. 4 and 5, respectively.

[0440] To further characterize the panel of LRP6 mAbs that bound to the first propeller domain of LRP6, competition experiments were carried by ELISA, using biotinylated mAb135.16. mAb135 was biotinylated using standard procedures and LRP6 antibodies were incubated on ELISA plates coated with LRP6-Fc as described above, in the presence of biontinylated mAb135 at 0.01 or 0.1 ug/ml. After three washes with TBST, wells were incubated with streptavidin-HRP (1:5000 in 0.5% BSA/PBS) for 1 hr at room temperature on a plate shaker. After five washes with TBST, 100 .mu.l TMB substrate (KPL #50-76-03) were added and color was allowed to develop for 10 min. Plates were read at 450 nm on a SpectraMax plate reader (Molecular Devices). Loss of binding of biotinylated mAb135.16 in the presence of the unlabeled test hybridoma supernatant and/or purified antibody, indicated that the test mAb bound to an epitope similar to or overlapping with the epitope recognized by mAb 135.16. Six LRP6 antibodies were found to bind an epitope similar to or overlapping with the epitope recognized by mAb 135, summarized in Table 5.

TABLE-US-00005 Activating Dkk1 mAb that bind mAb LRP6 antagonizing 135.16 overlapping mAb mAb epitope Domain mapping* 77.2 77.2 Propeller domain 2 213.7 213.7 Propeller domain 1 240.8 240.8 Propeller domain 1 421.1 421.1 421.1 Propeller domain 2 498.3 498.3 Propeller domain 1 606.4 606.4 Propeller domain 1 856.6 856.6 856.6 Propeller domain 1 923.3 923.3 Propeller domain 1 931.1 931.1 Propeller domain 1 993.9 Not determined 995.5 Not determined 1115.3 1115.3 Propeller domain 1 1213.2 1213.2 Propeller domain 1 1253.12 1253.12 1253.12 Propeller domain 1 1281.1 Propeller domain 1 1293.11 1293.11 1293.11 Propeller domain 1 1433.8 Propeller domain 1 1470.2 1470.2 Propeller domain 1 1903.1 1903.1 Propeller domain 1 135.16 135.16 Propeller domain 1 413.1 413.1 413.1 Propeller domain 1 620.1 620.1 620.1 Propeller domain 1 537.2 Not determined *Propeller domain 1 = SEQ ID NO: 16; Propeller domain 2 = SEQ ID NO: 370

[0441] To further define the epitope to which the anti-LRP6 mAbs bound, human/mouse chimeric constructs were made of propeller domain 1. Since mAb 135.16 did not bind to mouse LRP6, various portions of the human LRP6 propeller domain 1 were substituted with the corresponding mouse sequence (SEQ ID NO: 373 is the mouse LRP6 polypeptide). Binding of the anti-LRP6 mAb to the constructs was determined by FACS analysis as described above. As can be seen in FIG. 3D, the anti-LRP6 mAb 135.16 did not bind to construct 1c in which residues 252 to 283 were replaced by the mouse sequence. Analysis of the differences between the mouse and human sequences in region 1c showed that three amino acid residues differ between human and mouse: I236T, S243N and D264N. Each of these three residues in the human sequence was individually changed to the mouse residue and analyzed for binding. As can be seen in FIG. 3F, anti-LRP6 mAb 135.16 required Ser.sup.243 for binding.

Example 4

TCF-Luciferase Assay for Testing the Activity of Anti-LRP6 Monoclonal Antibodies

[0442] To investigate the effect of the anti-LRP6 mAbs disclosed herein on canonical Wnt signaling, a stable 293 cell line expressing a TCF luciferase reporter plasmid was used.

[0443] A 16TCF luciferase reporter construct was generated by cloning 16 repeats of a TCF consensus site (AGATCAAAGG (SEQ ID NO: 369) into the pTA-Luc vector (Clontech, Mountain View, Calif.). A geneticin selectable marker was inserted into the vector and used to select a stable clone (A6) exhibiting minimal basal reporter activation. 293 A6 cells were seeded in 96-well plates in DEMEM containing 10% fetal bovine serum (FBS) (Invitrogen, Carlsbad, Calif.), starved for 8 hours in DEMEM containing 0.1% FBS, treated in triplicate for 18 hours with the indicated antibodies (10 .mu.g/ml) in the presence or absence of recombinant Wnt3A (200 ng/ml; purchased from R&D Systems). Reporter activity was determined 18 hours post treatment, using a Veritas luminometer (Turner Biosystems, Sunnyvale, Calif.). As shown in FIG. 6 and summarized in Table 5, treatment with mAbs 77.2, 213.7, 240.8, 421.1, 498.3, 606.4, 856.6, 923.3, 931.1, 993.9, 995.5, 1115.3, 1213.2, 1253.12, 1281.1, 1293.11, 1433.8, 1470.2, 1903.1, 135.16, 413.1, 620.1, or 537.2 resulted in a 2-7 fold increase in Wnt3A dependent reporter activation, relative to cells treated with Wnt3A alone. LRP6 antibodies did not induce reporter activation in the absence of Wnt3A, indicating that LRP6 antibodies can not induce reporter activation by themselves (data not shown).

[0444] Canonical Wnt signaling can be inhibited by the soluble protein Dkk1, which prevents Wnt binding to LRP5/6 and causes Kremen-dependent internalization of LRP5/6 receptors (reviewed in He et al., supra, 2004). To determine whether LRP6 specific antibodies would affect Dkk1-dependent inhibition of LRP6 function, the effect of the antibodies on Dkk1-dependent inhibition of Wnt3a mediated reporter activation was determined in the TCF reporter assay, by treating Wnt3A stimulated reporter cells with Dkk1 (200 ng/ml, purchased from R&D Systems), in the presence or absence of LRP6 activating antibodies. As shown in FIG. 7 and summarized in Table 5, co-treatment of cells with Wnt3A and Dkk1 completely inhibited Wnt3A dependent reporter activation. However, in the presence of LRP6 mAbs 77.2, 213.7, 240.8, 421.1, 498.3, 606.4, 856.6, 923.3, 931.1, 1115.3, 1213.2, 1253.12, 1293.11, 1470.2, 1903.1, 135.16, 413.1 or 620.1, Wnt3A dependent reporter activation was restored to levels at or above those observed in cells treated with Wnt3A alone, indicating that these LRP6 mAb can inhibit Dkk1 function and can be used as Dkk1 antagonists.

[0445] To further characterize the activity of Wnt signaling activating anti-LRP6 antibodies, more detailed 16TCF luciferase reporter assays were carried out using the LRP6 activating antibody mAb135.16. 293 A6 reporter cells were treated with media only, recombinant Wnt3A, or recombinant Wnt3A plus recombinant Dkk1, in the presence or absence of a dose range of mAb135.16 or isotype control antibody. As shown in FIG. 8, co-treatment with mAb135.16 enhanced Wnt3A dependent reporter activation in a dose-dependent manner. Furthermore, Dkk1-dependent inhibition of Wnt3A dependent reporter activation was reversed by mAb 135.16 in a dose-dependent manner, to levels at or above observed with treatment with Wnt3A alone.

[0446] 293 A6 cells were then treated with a dose range of Wnt3A in the absence or presence of mAb135.16 or control antibody. As shown in FIG. 9, co-treatment with mAb135.16 not only enhanced the level of reporter activation in the presence of Wnt3A, but also made the cells more sensitive to Wnt3A treatment, as reporter activation could be observed at lower doses of Wnt3A. However, Fab fragments of mAb 135.16 were unable to enhance Wnt3A dependent 16TCF luciferase reporter activation. Finally, 293 A6 cells were treated with Wnt3A with or without Dkk1, in the presence or absence of Fab fragments of mAb135.16. As shown in FIG. 10, mAb 135.16 Fab fragments reversed inhibition of Wnt3A dependent reporter activity by Dkk1. These results imply that the enhancement of Wnt3A activity by activating LRP6 antibodies such as mAb 135.16, requires antibody-mediated dimerization of LRP6 through binding of bivalent antibody to the first domain of LRP6. In contrast, the ability of LRP6 antibodies to antagonize Dkk1 activity does not require antibody mediated dimerization of LRP6, suggesting that inhibition of Dkk1-dependent LRP6 functions by LRP6 activating antibodies such as mAb 135.16, may result from blocking an additional Dkk1 binding site in the first propeller domain of LRP6. In this context, it is interesting to note that the high bone mass mutations identified in LRP5 that render LRP5 insensitive to Dkk1, also reside in the first propeller domain of LRP5, suggesting that the first domains of LRP5/6 may regulate sensitivity of these receptors to the Dkk1 inhibitor.

Example 5

Effect of Anti-LRP6 mAbs on Dkk1-Dependent Internalization of LRP6

[0447] To assess the functional consequence of inhibiting Dkk1 binding to LRP6, Dkk1-dependent internalization of LRP6 was examined by immunofluoroescence microscopy as described in Binnerts et al (Proc. Natl. Acad. Sci. USA 104:14700-14705 (2007)). Briefly, HEK293 cells were co-transfected with HA-tagged LRP6 and wild-type Kremen1 proteins and analyzed using anti-HA antibodies to determine the location of the HA-tagged LRP6. LRP6 was mostly localized to the cell surface in untreated cells, whereas treatment with Dkk1 for 30 min caused LRP6 to internalize and localize into distinct intracellular punctae. In contrast, pre-treatment with anti-LRP6 mAb135 prevented Dkk1-dependent internalization of LRP6 and restored normal LRP6 cell surface levels (see FIG. 11). These results suggest that pre-incubation with anti-LRP6 mAbs result in functional inhibition of Dkk1/Kremen1-dependent internalization of LRP6.

Example 6

Models for Bone Diseases

[0448] Anti-LRP6 antibodies are tested for reduction of bone loss using a rat bone loss model as described in Kulkarni et al, J. Bone Miner. Res. 21:910-920 (2006). Briefly, 3- to 6-month old virgin Sprague-Dawley female rats are anesthetized by pentobarbital sodium and subjected to bilateral ovariectomy (OVX) or sham operation. Animals are caged in pairs and maintained on rodent chow and tap water ad libitum. OVX rats are permitted to lose bone for 1 month to establish osteopenia before the initiation of treatments. Anti-LRP6 antibodies are administered to the rats at 10 mg/kg three times a week intraperitoneally for 1-2 months. Animals are weighed every 2 weeks, and the dosing volumes are adjusted accordingly. One day after the last dose, the animals are sacrificed by CO.sub.2 inhalation. Femurs are removed and cleaned of soft tissue, and fixed in 10% formalin for 48 hours and stored at 4.degree. C. in 70% ethanol. Additionally, lumbar vertebrae L5 are removed and processed for biomechanical analyses.

[0449] Bone mineral density and geometric parameters of the harvested femurs are measured by peripheral quantitative computed tomography (pQCT). A two-dimensional scout view of the femur is obtained first and a distal growth plate of the femur is identified as a landmark. Measurements are performed at the metaphysic and mid-diaphysis of the femur, at 1.4 mm and 5.5 mm proximal to the growth plate, respectively. Analyses of the scans are performed using the manufacturer-supplied software.

[0450] Proximal tibias and tibial shaft are stained for 4 days in Villanueva osteochrome bone stain for osteoid staining (Polysciences, Warrington, Pa.) and dehydrated in a graded ethanol, defatted in acetone, and embedded in methyl methacrylate. Longitudinal sections of 210 .mu.m thickness are cut using a diamond wafering saw and further hand ground to 20 .mu.m sections of proximal tibia metaphysis (PTM) and 30 .mu.m of tibial shaft (TX). For PTM analyses, the measurements are performed on the entire marrow region within the cortical shell between 1 and 4 mm distal to the growth plate-metaphyseal junction using an Image Analysis System (Osteomeasure). Trabecular area, perimeter, single and double-labeling surfaces, eroded surface, osteoid surface, labeling and wall width are measured and trabecular number, thickness, separation, mineralizing surface, mineral appositional rate, bone formation rate/bone volume, surface reference and activation frequency are calculated. Osteoclast number is measured on the entire marrow region within the cortical shell between 0.67 and 2 mm under .times.20 magnification. The osteoclast number is normalized to trabecular bone surface. For analysis of cortical bone, TX, cross-sectional area, marrow area, eroded surface, single- and double-labeling surfaces, and labeling width are measured. These parameters are used to calculate the percent cortical bone are, marrow area, mineralizing surface, mineral appositional rate, and bone formation rate/surface reference as described in Parfitt et al., J. Bone Miner. Res. 2:595-610 (1987) and Ma et al, Bone 17:549-554 (1995).

[0451] Excised L5 vertebrae are used to evaluate the biomechanical properties of bones treated with anti-LRP6 antibodies. Mechanical properties of the L5 vertebrae are analyzed after the posterior processes are removed, and the ends of the centrum are made parallel using a diamond wafering saw. Veterbral specimens are loaded to failure in compression, using the materials testing device and analyzed using Test Works 4 software (MTS Corp., Minneapolis, Minn.). The compressive load is applied through a pivoting platen to correct for possible nonparallel alignment of the faces of the vertebral body. Specimens are tested in a saline solution at 37.degree. C. after equilibration. Parameters measured from the load-displacement curve include ultimate load (Fu), stiffness, and energy (area under the curve). The modulus of toughness is calculated by normalizing energy by the area.

Example 7

Murine Model for Wound Healing

[0452] Anti-LRP6 antibodies that cross-react with mouse LRP6 are tested for stimulating wound closure using a murine wound healing model (described in Fathke et al, BMC Cell Biology 7: (2006). All animal procedures are in accordance with the Institutional Animal Care and Use Committee. Briefly, either male C57Bl/6J mice (Jackson Labs), male TOPGAL mice (DasGupta and Fuchs, Development 126:4557-4568 (1999)) or male BATGAL mice (Maretto et al, Proc. Natl. Acad. Sci. USA 100:3299-3304 (2003)) between 8-12 weeks of age are used for the wounding experiments. Mice are anesthetized by intraperitoneal injection of a ketamine and xylazine mixture (15 mg/kg and 1 mg/kg, respectively, Phoenix Pharmaceuticals, Inc.). The dorsal hair is removed and skin is prepared for generation of a standardized 1.5 cm.sup.2 full thickness wound (including the panniculus carnosus muscle) on the midback. The wound is covered with a transparent semi-occlusive dressing (Tegaderm, 3M) to prevent dessication. Anti-LRP6 antibodies are administered to the mice via subcutaneous, intravenous or topical administration daily. On days 3, 7, 14, 21 and 30, wounds are excised and processed for histology and immunohistochemistry.

[0453] Wounds are digitally photographed at the time of generation (day 0) and again on days 3, 7, 14, 21 and 30, or until wound closure. Wound area is measured using NIH Image. Wound size is determined by using histologic sections cut at a right angle to the skin surface across the wound. Serial sections are observed, and the section at the center of the wound, with the largest wound diameter, is chosen to measure wound size. A grid is used to measure the size of the epidermal and mesenchymal (or dermal) component of each wound.

[0454] Wounds are excised, bisected along the cranial-caudal axis and either frozen in OCT (Tissue-Tek, Sakura) or placed in 10% formalin overnight. Frozen tissues are cut at 10 .mu.m sections, post fixed in 100% cold acetone, blocked for 1 hour with goat serum and then incubated with a PE-labeled anti-CD5 antibody (BD-Pharmigen, CA) for one hour. Tissues are counterstained for 5 min with DAPI (Molecular Probes, OR) to visualize nuclei. For tissues fixed in formalin, tissues are embedded, cut and stained with hematoxylin and eosin for further analysis.

Example 8

Murine Model for Osteolytic Lesions in Multiple Myeloma

[0455] Anti-LRP6 antibodies are tested for treatment of osteolytic lesions in multiple myeloma using a murine model such as the SCID-rab mouse model for human primary multiple myeloma (Yata and Yaccoby, Leukemia, 18:1891-1897 (2004)). Briefly, 4-week old rabbits are sacrificed and their femora and tibiae are cut into two pieces keeping the proximal and distal ends closed. The bone is inserted subcutaneously into 6- to 8-week old CB.17/Icr-SCID mice through a small (5 mm) incision. The incision is closed with sterile surgical staples and engraftment of the bones is allowed to take place for 6 to 8 weeks. For each experiment, 3 to 10.times.10.sup.6 unseparated human myeloma bone marrow cells containing more than 20% plasma cells in 100 .mu.l PBS are injected directly into the implanted rabbit bone. Mice are periodically bled from the tail vein and changes in levels of circulating human immunoglobulin (hlg) of the M-protein isotype is used as an indicator of multiple myeloma growth (determined by ELISA as described in Yaccoby et al, Blood 92:2908-2913 (1998) and Yaccoby and Epstein, Blood, 94:3576-3582 (1999), both of which are herein incorporated by reference in their entirety). When hlg levels reach 50 .mu.g/ml or higher, two mice injected with cells from the same patient are used for study. Mice are treated with the anti-LRP6 antibodies via subcutaneous injection at 100 .mu.g antibody in 100 .mu.l PBS into the surrounding area of the implanted bone. Mice receive treatment 5 days a week for 4 to 6 weeks.

[0456] Mice are anesthetized with ketamine plus xylazine. Radiographs taken with an AXR Minishot-100 beryllium source instrument (Associated X-Ray Imaging, Haverhill, Mass.) use a 10 second exposure at 40 kV. Changes in bone mineral density of the implanted bone and mouse femur are determined using a PIXImus DEXA (GE Medical Systems, LUNAR, Madison, Wis.).

[0457] For closer analysis of the bone structure, mice are sacrificed and the bones are fixed in 10% phosphate-buffered formalin for 24 hours. Rabbit and murine bones are further decalcified with 10% (w/v) EDTA, pH 7.0. The bones are embedded in paraffin for sectioning. Sections (5 .mu.m) are deparaffinized in xylene, rehydrated with ethanol, and rinsed in PBS, and then undergo antigen retrieval using microwave. After peroxidase quenching with 3% hydrogen peroxide for 10 min, sections are incubated with 5 .mu.g/ml mouse anti-bovine ostecalcin monoclonal antibody and mouse IgG control antibody (QED Bioscience, San Diego, Calif.) and the assay is completed with the use of the Dako immunoperoxidase kit (Dako, Carpinteria, Calif.). Sections are lightly counterstained with hematoxylin. According to the manufacturer, the osteocalcin antibody cross-reacts with human and rabbit tissues but not with mouse tissues. Tartrate-resistant acti phosphatase (TRAP) staining of deparaffinized bone sections are performed with an acid phosphatase kit (Sigma, St. Louis, Mo.). Osteocalcin-expressing osteoblasts and TRAP.sup.+ multinucleated osteoclasts in 4 nonoverlapping, millimeter-square areas are counted.

Sequence CWU 1

1

373110088DNAHomo sapiensCDS(143)..(4984) 1gtgccccttt ctttcttctc tcgctgggaa gctgggaagt atgagcgtgc agccctgccg 60ctgcggcggc cgccccggct cctcgcctcc cccacttctg gccacccctc gccggtgaga 120gaagagaacg cgagaaggga ag atg ggg gcc gtc ctg agg agc ctc ctg gcc 172 Met Gly Ala Val Leu Arg Ser Leu Leu Ala 1 5 10 tgc agc ttc tgt gtg ctc ctg aga gcg gcc cct ttg ttg ctt tat gca 220Cys Ser Phe Cys Val Leu Leu Arg Ala Ala Pro Leu Leu Leu Tyr Ala 15 20 25 aac aga cgg gac ttg cga ttg gtt gat gct aca aat ggc aaa gag aat 268Asn Arg Arg Asp Leu Arg Leu Val Asp Ala Thr Asn Gly Lys Glu Asn 30 35 40 gct acg att gta gtt gga ggc ttg gag gat gca gct gcg gtg gac ttt 316Ala Thr Ile Val Val Gly Gly Leu Glu Asp Ala Ala Ala Val Asp Phe 45 50 55 gtg ttt agt cat ggc ttg ata tac tgg agt gat gtc agc gaa gaa gcc 364Val Phe Ser His Gly Leu Ile Tyr Trp Ser Asp Val Ser Glu Glu Ala 60 65 70 att aaa cga aca gaa ttt aac aaa act gag agt gtg cag aat gtt gtt 412Ile Lys Arg Thr Glu Phe Asn Lys Thr Glu Ser Val Gln Asn Val Val 75 80 85 90 gtt tct gga tta ttg tcc ccc gat ggg ctg gca tgt gat tgg ctt gga 460Val Ser Gly Leu Leu Ser Pro Asp Gly Leu Ala Cys Asp Trp Leu Gly 95 100 105 gaa aaa ttg tac tgg aca gat tct gaa act aat cgg att gaa gtt tct 508Glu Lys Leu Tyr Trp Thr Asp Ser Glu Thr Asn Arg Ile Glu Val Ser 110 115 120 aat tta gat gga tct tta cga aaa gtt tta ttt tgg caa gag ttg gat 556Asn Leu Asp Gly Ser Leu Arg Lys Val Leu Phe Trp Gln Glu Leu Asp 125 130 135 caa ccc aga gct att gcc tta gat cct tca agt ggg ttc atg tac tgg 604Gln Pro Arg Ala Ile Ala Leu Asp Pro Ser Ser Gly Phe Met Tyr Trp 140 145 150 aca gac tgg gga gaa gtg cca aag ata gaa cgt gct gga atg gat ggt 652Thr Asp Trp Gly Glu Val Pro Lys Ile Glu Arg Ala Gly Met Asp Gly 155 160 165 170 tca agt cgc ttc att ata ata aac agt gaa att tac tgg cca aat gga 700Ser Ser Arg Phe Ile Ile Ile Asn Ser Glu Ile Tyr Trp Pro Asn Gly 175 180 185 ctg act ttg gat tat gaa gaa caa aag ctt tat tgg gca gat gca aaa 748Leu Thr Leu Asp Tyr Glu Glu Gln Lys Leu Tyr Trp Ala Asp Ala Lys 190 195 200 ctt aat ttc atc cac aaa tca aat ctg gat gga aca aat cgg cag gca 796Leu Asn Phe Ile His Lys Ser Asn Leu Asp Gly Thr Asn Arg Gln Ala 205 210 215 gtg gtt aaa ggt tcc ctt cca cat cct ttt gcc ttg acg tta ttt gag 844Val Val Lys Gly Ser Leu Pro His Pro Phe Ala Leu Thr Leu Phe Glu 220 225 230 gac ata ttg tac tgg act gac tgg agc aca cac tcc att ttg gct tgc 892Asp Ile Leu Tyr Trp Thr Asp Trp Ser Thr His Ser Ile Leu Ala Cys 235 240 245 250 aac aag tat act ggt gag ggt ctg cgt gaa atc cat tct gac atc ttc 940Asn Lys Tyr Thr Gly Glu Gly Leu Arg Glu Ile His Ser Asp Ile Phe 255 260 265 tct ccc atg gat ata cat gcc ttc agc caa cag agg cag cca aat gcc 988Ser Pro Met Asp Ile His Ala Phe Ser Gln Gln Arg Gln Pro Asn Ala 270 275 280 aca aat cca tgt gga att gac aat ggg ggt tgt tcc cat ttg tgt ttg 1036Thr Asn Pro Cys Gly Ile Asp Asn Gly Gly Cys Ser His Leu Cys Leu 285 290 295 atg tct cca gtc aag cct ttt tat cag tgt gct tgc ccc act ggg gtc 1084Met Ser Pro Val Lys Pro Phe Tyr Gln Cys Ala Cys Pro Thr Gly Val 300 305 310 aaa ctc ctg gag aat gga aaa acc tgc aaa gat ggt gcc aca gaa tta 1132Lys Leu Leu Glu Asn Gly Lys Thr Cys Lys Asp Gly Ala Thr Glu Leu 315 320 325 330 ttg ctt tta gct cga agg aca gac ttg aga cgc att tct ttg gat aca 1180Leu Leu Leu Ala Arg Arg Thr Asp Leu Arg Arg Ile Ser Leu Asp Thr 335 340 345 cca gat ttt aca gac att gtt ctg cag tta gaa gac atc cgt cat gcc 1228Pro Asp Phe Thr Asp Ile Val Leu Gln Leu Glu Asp Ile Arg His Ala 350 355 360 att gcc ata gat tac gat cct gtg gaa ggc tac atc tac tgg act gat 1276Ile Ala Ile Asp Tyr Asp Pro Val Glu Gly Tyr Ile Tyr Trp Thr Asp 365 370 375 gat gaa gtg agg gcc ata cgc cgt tca ttt ata gat gga tct ggc agt 1324Asp Glu Val Arg Ala Ile Arg Arg Ser Phe Ile Asp Gly Ser Gly Ser 380 385 390 cag ttt gtg gtc act gct caa att gcc cat cct gat ggt att gct gtg 1372Gln Phe Val Val Thr Ala Gln Ile Ala His Pro Asp Gly Ile Ala Val 395 400 405 410 gac tgg gtt gca cga aat ctt tat tgg aca gac act ggc act gat cga 1420Asp Trp Val Ala Arg Asn Leu Tyr Trp Thr Asp Thr Gly Thr Asp Arg 415 420 425 ata gaa gtg aca agg ctc aat ggg acc atg agg aag atc ttg att tca 1468Ile Glu Val Thr Arg Leu Asn Gly Thr Met Arg Lys Ile Leu Ile Ser 430 435 440 gag gac tta gag gaa ccc cgg gct att gtg tta gat ccc atg gtt ggg 1516Glu Asp Leu Glu Glu Pro Arg Ala Ile Val Leu Asp Pro Met Val Gly 445 450 455 tac atg tat tgg act gac tgg gga gaa att ccg aaa att gag cga gca 1564Tyr Met Tyr Trp Thr Asp Trp Gly Glu Ile Pro Lys Ile Glu Arg Ala 460 465 470 gct ctg gat ggt tct gac cgt gta gta ttg gtt aac act tct ctt ggt 1612Ala Leu Asp Gly Ser Asp Arg Val Val Leu Val Asn Thr Ser Leu Gly 475 480 485 490 tgg cca aat ggt tta gcc ttg gat tat gat gaa ggc aaa ata tac tgg 1660Trp Pro Asn Gly Leu Ala Leu Asp Tyr Asp Glu Gly Lys Ile Tyr Trp 495 500 505 gga gat gcc aaa aca gac aag att gag gtt atg aat act gat ggc act 1708Gly Asp Ala Lys Thr Asp Lys Ile Glu Val Met Asn Thr Asp Gly Thr 510 515 520 ggg aga cga gta cta gtg gaa gac aaa att cct cac ata ttt gga ttt 1756Gly Arg Arg Val Leu Val Glu Asp Lys Ile Pro His Ile Phe Gly Phe 525 530 535 act ttg ttg ggt gac tat gtt tac tgg act gac tgg cag agg cgt agc 1804Thr Leu Leu Gly Asp Tyr Val Tyr Trp Thr Asp Trp Gln Arg Arg Ser 540 545 550 att gaa aga gtt cat aaa cga agt gca gag agg gaa gtg atc ata gat 1852Ile Glu Arg Val His Lys Arg Ser Ala Glu Arg Glu Val Ile Ile Asp 555 560 565 570 cag ctg cct gac ctc atg ggc cta aag gct aca aat gtt cat cga gtg 1900Gln Leu Pro Asp Leu Met Gly Leu Lys Ala Thr Asn Val His Arg Val 575 580 585 att ggt tcc aac ccc tgt gct gag gaa aac ggg gga tgt agc cat ctc 1948Ile Gly Ser Asn Pro Cys Ala Glu Glu Asn Gly Gly Cys Ser His Leu 590 595 600 tgc ctc tat aga cct cag ggc ctt cgc tgt gct tgc cct att ggc ttt 1996Cys Leu Tyr Arg Pro Gln Gly Leu Arg Cys Ala Cys Pro Ile Gly Phe 605 610 615 gaa ctc atc agt gac atg aag acc tgc att gtc cca gag gct ttc ctt 2044Glu Leu Ile Ser Asp Met Lys Thr Cys Ile Val Pro Glu Ala Phe Leu 620 625 630 ttg ttt tca cgg aga gca gat atc aga cga att tct ctg gaa aca aac 2092Leu Phe Ser Arg Arg Ala Asp Ile Arg Arg Ile Ser Leu Glu Thr Asn 635 640 645 650 aat aat aat gtg gct att cca ctc act ggt gtc aaa gaa gct tct gct 2140Asn Asn Asn Val Ala Ile Pro Leu Thr Gly Val Lys Glu Ala Ser Ala 655 660 665 ttg gat ttt gat gtg aca gac aac cga att tat tgg act gat ata tca 2188Leu Asp Phe Asp Val Thr Asp Asn Arg Ile Tyr Trp Thr Asp Ile Ser 670 675 680 ctc aag acc atc agc aga gcc ttt atg aat ggc agt gca ctg gaa cat 2236Leu Lys Thr Ile Ser Arg Ala Phe Met Asn Gly Ser Ala Leu Glu His 685 690 695 gtg gta gaa ttc ggc tta gat tat cca gaa ggc atg gca gta gac tgg 2284Val Val Glu Phe Gly Leu Asp Tyr Pro Glu Gly Met Ala Val Asp Trp 700 705 710 ctt ggg aag aac ttg tac tgg gca gac aca gga acg aat cga att gag 2332Leu Gly Lys Asn Leu Tyr Trp Ala Asp Thr Gly Thr Asn Arg Ile Glu 715 720 725 730 gtg tca aag ttg gat ggg cag cac cga caa gtt ttg gtg tgg aaa gac 2380Val Ser Lys Leu Asp Gly Gln His Arg Gln Val Leu Val Trp Lys Asp 735 740 745 cta gat agt ccc aga gct ctc gcg ttg gac cct gcc gaa gga ttt atg 2428Leu Asp Ser Pro Arg Ala Leu Ala Leu Asp Pro Ala Glu Gly Phe Met 750 755 760 tat tgg act gaa tgg ggt gga aaa cct aag ata gac aga gct gca atg 2476Tyr Trp Thr Glu Trp Gly Gly Lys Pro Lys Ile Asp Arg Ala Ala Met 765 770 775 gat gga agt gaa cgt act acc tta gtt cca aat gtg ggg cgg gca aac 2524Asp Gly Ser Glu Arg Thr Thr Leu Val Pro Asn Val Gly Arg Ala Asn 780 785 790 ggc cta act att gat tat gct aaa agg agg ctt tat tgg aca gac ctg 2572Gly Leu Thr Ile Asp Tyr Ala Lys Arg Arg Leu Tyr Trp Thr Asp Leu 795 800 805 810 gac acc aac tta ata gaa tct tca aat atg ctt ggg ctc aac cgt gaa 2620Asp Thr Asn Leu Ile Glu Ser Ser Asn Met Leu Gly Leu Asn Arg Glu 815 820 825 gtt ata gca gat gac ttg cct cat cct ttt ggc tta act cag tac caa 2668Val Ile Ala Asp Asp Leu Pro His Pro Phe Gly Leu Thr Gln Tyr Gln 830 835 840 gat tat atc tac tgg acg gac tgg agc cga cgc agc att gag cgt gcc 2716Asp Tyr Ile Tyr Trp Thr Asp Trp Ser Arg Arg Ser Ile Glu Arg Ala 845 850 855 aac aaa acc agt ggc caa aac cgc acc atc att cag ggc cat ttg gat 2764Asn Lys Thr Ser Gly Gln Asn Arg Thr Ile Ile Gln Gly His Leu Asp 860 865 870 tat gtg atg gac atc ctc gtc ttt cac tca tct cga cag tca ggg tgg 2812Tyr Val Met Asp Ile Leu Val Phe His Ser Ser Arg Gln Ser Gly Trp 875 880 885 890 aat gaa tgt gct tcc agc aat ggg cac tgc tcc cac ctc tgc ttg gct 2860Asn Glu Cys Ala Ser Ser Asn Gly His Cys Ser His Leu Cys Leu Ala 895 900 905 gtg cca gtt ggg ggt ttt gtt tgt gga tgc cct gcc cac tac tct ctt 2908Val Pro Val Gly Gly Phe Val Cys Gly Cys Pro Ala His Tyr Ser Leu 910 915 920 aat gct gac aac agg act tgt agt gct cct acg act ttc ctg ctc ttc 2956Asn Ala Asp Asn Arg Thr Cys Ser Ala Pro Thr Thr Phe Leu Leu Phe 925 930 935 agt caa aag agt gcc atc aac cgc atg gtg att gat gaa caa cag agc 3004Ser Gln Lys Ser Ala Ile Asn Arg Met Val Ile Asp Glu Gln Gln Ser 940 945 950 ccc gac atc atc ctt ccc atc cac agc ctt cgg aat gtc cgg gcc att 3052Pro Asp Ile Ile Leu Pro Ile His Ser Leu Arg Asn Val Arg Ala Ile 955 960 965 970 gac tat gac cca ctg gac aag caa ctc tat tgg att gac tca cga caa 3100Asp Tyr Asp Pro Leu Asp Lys Gln Leu Tyr Trp Ile Asp Ser Arg Gln 975 980 985 aac atg atc cga aag gca caa gaa gat ggc agc cag ggc ttt act gtg 3148Asn Met Ile Arg Lys Ala Gln Glu Asp Gly Ser Gln Gly Phe Thr Val 990 995 1000 gtt gtg agc tca gtt ccg agt cag aac ctg gaa ata caa ccc tat 3193Val Val Ser Ser Val Pro Ser Gln Asn Leu Glu Ile Gln Pro Tyr 1005 1010 1015 gac ctc agc att gat att tac agc cgc tac atc tac tgg act tgt 3238Asp Leu Ser Ile Asp Ile Tyr Ser Arg Tyr Ile Tyr Trp Thr Cys 1020 1025 1030 gag gct acc aat gtc att aat gtg aca aga tta gat ggg aga tca 3283Glu Ala Thr Asn Val Ile Asn Val Thr Arg Leu Asp Gly Arg Ser 1035 1040 1045 gtt gga gtg gtg ctg aaa ggc gag cag gac aga cct cga gcc gtt 3328Val Gly Val Val Leu Lys Gly Glu Gln Asp Arg Pro Arg Ala Val 1050 1055 1060 gtg gta aac cca gag aaa ggg tat atg tat ttt acc aat ctt cag 3373Val Val Asn Pro Glu Lys Gly Tyr Met Tyr Phe Thr Asn Leu Gln 1065 1070 1075 gaa agg tct cct aaa att gaa cgg gct gct ttg gat ggg aca gaa 3418Glu Arg Ser Pro Lys Ile Glu Arg Ala Ala Leu Asp Gly Thr Glu 1080 1085 1090 cgg gag gtc ctc ttt ttc agt ggc tta agt aaa cca att gct tta 3463Arg Glu Val Leu Phe Phe Ser Gly Leu Ser Lys Pro Ile Ala Leu 1095 1100 1105 gcc ctt gat agc agg ctg ggc aag ctc ttt tgg gct gat tca gat 3508Ala Leu Asp Ser Arg Leu Gly Lys Leu Phe Trp Ala Asp Ser Asp 1110 1115 1120 ctc cgg cga att gaa agc agt gat ctc tca ggt gct aac cgg ata 3553Leu Arg Arg Ile Glu Ser Ser Asp Leu Ser Gly Ala Asn Arg Ile 1125 1130 1135 gta tta gaa gac tcc aat atc ttg cag cct gtg gga ctt act gtg 3598Val Leu Glu Asp Ser Asn Ile Leu Gln Pro Val Gly Leu Thr Val 1140 1145 1150 ttt gaa aac tgg ctc tat tgg att gat aaa cag cag caa atg att 3643Phe Glu Asn Trp Leu Tyr Trp Ile Asp Lys Gln Gln Gln Met Ile 1155 1160 1165 gaa aaa att gac atg aca ggt cga gag ggt aga acc aaa gtc caa 3688Glu Lys Ile Asp Met Thr Gly Arg Glu Gly Arg Thr Lys Val Gln 1170 1175 1180 gct cga att gcc cag ctt agt gac att cat gca gta aag gag ctg 3733Ala Arg Ile Ala Gln Leu Ser Asp Ile His Ala Val Lys Glu Leu 1185 1190 1195 aac ctt caa gaa tac aga cag cac cct tgt gct cag gat aat ggt 3778Asn Leu Gln Glu Tyr Arg Gln His Pro Cys Ala Gln Asp Asn Gly 1200 1205 1210 ggc tgt tca cat att tgt ctt gta aag ggg gat ggt act aca agg 3823Gly Cys Ser His Ile Cys Leu Val Lys Gly Asp Gly Thr Thr Arg 1215 1220 1225 tgt tct tgc ccc atg cac ctg gtt cta ctt caa gat gag cta tca 3868Cys Ser Cys Pro Met His Leu Val Leu Leu Gln Asp Glu Leu Ser 1230 1235 1240 tgt gga gaa cct cca aca tgt tct cct cag cag ttt act tgt ttc 3913Cys Gly Glu Pro Pro Thr Cys Ser Pro Gln Gln Phe Thr Cys Phe 1245 1250 1255 acg ggg gaa att gac tgt atc cct gtg gct tgg cgg tgc gat ggg 3958Thr Gly Glu Ile Asp Cys Ile Pro Val Ala Trp Arg Cys Asp Gly 1260 1265 1270 ttt act gaa tgt gaa gac cac agt gat gaa ctc aat tgt cct gta 4003Phe Thr Glu Cys Glu Asp His Ser Asp Glu Leu Asn Cys Pro Val 1275 1280 1285 tgc tca gag tcc cag ttc cag tgt gcc agt ggg cag tgt att gat 4048Cys Ser Glu Ser Gln Phe Gln Cys Ala Ser Gly Gln Cys Ile Asp 1290 1295 1300

ggt gcc ctc cga tgc aat gga gat gca aac tgc cag gac aaa tca 4093Gly Ala Leu Arg Cys Asn Gly Asp Ala Asn Cys Gln Asp Lys Ser 1305 1310 1315 gat gag aag aac tgt gaa gtg ctt tgt tta att gat cag ttc cgc 4138Asp Glu Lys Asn Cys Glu Val Leu Cys Leu Ile Asp Gln Phe Arg 1320 1325 1330 tgt gcc aat ggt cag tgc att gga aag cac aag aag tgt gat cat 4183Cys Ala Asn Gly Gln Cys Ile Gly Lys His Lys Lys Cys Asp His 1335 1340 1345 aat gtg gat tgc agt gac aag tca gat gaa ctg gat tgt tat ccg 4228Asn Val Asp Cys Ser Asp Lys Ser Asp Glu Leu Asp Cys Tyr Pro 1350 1355 1360 act gaa gaa cca gca cca cag gcc acc aat aca gtt ggt tct gtt 4273Thr Glu Glu Pro Ala Pro Gln Ala Thr Asn Thr Val Gly Ser Val 1365 1370 1375 att ggc gta att gtc acc att ttt gtg tct gga act gta tac ttt 4318Ile Gly Val Ile Val Thr Ile Phe Val Ser Gly Thr Val Tyr Phe 1380 1385 1390 atc tgc cag agg atg ttg tgt cca cgt atg aag gga gat ggg gaa 4363Ile Cys Gln Arg Met Leu Cys Pro Arg Met Lys Gly Asp Gly Glu 1395 1400 1405 act atg act aat gac tat gta gtt cat gga cca gct tct gtg cct 4408Thr Met Thr Asn Asp Tyr Val Val His Gly Pro Ala Ser Val Pro 1410 1415 1420 ctt ggt tat gtg cca cac cca agt tct ttg tca gga tct ctt cca 4453Leu Gly Tyr Val Pro His Pro Ser Ser Leu Ser Gly Ser Leu Pro 1425 1430 1435 gga atg tct cga ggt aaa tca atg atc agc tcc ctc agt atc atg 4498Gly Met Ser Arg Gly Lys Ser Met Ile Ser Ser Leu Ser Ile Met 1440 1445 1450 ggg gga agc agt gga ccc ccc tat gac cga gcc cat gtt aca gga 4543Gly Gly Ser Ser Gly Pro Pro Tyr Asp Arg Ala His Val Thr Gly 1455 1460 1465 gca tca tca agt agt tct tca agc acc aaa ggc act tac ttc cct 4588Ala Ser Ser Ser Ser Ser Ser Ser Thr Lys Gly Thr Tyr Phe Pro 1470 1475 1480 gca att ttg aac cct cca cca tcc cca gcc aca gag cga tca cat 4633Ala Ile Leu Asn Pro Pro Pro Ser Pro Ala Thr Glu Arg Ser His 1485 1490 1495 tac act atg gaa ttt gga tat tct tca aac agt cct tcc act cat 4678Tyr Thr Met Glu Phe Gly Tyr Ser Ser Asn Ser Pro Ser Thr His 1500 1505 1510 agg tca tac agc tac agg cca tat agc tac cgg cac ttt gca ccc 4723Arg Ser Tyr Ser Tyr Arg Pro Tyr Ser Tyr Arg His Phe Ala Pro 1515 1520 1525 ccc acc aca ccc tgc agc aca gat gtt tgt gac agt gac tat gct 4768Pro Thr Thr Pro Cys Ser Thr Asp Val Cys Asp Ser Asp Tyr Ala 1530 1535 1540 cct agt cgg aga atg acc tca gtg gca aca gcc aag ggc tat acc 4813Pro Ser Arg Arg Met Thr Ser Val Ala Thr Ala Lys Gly Tyr Thr 1545 1550 1555 agt gac ttg aac tat gat tca gaa cct gtg ccc cca cct ccc aca 4858Ser Asp Leu Asn Tyr Asp Ser Glu Pro Val Pro Pro Pro Pro Thr 1560 1565 1570 ccc cga agc caa tac ttg tca gca gag gag aac tat gaa agc tgc 4903Pro Arg Ser Gln Tyr Leu Ser Ala Glu Glu Asn Tyr Glu Ser Cys 1575 1580 1585 cca cct tct cca tac aca gag agg agc tat tct cat cac ctc tac 4948Pro Pro Ser Pro Tyr Thr Glu Arg Ser Tyr Ser His His Leu Tyr 1590 1595 1600 cca ccg cca ccc tct ccc tgt aca gac tcc tcc tga ggaggggccc 4994Pro Pro Pro Pro Ser Pro Cys Thr Asp Ser Ser 1605 1610 tcctcctctg actgcctcca acgtaaaaat gtaaatataa atttggttga gatctggagg 5054gggggaggga gctattagag aaggatgagg cagaccatgt acagttaaaa ttataaaatg 5114gggtagggaa tactggagat atttgtacag aagaaaagga tatttatata ttttcttaaa 5174acagcagatt tgctgcttgt gccataaaag tttgtataaa aaaaatttgt actaaaagtt 5234ttatttttgc aaactaaata cacaaagcat gccttaaacc cagtgaagca actgagtaca 5294aaggaaacag gaataataaa ggcatcactg accaggaata tctgggcttt attgatacca 5354aaaataaaaa agaggaagaa gaaaaattaa gtccatctca gagcagcaaa ccatagatac 5414atggatgtag ccagatagcc ttcagttaac taacatttga gggccaacaa gtaagaaatg 5474atgaaaggaa aaaaatgcaa ttaatactaa ccttggacga agggctttgt tttctctagg 5534aatccaacag tgctagtgag gaaagtagat atttctaaaa acccattctg ggtgttgctg 5594ttgtaggaga gatcagccct ctggtaagat gccatgaagc tgtgtgtgtg tgcaagtctc 5654tgtccctacc tttagaatcc atacctctgt caaaatgaat ttttttctct aggtatgttt 5714accttgctgc ctcctccagc aacttggtaa gtcattttgc taagatacca tgattttttt 5774aagctgaagc attgactaaa tggaattttc taaattaaac ttgattttaa tatttcttct 5834agctccattc cccagtaggc ttagctcttc aatttgactg ctgtttttgc ataatgatca 5894aaagttagac atattatttc tcttcttcca agattgtttt aatgctcatt aaaatgtctt 5954tttacaacac atatagacaa tgtttaagaa ttaaaaattt aaccattatg tttttgttgt 6014aaatctcata tccttgcact actttcagca tatatcacag tacgaaatca tttatatata 6074tatatatata tatatatata tatatatata tatatatata tatattttgt ttgtttgttt 6134gttttctgag taaaacattt aaatatgttc tggttagaga caatctattt aaaaagattt 6194ttttcttatt aggattttcc ctatattaac agtttgtgat gttttcatgt tctttagacc 6254ggtttttctc agaataatgt ctacatacat acctcttcta atgtgtgaca tgaatttaat 6314atctttctgt tacccactgt gaatgttagg ctgttttcaa attatccaca aattattctt 6374gtaatcaccc aatattttta tgtgggtcct ctcttaccca ttatggatta agatagttta 6434acaaatttaa caatgaggat taaatgagaa ggcaaactgt taacttctca gctgtcagaa 6494tttgggtgga agggaataat ggaagcctct tttgtgatct gcctgacctg ctgtcatgta 6554tggtactggg gctgctacat cttgagctat cagggctgac ctgtggaatg attctagcac 6614ttgctctgcc accttgccag aagttcgttt cctgcttttt acacatgtgt agcacttctc 6674tgctaaaatt gaatggtttt aaactaatgt atttttagct taagaggtgt tggtcagtta 6734attattgaat tttttttttt tcttttttaa ttctgtcttg ccaaggcctc tctgggtttc 6794agggcccaag agaaaacagt ggaagaaagg attcagaatt tgggcaaggg tgaagtaact 6854gttcatgcaa gttaaaaata cctaagtaaa gtttttgaag ataaaattgt ggtttcagaa 6914taatgctgat tgttggagac tgtaagaatc aggtgcactt gattttgcat ataagcaaat 6974ggtaaatcta tcagaatcct aaaacagaca agcatgaact cttcccattg ctggaactaa 7034gtgcccacag tgtcagacaa aatggacatt gaacttggat tctgtgatac acagggcact 7094tgatgcttaa atgaagatgg aaaggttagc aatacctggg tgtcagttag aatttgagaa 7154ttctatatgt ttacatattt aaatgtgcat cttgatctgg tgggcttccc atgtggagac 7214ttgcactcta attaactaag aagaatattg ccttgttgga tctcagtcca cgtgcttgca 7274ctgcgatggc aatggcctct tcttcaaaat actaatttgt gtgccaattt gtttaaaatt 7334atttgaaggc agttcagcct aatctcagtg ttctctttct ggggtagatg agatggattc 7394ttaatatttc tgggagtact ttttaatgag agaattgtca aatttggaaa gatttattga 7454gccttaggtt acatggacag ttaagcttaa gtaaactgta tattgattat caaacacaag 7514ctgtaattgg aaaagttgag aggaaaagca tgagatcaca aattaggggg aaaaaagaaa 7574agggattttt aaatttggtg tattaaattc attgtccaag ggggaaaatg aataatgttt 7634cattagattc cttatatgca aaagtattta ttttgaacat gtgtcctaaa atatatgcac 7694taactgatgt gattaaaatt gtccaagaaa taaacttgag cataacatac tttgtgtgca 7754ccacagtaag ctattctgca ttgaagtggt cttttataac taaggcctgg actttgctcc 7814aacagagtcg tggtcttctg aatagtgact taaggagttt tgtttgctta agtcagataa 7874tagcacattc acagggaaac aaagagagtt ggtggataga attttctgac tattaatttt 7934tcttccatga aattttatta tgcctttggc actttctgcc actcttacag catatcacaa 7994gatatctgtt tagcagaaga ttatgtagtt actttaattt taatataaaa gtagcttgtg 8054atacattacc aagagatctc tgattcttta gtaagtttga gaacacctat tctacagaga 8114tgataggtac ttagaaatga agactttaaa gtacatttta atctaatata ggccagtaat 8174tgggggaagg ggctttgagc agtacaattt taagatgatt ttgagggttg tatttcttta 8234tcatttaaaa atatcctaaa gtcagtaatt tatatgaagg aaactcattc attattgaag 8294gtattaaaaa tagccatcat ctgtattagg tagcagtttt ggaggatcat ctttttcttt 8354tgctataaag ccctattaat gaagaatact tccagtagag ttaatagctg tagcttacct 8414agtgtgttaa tgaagtgtgt ttatttatgt gacttgatac cagtagtcat aatagagact 8474gaagaggtat gcgttaagca cgcctacttc tatgcagtaa acaggctgca gctgcctaga 8534ttagattctt agaaatgtca tattttgaat tgttttattt cttgtagggg aagctttgtc 8594ccacttcatt catttgcatg ccataggaat tacatattgg ttatcattac gtatctaaca 8654agattcagaa acaaaaatct tggacttttc acatccgaaa tatgtcagct cttaataaat 8714gtgtggtgct taagtctaca tatggcatcc atagttgatt tagagtatgg atatgagtgt 8774gttgaccagt tatcagtagg tggacaaata tttgggcatc tacagatgag actatgcact 8834aagtgtggac tgagtcctaa agaagcttat agtcaggtgt tgtttaaaac attatcagaa 8894ttcttaaacc caaggaattt aattttattt ggtatttctt aagcctaaaa tgaaccaaga 8954gaaagatgat tttagaaagt acttgtagtg aaagatgatt ttagaaagta cttgtagtgc 9014atgtgtggct tctgactttt gggatggcac cattttataa tagtttcaaa atttagcttt 9074tgaaattctc aacattttat ggtagaagac tttggacctc aagtataaaa ttatacgttt 9134ataatttttt taaaatttaa attataagta ttgtgaattc acactctcag gctattgtct 9194gacttgatct acgtctcata aagcctgtac ctgagtggag tggaaggtgg agtcttaggt 9254taatcagtta ctgactctac cctcaccctc tttcaattga ggtaaacttt gctgtttttc 9314tttttcataa agcattctca aattgttgag tttattgctg aaaaaaatct ccatgacttt 9374acagatagaa ttacaaacta aatgatgtct tgtatttaga agcagagtac agacctaacg 9434aactgttaga ttctccacca tcacttaggg tttgcccaga agcaacacca gagaattaca 9494gacaacgcgc ttttgctgaa ctgtccattt tggtggttgt gtttttcagt caaatataag 9554caggatgggc gatagagata tatttatata tagatacata ttctatatat ctaatgccta 9614aatatgggta ttaaagggaa aatttttaaa gtctgattaa atccaatatg acatgaaatt 9674aaatatatgg attagtaagg aaaaatgtta aaaagtagag aggataccaa gaagattaaa 9734ctggactagc cttatttgca agtgaaggat ctggtgctgc tttcagatgt ttatctttta 9794tttttttccc ttaagcttta atcttcgtca ttgtcttaaa gtcaactggt gtttcttgtt 9854cattgacttt ggtacgatgg tgctttgcaa ggatgtattt atgttataat ggccaacatt 9914tggtcagccc ttgtccactt attcacttcc ctccttttgt aaaataagtg ctttaattat 9974aaactgtata aaaatacctt gtataaaccc cttttttgat tattacaata aataagctga 10034attgtaacaa atgaaatttg atttttgtaa taaaacagtg gaaaagtaaa aaaa 1008821613PRTHomo sapiens 2Met Gly Ala Val Leu Arg Ser Leu Leu Ala Cys Ser Phe Cys Val Leu 1 5 10 15 Leu Arg Ala Ala Pro Leu Leu Leu Tyr Ala Asn Arg Arg Asp Leu Arg 20 25 30 Leu Val Asp Ala Thr Asn Gly Lys Glu Asn Ala Thr Ile Val Val Gly 35 40 45 Gly Leu Glu Asp Ala Ala Ala Val Asp Phe Val Phe Ser His Gly Leu 50 55 60 Ile Tyr Trp Ser Asp Val Ser Glu Glu Ala Ile Lys Arg Thr Glu Phe 65 70 75 80 Asn Lys Thr Glu Ser Val Gln Asn Val Val Val Ser Gly Leu Leu Ser 85 90 95 Pro Asp Gly Leu Ala Cys Asp Trp Leu Gly Glu Lys Leu Tyr Trp Thr 100 105 110 Asp Ser Glu Thr Asn Arg Ile Glu Val Ser Asn Leu Asp Gly Ser Leu 115 120 125 Arg Lys Val Leu Phe Trp Gln Glu Leu Asp Gln Pro Arg Ala Ile Ala 130 135 140 Leu Asp Pro Ser Ser Gly Phe Met Tyr Trp Thr Asp Trp Gly Glu Val 145 150 155 160 Pro Lys Ile Glu Arg Ala Gly Met Asp Gly Ser Ser Arg Phe Ile Ile 165 170 175 Ile Asn Ser Glu Ile Tyr Trp Pro Asn Gly Leu Thr Leu Asp Tyr Glu 180 185 190 Glu Gln Lys Leu Tyr Trp Ala Asp Ala Lys Leu Asn Phe Ile His Lys 195 200 205 Ser Asn Leu Asp Gly Thr Asn Arg Gln Ala Val Val Lys Gly Ser Leu 210 215 220 Pro His Pro Phe Ala Leu Thr Leu Phe Glu Asp Ile Leu Tyr Trp Thr 225 230 235 240 Asp Trp Ser Thr His Ser Ile Leu Ala Cys Asn Lys Tyr Thr Gly Glu 245 250 255 Gly Leu Arg Glu Ile His Ser Asp Ile Phe Ser Pro Met Asp Ile His 260 265 270 Ala Phe Ser Gln Gln Arg Gln Pro Asn Ala Thr Asn Pro Cys Gly Ile 275 280 285 Asp Asn Gly Gly Cys Ser His Leu Cys Leu Met Ser Pro Val Lys Pro 290 295 300 Phe Tyr Gln Cys Ala Cys Pro Thr Gly Val Lys Leu Leu Glu Asn Gly 305 310 315 320 Lys Thr Cys Lys Asp Gly Ala Thr Glu Leu Leu Leu Leu Ala Arg Arg 325 330 335 Thr Asp Leu Arg Arg Ile Ser Leu Asp Thr Pro Asp Phe Thr Asp Ile 340 345 350 Val Leu Gln Leu Glu Asp Ile Arg His Ala Ile Ala Ile Asp Tyr Asp 355 360 365 Pro Val Glu Gly Tyr Ile Tyr Trp Thr Asp Asp Glu Val Arg Ala Ile 370 375 380 Arg Arg Ser Phe Ile Asp Gly Ser Gly Ser Gln Phe Val Val Thr Ala 385 390 395 400 Gln Ile Ala His Pro Asp Gly Ile Ala Val Asp Trp Val Ala Arg Asn 405 410 415 Leu Tyr Trp Thr Asp Thr Gly Thr Asp Arg Ile Glu Val Thr Arg Leu 420 425 430 Asn Gly Thr Met Arg Lys Ile Leu Ile Ser Glu Asp Leu Glu Glu Pro 435 440 445 Arg Ala Ile Val Leu Asp Pro Met Val Gly Tyr Met Tyr Trp Thr Asp 450 455 460 Trp Gly Glu Ile Pro Lys Ile Glu Arg Ala Ala Leu Asp Gly Ser Asp 465 470 475 480 Arg Val Val Leu Val Asn Thr Ser Leu Gly Trp Pro Asn Gly Leu Ala 485 490 495 Leu Asp Tyr Asp Glu Gly Lys Ile Tyr Trp Gly Asp Ala Lys Thr Asp 500 505 510 Lys Ile Glu Val Met Asn Thr Asp Gly Thr Gly Arg Arg Val Leu Val 515 520 525 Glu Asp Lys Ile Pro His Ile Phe Gly Phe Thr Leu Leu Gly Asp Tyr 530 535 540 Val Tyr Trp Thr Asp Trp Gln Arg Arg Ser Ile Glu Arg Val His Lys 545 550 555 560 Arg Ser Ala Glu Arg Glu Val Ile Ile Asp Gln Leu Pro Asp Leu Met 565 570 575 Gly Leu Lys Ala Thr Asn Val His Arg Val Ile Gly Ser Asn Pro Cys 580 585 590 Ala Glu Glu Asn Gly Gly Cys Ser His Leu Cys Leu Tyr Arg Pro Gln 595 600 605 Gly Leu Arg Cys Ala Cys Pro Ile Gly Phe Glu Leu Ile Ser Asp Met 610 615 620 Lys Thr Cys Ile Val Pro Glu Ala Phe Leu Leu Phe Ser Arg Arg Ala 625 630 635 640 Asp Ile Arg Arg Ile Ser Leu Glu Thr Asn Asn Asn Asn Val Ala Ile 645 650 655 Pro Leu Thr Gly Val Lys Glu Ala Ser Ala Leu Asp Phe Asp Val Thr 660 665 670 Asp Asn Arg Ile Tyr Trp Thr Asp Ile Ser Leu Lys Thr Ile Ser Arg 675 680 685 Ala Phe Met Asn Gly Ser Ala Leu Glu His Val Val Glu Phe Gly Leu 690 695 700 Asp Tyr Pro Glu Gly Met Ala Val Asp Trp Leu Gly Lys Asn Leu Tyr 705 710 715 720 Trp Ala Asp Thr Gly Thr Asn Arg Ile Glu Val Ser Lys Leu Asp Gly 725 730 735 Gln His Arg Gln Val Leu Val Trp Lys Asp Leu Asp Ser Pro Arg Ala 740 745 750 Leu Ala Leu Asp Pro Ala Glu Gly Phe Met Tyr Trp Thr Glu Trp Gly 755 760 765 Gly Lys Pro Lys Ile Asp Arg Ala Ala Met Asp Gly Ser Glu Arg Thr 770 775 780 Thr Leu Val Pro Asn Val Gly Arg Ala Asn Gly Leu Thr Ile Asp Tyr 785 790 795 800 Ala Lys Arg Arg Leu Tyr Trp Thr Asp Leu Asp Thr Asn Leu Ile Glu 805 810 815 Ser Ser Asn Met Leu Gly Leu Asn Arg Glu Val Ile Ala Asp Asp Leu 820 825 830 Pro His Pro Phe Gly Leu Thr Gln Tyr Gln Asp Tyr Ile Tyr Trp Thr 835 840 845 Asp Trp Ser Arg Arg Ser Ile Glu Arg Ala Asn Lys Thr Ser Gly Gln 850 855 860 Asn Arg Thr Ile Ile Gln Gly His Leu Asp Tyr Val Met Asp Ile Leu 865 870 875 880 Val Phe His Ser Ser Arg Gln Ser Gly Trp Asn Glu Cys Ala Ser Ser 885 890 895 Asn Gly His Cys Ser His Leu Cys Leu Ala Val Pro Val Gly Gly Phe 900 905 910 Val Cys Gly Cys Pro Ala His Tyr Ser Leu Asn Ala Asp Asn Arg Thr 915 920 925 Cys Ser Ala Pro Thr Thr Phe Leu Leu Phe Ser Gln Lys Ser Ala Ile 930 935 940

Asn Arg Met Val Ile Asp Glu Gln Gln Ser Pro Asp Ile Ile Leu Pro 945 950 955 960 Ile His Ser Leu Arg Asn Val Arg Ala Ile Asp Tyr Asp Pro Leu Asp 965 970 975 Lys Gln Leu Tyr Trp Ile Asp Ser Arg Gln Asn Met Ile Arg Lys Ala 980 985 990 Gln Glu Asp Gly Ser Gln Gly Phe Thr Val Val Val Ser Ser Val Pro 995 1000 1005 Ser Gln Asn Leu Glu Ile Gln Pro Tyr Asp Leu Ser Ile Asp Ile 1010 1015 1020 Tyr Ser Arg Tyr Ile Tyr Trp Thr Cys Glu Ala Thr Asn Val Ile 1025 1030 1035 Asn Val Thr Arg Leu Asp Gly Arg Ser Val Gly Val Val Leu Lys 1040 1045 1050 Gly Glu Gln Asp Arg Pro Arg Ala Val Val Val Asn Pro Glu Lys 1055 1060 1065 Gly Tyr Met Tyr Phe Thr Asn Leu Gln Glu Arg Ser Pro Lys Ile 1070 1075 1080 Glu Arg Ala Ala Leu Asp Gly Thr Glu Arg Glu Val Leu Phe Phe 1085 1090 1095 Ser Gly Leu Ser Lys Pro Ile Ala Leu Ala Leu Asp Ser Arg Leu 1100 1105 1110 Gly Lys Leu Phe Trp Ala Asp Ser Asp Leu Arg Arg Ile Glu Ser 1115 1120 1125 Ser Asp Leu Ser Gly Ala Asn Arg Ile Val Leu Glu Asp Ser Asn 1130 1135 1140 Ile Leu Gln Pro Val Gly Leu Thr Val Phe Glu Asn Trp Leu Tyr 1145 1150 1155 Trp Ile Asp Lys Gln Gln Gln Met Ile Glu Lys Ile Asp Met Thr 1160 1165 1170 Gly Arg Glu Gly Arg Thr Lys Val Gln Ala Arg Ile Ala Gln Leu 1175 1180 1185 Ser Asp Ile His Ala Val Lys Glu Leu Asn Leu Gln Glu Tyr Arg 1190 1195 1200 Gln His Pro Cys Ala Gln Asp Asn Gly Gly Cys Ser His Ile Cys 1205 1210 1215 Leu Val Lys Gly Asp Gly Thr Thr Arg Cys Ser Cys Pro Met His 1220 1225 1230 Leu Val Leu Leu Gln Asp Glu Leu Ser Cys Gly Glu Pro Pro Thr 1235 1240 1245 Cys Ser Pro Gln Gln Phe Thr Cys Phe Thr Gly Glu Ile Asp Cys 1250 1255 1260 Ile Pro Val Ala Trp Arg Cys Asp Gly Phe Thr Glu Cys Glu Asp 1265 1270 1275 His Ser Asp Glu Leu Asn Cys Pro Val Cys Ser Glu Ser Gln Phe 1280 1285 1290 Gln Cys Ala Ser Gly Gln Cys Ile Asp Gly Ala Leu Arg Cys Asn 1295 1300 1305 Gly Asp Ala Asn Cys Gln Asp Lys Ser Asp Glu Lys Asn Cys Glu 1310 1315 1320 Val Leu Cys Leu Ile Asp Gln Phe Arg Cys Ala Asn Gly Gln Cys 1325 1330 1335 Ile Gly Lys His Lys Lys Cys Asp His Asn Val Asp Cys Ser Asp 1340 1345 1350 Lys Ser Asp Glu Leu Asp Cys Tyr Pro Thr Glu Glu Pro Ala Pro 1355 1360 1365 Gln Ala Thr Asn Thr Val Gly Ser Val Ile Gly Val Ile Val Thr 1370 1375 1380 Ile Phe Val Ser Gly Thr Val Tyr Phe Ile Cys Gln Arg Met Leu 1385 1390 1395 Cys Pro Arg Met Lys Gly Asp Gly Glu Thr Met Thr Asn Asp Tyr 1400 1405 1410 Val Val His Gly Pro Ala Ser Val Pro Leu Gly Tyr Val Pro His 1415 1420 1425 Pro Ser Ser Leu Ser Gly Ser Leu Pro Gly Met Ser Arg Gly Lys 1430 1435 1440 Ser Met Ile Ser Ser Leu Ser Ile Met Gly Gly Ser Ser Gly Pro 1445 1450 1455 Pro Tyr Asp Arg Ala His Val Thr Gly Ala Ser Ser Ser Ser Ser 1460 1465 1470 Ser Ser Thr Lys Gly Thr Tyr Phe Pro Ala Ile Leu Asn Pro Pro 1475 1480 1485 Pro Ser Pro Ala Thr Glu Arg Ser His Tyr Thr Met Glu Phe Gly 1490 1495 1500 Tyr Ser Ser Asn Ser Pro Ser Thr His Arg Ser Tyr Ser Tyr Arg 1505 1510 1515 Pro Tyr Ser Tyr Arg His Phe Ala Pro Pro Thr Thr Pro Cys Ser 1520 1525 1530 Thr Asp Val Cys Asp Ser Asp Tyr Ala Pro Ser Arg Arg Met Thr 1535 1540 1545 Ser Val Ala Thr Ala Lys Gly Tyr Thr Ser Asp Leu Asn Tyr Asp 1550 1555 1560 Ser Glu Pro Val Pro Pro Pro Pro Thr Pro Arg Ser Gln Tyr Leu 1565 1570 1575 Ser Ala Glu Glu Asn Tyr Glu Ser Cys Pro Pro Ser Pro Tyr Thr 1580 1585 1590 Glu Arg Ser Tyr Ser His His Leu Tyr Pro Pro Pro Pro Ser Pro 1595 1600 1605 Cys Thr Asp Ser Ser 1610 31354PRTHomo sapiens 3Ala Pro Leu Leu Leu Tyr Ala Asn Arg Arg Asp Leu Arg Leu Val Asp 1 5 10 15 Ala Thr Asn Gly Lys Glu Asn Ala Thr Ile Val Val Gly Gly Leu Glu 20 25 30 Asp Ala Ala Ala Val Asp Phe Val Phe Ser His Gly Leu Ile Tyr Trp 35 40 45 Ser Asp Val Ser Glu Glu Ala Ile Lys Arg Thr Glu Phe Asn Lys Thr 50 55 60 Glu Ser Val Gln Asn Val Val Val Ser Gly Leu Leu Ser Pro Asp Gly 65 70 75 80 Leu Ala Cys Asp Trp Leu Gly Glu Lys Leu Tyr Trp Thr Asp Ser Glu 85 90 95 Thr Asn Arg Ile Glu Val Ser Asn Leu Asp Gly Ser Leu Arg Lys Val 100 105 110 Leu Phe Trp Gln Glu Leu Asp Gln Pro Arg Ala Ile Ala Leu Asp Pro 115 120 125 Ser Ser Gly Phe Met Tyr Trp Thr Asp Trp Gly Glu Val Pro Lys Ile 130 135 140 Glu Arg Ala Gly Met Asp Gly Ser Ser Arg Phe Ile Ile Ile Asn Ser 145 150 155 160 Glu Ile Tyr Trp Pro Asn Gly Leu Thr Leu Asp Tyr Glu Glu Gln Lys 165 170 175 Leu Tyr Trp Ala Asp Ala Lys Leu Asn Phe Ile His Lys Ser Asn Leu 180 185 190 Asp Gly Thr Asn Arg Gln Ala Val Val Lys Gly Ser Leu Pro His Pro 195 200 205 Phe Ala Leu Thr Leu Phe Glu Asp Ile Leu Tyr Trp Thr Asp Trp Ser 210 215 220 Thr His Ser Ile Leu Ala Cys Asn Lys Tyr Thr Gly Glu Gly Leu Arg 225 230 235 240 Glu Ile His Ser Asp Ile Phe Ser Pro Met Asp Ile His Ala Phe Ser 245 250 255 Gln Gln Arg Gln Pro Asn Ala Thr Asn Pro Cys Gly Ile Asp Asn Gly 260 265 270 Gly Cys Ser His Leu Cys Leu Met Ser Pro Val Lys Pro Phe Tyr Gln 275 280 285 Cys Ala Cys Pro Thr Gly Val Lys Leu Leu Glu Asn Gly Lys Thr Cys 290 295 300 Lys Asp Gly Ala Thr Glu Leu Leu Leu Leu Ala Arg Arg Thr Asp Leu 305 310 315 320 Arg Arg Ile Ser Leu Asp Thr Pro Asp Phe Thr Asp Ile Val Leu Gln 325 330 335 Leu Glu Asp Ile Arg His Ala Ile Ala Ile Asp Tyr Asp Pro Val Glu 340 345 350 Gly Tyr Ile Tyr Trp Thr Asp Asp Glu Val Arg Ala Ile Arg Arg Ser 355 360 365 Phe Ile Asp Gly Ser Gly Ser Gln Phe Val Val Thr Ala Gln Ile Ala 370 375 380 His Pro Asp Gly Ile Ala Val Asp Trp Val Ala Arg Asn Leu Tyr Trp 385 390 395 400 Thr Asp Thr Gly Thr Asp Arg Ile Glu Val Thr Arg Leu Asn Gly Thr 405 410 415 Met Arg Lys Ile Leu Ile Ser Glu Asp Leu Glu Glu Pro Arg Ala Ile 420 425 430 Val Leu Asp Pro Met Val Gly Tyr Met Tyr Trp Thr Asp Trp Gly Glu 435 440 445 Ile Pro Lys Ile Glu Arg Ala Ala Leu Asp Gly Ser Asp Arg Val Val 450 455 460 Leu Val Asn Thr Ser Leu Gly Trp Pro Asn Gly Leu Ala Leu Asp Tyr 465 470 475 480 Asp Glu Gly Lys Ile Tyr Trp Gly Asp Ala Lys Thr Asp Lys Ile Glu 485 490 495 Val Met Asn Thr Asp Gly Thr Gly Arg Arg Val Leu Val Glu Asp Lys 500 505 510 Ile Pro His Ile Phe Gly Phe Thr Leu Leu Gly Asp Tyr Val Tyr Trp 515 520 525 Thr Asp Trp Gln Arg Arg Ser Ile Glu Arg Val His Lys Arg Ser Ala 530 535 540 Glu Arg Glu Val Ile Ile Asp Gln Leu Pro Asp Leu Met Gly Leu Lys 545 550 555 560 Ala Thr Asn Val His Arg Val Ile Gly Ser Asn Pro Cys Ala Glu Glu 565 570 575 Asn Gly Gly Cys Ser His Leu Cys Leu Tyr Arg Pro Gln Gly Leu Arg 580 585 590 Cys Ala Cys Pro Ile Gly Phe Glu Leu Ile Ser Asp Met Lys Thr Cys 595 600 605 Ile Val Pro Glu Ala Phe Leu Leu Phe Ser Arg Arg Ala Asp Ile Arg 610 615 620 Arg Ile Ser Leu Glu Thr Asn Asn Asn Asn Val Ala Ile Pro Leu Thr 625 630 635 640 Gly Val Lys Glu Ala Ser Ala Leu Asp Phe Asp Val Thr Asp Asn Arg 645 650 655 Ile Tyr Trp Thr Asp Ile Ser Leu Lys Thr Ile Ser Arg Ala Phe Met 660 665 670 Asn Gly Ser Ala Leu Glu His Val Val Glu Phe Gly Leu Asp Tyr Pro 675 680 685 Glu Gly Met Ala Val Asp Trp Leu Gly Lys Asn Leu Tyr Trp Ala Asp 690 695 700 Thr Gly Thr Asn Arg Ile Glu Val Ser Lys Leu Asp Gly Gln His Arg 705 710 715 720 Gln Val Leu Val Trp Lys Asp Leu Asp Ser Pro Arg Ala Leu Ala Leu 725 730 735 Asp Pro Ala Glu Gly Phe Met Tyr Trp Thr Glu Trp Gly Gly Lys Pro 740 745 750 Lys Ile Asp Arg Ala Ala Met Asp Gly Ser Glu Arg Thr Thr Leu Val 755 760 765 Pro Asn Val Gly Arg Ala Asn Gly Leu Thr Ile Asp Tyr Ala Lys Arg 770 775 780 Arg Leu Tyr Trp Thr Asp Leu Asp Thr Asn Leu Ile Glu Ser Ser Asn 785 790 795 800 Met Leu Gly Leu Asn Arg Glu Val Ile Ala Asp Asp Leu Pro His Pro 805 810 815 Phe Gly Leu Thr Gln Tyr Gln Asp Tyr Ile Tyr Trp Thr Asp Trp Ser 820 825 830 Arg Arg Ser Ile Glu Arg Ala Asn Lys Thr Ser Gly Gln Asn Arg Thr 835 840 845 Ile Ile Gln Gly His Leu Asp Tyr Val Met Asp Ile Leu Val Phe His 850 855 860 Ser Ser Arg Gln Ser Gly Trp Asn Glu Cys Ala Ser Ser Asn Gly His 865 870 875 880 Cys Ser His Leu Cys Leu Ala Val Pro Val Gly Gly Phe Val Cys Gly 885 890 895 Cys Pro Ala His Tyr Ser Leu Asn Ala Asp Asn Arg Thr Cys Ser Ala 900 905 910 Pro Thr Thr Phe Leu Leu Phe Ser Gln Lys Ser Ala Ile Asn Arg Met 915 920 925 Val Ile Asp Glu Gln Gln Ser Pro Asp Ile Ile Leu Pro Ile His Ser 930 935 940 Leu Arg Asn Val Arg Ala Ile Asp Tyr Asp Pro Leu Asp Lys Gln Leu 945 950 955 960 Tyr Trp Ile Asp Ser Arg Gln Asn Met Ile Arg Lys Ala Gln Glu Asp 965 970 975 Gly Ser Gln Gly Phe Thr Val Val Val Ser Ser Val Pro Ser Gln Asn 980 985 990 Leu Glu Ile Gln Pro Tyr Asp Leu Ser Ile Asp Ile Tyr Ser Arg Tyr 995 1000 1005 Ile Tyr Trp Thr Cys Glu Ala Thr Asn Val Ile Asn Val Thr Arg 1010 1015 1020 Leu Asp Gly Arg Ser Val Gly Val Val Leu Lys Gly Glu Gln Asp 1025 1030 1035 Arg Pro Arg Ala Ile Val Val Asn Pro Glu Lys Gly Tyr Met Tyr 1040 1045 1050 Phe Thr Asn Leu Gln Glu Arg Ser Pro Lys Ile Glu Arg Ala Ala 1055 1060 1065 Leu Asp Gly Thr Glu Arg Glu Val Leu Phe Phe Ser Gly Leu Ser 1070 1075 1080 Lys Pro Ile Ala Leu Ala Leu Asp Ser Arg Leu Gly Lys Leu Phe 1085 1090 1095 Trp Ala Asp Ser Asp Leu Arg Arg Ile Glu Ser Ser Asp Leu Ser 1100 1105 1110 Gly Ala Asn Arg Ile Val Leu Glu Asp Ser Asn Ile Leu Gln Pro 1115 1120 1125 Val Gly Leu Thr Val Phe Glu Asn Trp Leu Tyr Trp Ile Asp Lys 1130 1135 1140 Gln Gln Gln Met Ile Glu Lys Ile Asp Met Thr Gly Arg Glu Gly 1145 1150 1155 Arg Thr Lys Val Gln Ala Arg Ile Ala Gln Leu Ser Asp Ile His 1160 1165 1170 Ala Val Lys Glu Leu Asn Leu Gln Glu Tyr Arg Gln His Pro Cys 1175 1180 1185 Ala Gln Asp Asn Gly Gly Cys Ser His Ile Cys Leu Val Lys Gly 1190 1195 1200 Asp Gly Thr Thr Arg Cys Ser Cys Pro Met His Leu Val Leu Leu 1205 1210 1215 Gln Asp Glu Leu Ser Cys Gly Glu Pro Pro Thr Cys Ser Pro Gln 1220 1225 1230 Gln Phe Thr Cys Phe Thr Gly Glu Ile Asp Cys Ile Pro Val Ala 1235 1240 1245 Trp Arg Cys Asp Gly Phe Thr Glu Cys Glu Asp His Ser Asp Glu 1250 1255 1260 Leu Asn Cys Pro Val Cys Ser Glu Ser Gln Phe Gln Cys Ala Ser 1265 1270 1275 Gly Gln Cys Ile Asp Gly Ala Leu Arg Cys Asn Gly Asp Ala Asn 1280 1285 1290 Cys Gln Asp Lys Ser Asp Glu Lys Asn Cys Glu Val Leu Cys Leu 1295 1300 1305 Ile Asp Gln Phe Arg Cys Ala Asn Gly Gln Cys Ile Gly Lys His 1310 1315 1320 Lys Lys Cys Asp His Asn Val Asp Cys Ser Asp Lys Ser Asp Glu 1325 1330 1335 Leu Asp Cys Tyr Pro Thr Glu Glu Pro Ala Pro Gln Ala Thr Asn 1340 1345 1350 Thr 44896DNAArtificialHA-tagged LRP6 4atggagacag acacactcct gctatgggta ctgctgctct gggttccagg ttccactggt 60gacgctagat acccatacga tgttccagat tacgctggag gtggcgctag cgcccctttg 120ttgctttatg caaacagacg ggacttgcga ttggttgatg ctacaaatgg caaagagaat 180gctacgattg tagttggagg cttggaggat gcagctgcgg tggactttgt gtttagtcat 240ggcttgatat actggagtga tgtcagcgaa gaagccatta aacgaacaga atttaacaaa 300actgagagtg tgcagaatgt tgttgtttct ggattattgt cccccgatgg gctggcatgt 360gattggcttg gagaaaaatt gtactggaca gattctgaaa ctaatcggat tgaagtttct 420aatttagatg gatctttacg aaaagtttta ttttggcaag agttggatca acccagagct 480attgccttag atccttcaag tgggttcatg tactggacag actggggaga agtgccaaag 540atagaacgtg ctggaatgga tggttcaagt cgcttcatta taataaacag tgaaatttac 600tggccaaatg gactgacttt ggattatgaa gaacaaaagc tttattgggc agatgcaaaa 660cttaatttca tccacaaatc aaatctggat ggaacaaatc ggcaggcagt ggttaaaggt 720tcccttccac atccttttgc cttgacgtta tttgaggaca tattgtactg gactgactgg 780agcacacact ccattttggc ttgcaacaag tatactggtg agggtctgcg tgaaatccat 840tctgacatct tctctcccat ggatatacat gccttcagcc aacagaggca gccaaatgcc 900acaaatccat gtggaattga caatgggggt tgttcccatt tgtgtttgat gtctccagtc 960aagccttttt atcagtgtgc ttgccccact ggggtcaaac tcctggagaa tggaaaaacc 1020tgcaaagatg gtgccacaga attattgctt ttagctcgaa ggacagactt gagacgcatt 1080tctttggata caccagattt tacagacatt gttctgcagt tagaagacat ccgtcatgcc 1140attgccatag attacgatcc tgtggaaggc tacatctact ggactgatga tgaagtgagg 1200gccatacgcc gttcatttat agatggatct ggcagtcagt ttgtggtcac tgctcaaatt 1260gcccatcctg atggtattgc tgtggactgg gttgcacgaa atctttattg gacagacact 1320ggcactgatc gaatagaagt gacaaggctc aatgggacca tgaggaagat cttgatttca 1380gaggacttag aggaaccccg ggctattgtg ttagatccca tggttgggta catgtattgg 1440actgactggg gagaaattcc gaaaattgag

cgagcagctc tggatggttc tgaccgtgta 1500gtattggtta acacttctct tggttggcca aatggtttag ccttggatta tgatgaaggc 1560aaaatatact ggggagatgc caaaacagac aagattgagg ttatgaatac tgatggcact 1620gggagacgag tactagtgga agacaaaatt cctcacatat ttggatttac tttgttgggt 1680gactatgttt actggactga ctggcagagg cgtagcattg aaagagttca taaacgaagt 1740gcagagaggg aagtgatcat agatcagctg cctgacctca tgggcctaaa ggctacaaat 1800gttcatcgag tgattggttc caacccctgt gctgaggaaa acgggggatg tagccatctc 1860tgcctctata gacctcaggg ccttcgctgt gcttgcccta ttggctttga actcatcagt 1920gacatgaaga cctgcattgt cccagaggct ttccttttgt tttcacggag agcagatatc 1980agacgaattt ctctggaaac aaacaataat aatgtggcta ttccactcac tggtgtcaaa 2040gaagcttctg ctttggattt tgatgtgaca gacaaccgaa tttattggac tgatatatca 2100ctcaagacca tcagcagagc ctttatgaat ggcagtgcac tggaacatgt ggtagaattc 2160ggcttagatt atccagaagg catggcagta gactggcttg ggaagaactt gtactgggca 2220gacacaggaa cgaatcgaat tgaggtgtca aagttggatg ggcagcaccg acaagttttg 2280gtgtggaaag acctagatag tcccagagct ctcgcgttgg accctgccga aggatttatg 2340tattggactg aatggggtgg aaaacctaag atagacagag ctgcaatgga tggaagtgaa 2400cgtactacct tagttccaaa tgtggggcgg gcaaacggcc taactattga ttatgctaaa 2460aggaggcttt attggacaga cctggacacc aacttaatag aatcttcaaa tatgcttggg 2520ctcaaccgtg aagttatagc agatgacttg cctcatcctt ttggcttaac tcagtaccaa 2580gattatatct actggacgga ctggagccga cgcagcattg agcgtgccaa caaaaccagt 2640ggccaaaacc gcaccatcat tcagggccat ttggattatg tgatggacat cctcgtcttt 2700cactcatctc gacagtcagg gtggaatgaa tgtgcttcca gcaatgggca ctgctcccac 2760ctctgcttgg ctgtgccagt tgggggtttt gtttgtggat gccctgccca ctactctctt 2820aatgctgaca acaggacttg tagtgctcct acgactttcc tgctcttcag tcaaaagagt 2880gccatcaacc gcatggtgat tgatgaacaa cagagccccg acatcatcct tcccatccac 2940agccttcgga atgtccgggc cattgactat gacccactgg acaagcaact ctattggatt 3000gactcacgac aaaacatgat ccgaaaggca caagaagatg gcagccaggg ctttactgtg 3060gttgtgagct cagttccgag tcagaacctg gaaatacaac cctatgacct cagcattgat 3120atttacagcc gctacatcta ctggacttgt gaggctacca atgtcattaa tgtgacaaga 3180ttagatggga gatcagttgg agtggtgctg aaaggcgagc aggacagacc tcgagccgtt 3240gtggtaaacc cagagaaagg gtatatgtat tttaccaatc ttcaggaaag gtctcctaaa 3300attgaacggg ctgctttgga tgggacagaa cgggaggtcc tctttttcag tggcttaagt 3360aaaccaattg ctttagccct tgatagcagg ctgggcaagc tcttttgggc tgattcagat 3420ctccggcgaa ttgaaagcag tgatctctca ggtgctaacc ggatagtatt agaagactcc 3480aatatcttgc agcctgtggg acttactgtg tttgaaaact ggctctattg gattgataaa 3540cagcagcaaa tgattgaaaa aattgacatg acaggtcgag agggtagaac caaagtccaa 3600gctcgaattg cccagcttag tgacattcat gcagtaaagg agctgaacct tcaagaatac 3660agacagcacc cttgtgctca ggataatggt ggctgttcac atatttgtct tgtaaagggg 3720gatggtacta caaggtgttc ttgccccatg cacctggttc tacttcaaga tgagctatca 3780tgtggagaac ctccaacatg ttctcctcag cagtttactt gtttcacggg ggaaattgac 3840tgtatccctg tggcttggcg gtgcgatggg tttactgaat gtgaagacca cagtgatgaa 3900ctcaattgtc ctgtatgctc agagtcccag ttccagtgtg ccagtgggca gtgtattgat 3960ggtgccctcc gatgcaatgg agatgcaaac tgccaggaca aatcagatga gaagaactgt 4020gaagtgcttt gtttaattga tcagttccgc tgtgccaatg gtcagtgcat tggaaagcac 4080aagaagtgtg atcataatgt ggattgcagt gacaagtcag atgaactgga ttgttatccg 4140actgaagaac cagcaccaca ggccaccaat acagttggtt ctgttattgg cgtaattgtc 4200accatttttg tgtctggaac tgtatacttt atctgccaga ggatgttgtg tccacgtatg 4260aagggagatg gggaaactat gactaatgac tatgtagttc atggaccagc ttctgtgcct 4320cttggttatg tgccacaccc aagttctttg tcaggatctc ttccaggaat gtctcgaggt 4380aaatcaatga tcagctccct cagtatcatg gggggaagca gtggaccccc ctatgaccga 4440gcccatgtta caggagcatc atcaagtagt tcttcaagca ccaaaggcac ttacttccct 4500gcaattttga accctccacc atccccagcc acagagcgat cacattacac tatggaattt 4560ggatattctt caaacagtcc ttccactcat aggtcataca gctacaggcc atatagctac 4620cggcactttg caccccccac cacaccctgc agcacagatg tttgtgacag tgactatgct 4680cctagtcgga gaatgacctc agtggcaaca gccaagggct ataccagtga cttgaactat 4740gattcagaac ctgtgccccc acctcccaca ccccgaagcc aatacttgtc agcagaggag 4800aactatgaaa gctgcccacc ttctccatac acagagagga gctattctca tcacctctac 4860ccaccgccac cctctccctg tacagactcc tcctga 489651631PRTArtificialHA-tagged LRP6 5Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Asp Ala Arg Tyr Pro Tyr Asp Val Pro Asp Tyr Ala 20 25 30 Gly Gly Gly Ala Ser Ala Pro Leu Leu Leu Tyr Ala Asn Arg Arg Asp 35 40 45 Leu Arg Leu Val Asp Ala Thr Asn Gly Lys Glu Asn Ala Thr Ile Val 50 55 60 Val Gly Gly Leu Glu Asp Ala Ala Ala Val Asp Phe Val Phe Ser His 65 70 75 80 Gly Leu Ile Tyr Trp Ser Asp Val Ser Glu Glu Ala Ile Lys Arg Thr 85 90 95 Glu Phe Asn Lys Thr Glu Ser Val Gln Asn Val Val Val Ser Gly Leu 100 105 110 Leu Ser Pro Asp Gly Leu Ala Cys Asp Trp Leu Gly Glu Lys Leu Tyr 115 120 125 Trp Thr Asp Ser Glu Thr Asn Arg Ile Glu Val Ser Asn Leu Asp Gly 130 135 140 Ser Leu Arg Lys Val Leu Phe Trp Gln Glu Leu Asp Gln Pro Arg Ala 145 150 155 160 Ile Ala Leu Asp Pro Ser Ser Gly Phe Met Tyr Trp Thr Asp Trp Gly 165 170 175 Glu Val Pro Lys Ile Glu Arg Ala Gly Met Asp Gly Ser Ser Arg Phe 180 185 190 Ile Ile Ile Asn Ser Glu Ile Tyr Trp Pro Asn Gly Leu Thr Leu Asp 195 200 205 Tyr Glu Glu Gln Lys Leu Tyr Trp Ala Asp Ala Lys Leu Asn Phe Ile 210 215 220 His Lys Ser Asn Leu Asp Gly Thr Asn Arg Gln Ala Val Val Lys Gly 225 230 235 240 Ser Leu Pro His Pro Phe Ala Leu Thr Leu Phe Glu Asp Ile Leu Tyr 245 250 255 Trp Thr Asp Trp Ser Thr His Ser Ile Leu Ala Cys Asn Lys Tyr Thr 260 265 270 Gly Glu Gly Leu Arg Glu Ile His Ser Asp Ile Phe Ser Pro Met Asp 275 280 285 Ile His Ala Phe Ser Gln Gln Arg Gln Pro Asn Ala Thr Asn Pro Cys 290 295 300 Gly Ile Asp Asn Gly Gly Cys Ser His Leu Cys Leu Met Ser Pro Val 305 310 315 320 Lys Pro Phe Tyr Gln Cys Ala Cys Pro Thr Gly Val Lys Leu Leu Glu 325 330 335 Asn Gly Lys Thr Cys Lys Asp Gly Ala Thr Glu Leu Leu Leu Leu Ala 340 345 350 Arg Arg Thr Asp Leu Arg Arg Ile Ser Leu Asp Thr Pro Asp Phe Thr 355 360 365 Asp Ile Val Leu Gln Leu Glu Asp Ile Arg His Ala Ile Ala Ile Asp 370 375 380 Tyr Asp Pro Val Glu Gly Tyr Ile Tyr Trp Thr Asp Asp Glu Val Arg 385 390 395 400 Ala Ile Arg Arg Ser Phe Ile Asp Gly Ser Gly Ser Gln Phe Val Val 405 410 415 Thr Ala Gln Ile Ala His Pro Asp Gly Ile Ala Val Asp Trp Val Ala 420 425 430 Arg Asn Leu Tyr Trp Thr Asp Thr Gly Thr Asp Arg Ile Glu Val Thr 435 440 445 Arg Leu Asn Gly Thr Met Arg Lys Ile Leu Ile Ser Glu Asp Leu Glu 450 455 460 Glu Pro Arg Ala Ile Val Leu Asp Pro Met Val Gly Tyr Met Tyr Trp 465 470 475 480 Thr Asp Trp Gly Glu Ile Pro Lys Ile Glu Arg Ala Ala Leu Asp Gly 485 490 495 Ser Asp Arg Val Val Leu Val Asn Thr Ser Leu Gly Trp Pro Asn Gly 500 505 510 Leu Ala Leu Asp Tyr Asp Glu Gly Lys Ile Tyr Trp Gly Asp Ala Lys 515 520 525 Thr Asp Lys Ile Glu Val Met Asn Thr Asp Gly Thr Gly Arg Arg Val 530 535 540 Leu Val Glu Asp Lys Ile Pro His Ile Phe Gly Phe Thr Leu Leu Gly 545 550 555 560 Asp Tyr Val Tyr Trp Thr Asp Trp Gln Arg Arg Ser Ile Glu Arg Val 565 570 575 His Lys Arg Ser Ala Glu Arg Glu Val Ile Ile Asp Gln Leu Pro Asp 580 585 590 Leu Met Gly Leu Lys Ala Thr Asn Val His Arg Val Ile Gly Ser Asn 595 600 605 Pro Cys Ala Glu Glu Asn Gly Gly Cys Ser His Leu Cys Leu Tyr Arg 610 615 620 Pro Gln Gly Leu Arg Cys Ala Cys Pro Ile Gly Phe Glu Leu Ile Ser 625 630 635 640 Asp Met Lys Thr Cys Ile Val Pro Glu Ala Phe Leu Leu Phe Ser Arg 645 650 655 Arg Ala Asp Ile Arg Arg Ile Ser Leu Glu Thr Asn Asn Asn Asn Val 660 665 670 Ala Ile Pro Leu Thr Gly Val Lys Glu Ala Ser Ala Leu Asp Phe Asp 675 680 685 Val Thr Asp Asn Arg Ile Tyr Trp Thr Asp Ile Ser Leu Lys Thr Ile 690 695 700 Ser Arg Ala Phe Met Asn Gly Ser Ala Leu Glu His Val Val Glu Phe 705 710 715 720 Gly Leu Asp Tyr Pro Glu Gly Met Ala Val Asp Trp Leu Gly Lys Asn 725 730 735 Leu Tyr Trp Ala Asp Thr Gly Thr Asn Arg Ile Glu Val Ser Lys Leu 740 745 750 Asp Gly Gln His Arg Gln Val Leu Val Trp Lys Asp Leu Asp Ser Pro 755 760 765 Arg Ala Leu Ala Leu Asp Pro Ala Glu Gly Phe Met Tyr Trp Thr Glu 770 775 780 Trp Gly Gly Lys Pro Lys Ile Asp Arg Ala Ala Met Asp Gly Ser Glu 785 790 795 800 Arg Thr Thr Leu Val Pro Asn Val Gly Arg Ala Asn Gly Leu Thr Ile 805 810 815 Asp Tyr Ala Lys Arg Arg Leu Tyr Trp Thr Asp Leu Asp Thr Asn Leu 820 825 830 Ile Glu Ser Ser Asn Met Leu Gly Leu Asn Arg Glu Val Ile Ala Asp 835 840 845 Asp Leu Pro His Pro Phe Gly Leu Thr Gln Tyr Gln Asp Tyr Ile Tyr 850 855 860 Trp Thr Asp Trp Ser Arg Arg Ser Ile Glu Arg Ala Asn Lys Thr Ser 865 870 875 880 Gly Gln Asn Arg Thr Ile Ile Gln Gly His Leu Asp Tyr Val Met Asp 885 890 895 Ile Leu Val Phe His Ser Ser Arg Gln Ser Gly Trp Asn Glu Cys Ala 900 905 910 Ser Ser Asn Gly His Cys Ser His Leu Cys Leu Ala Val Pro Val Gly 915 920 925 Gly Phe Val Cys Gly Cys Pro Ala His Tyr Ser Leu Asn Ala Asp Asn 930 935 940 Arg Thr Cys Ser Ala Pro Thr Thr Phe Leu Leu Phe Ser Gln Lys Ser 945 950 955 960 Ala Ile Asn Arg Met Val Ile Asp Glu Gln Gln Ser Pro Asp Ile Ile 965 970 975 Leu Pro Ile His Ser Leu Arg Asn Val Arg Ala Ile Asp Tyr Asp Pro 980 985 990 Leu Asp Lys Gln Leu Tyr Trp Ile Asp Ser Arg Gln Asn Met Ile Arg 995 1000 1005 Lys Ala Gln Glu Asp Gly Ser Gln Gly Phe Thr Val Val Val Ser 1010 1015 1020 Ser Val Pro Ser Gln Asn Leu Glu Ile Gln Pro Tyr Asp Leu Ser 1025 1030 1035 Ile Asp Ile Tyr Ser Arg Tyr Ile Tyr Trp Thr Cys Glu Ala Thr 1040 1045 1050 Asn Val Ile Asn Val Thr Arg Leu Asp Gly Arg Ser Val Gly Val 1055 1060 1065 Val Leu Lys Gly Glu Gln Asp Arg Pro Arg Ala Val Val Val Asn 1070 1075 1080 Pro Glu Lys Gly Tyr Met Tyr Phe Thr Asn Leu Gln Glu Arg Ser 1085 1090 1095 Pro Lys Ile Glu Arg Ala Ala Leu Asp Gly Thr Glu Arg Glu Val 1100 1105 1110 Leu Phe Phe Ser Gly Leu Ser Lys Pro Ile Ala Leu Ala Leu Asp 1115 1120 1125 Ser Arg Leu Gly Lys Leu Phe Trp Ala Asp Ser Asp Leu Arg Arg 1130 1135 1140 Ile Glu Ser Ser Asp Leu Ser Gly Ala Asn Arg Ile Val Leu Glu 1145 1150 1155 Asp Ser Asn Ile Leu Gln Pro Val Gly Leu Thr Val Phe Glu Asn 1160 1165 1170 Trp Leu Tyr Trp Ile Asp Lys Gln Gln Gln Met Ile Glu Lys Ile 1175 1180 1185 Asp Met Thr Gly Arg Glu Gly Arg Thr Lys Val Gln Ala Arg Ile 1190 1195 1200 Ala Gln Leu Ser Asp Ile His Ala Val Lys Glu Leu Asn Leu Gln 1205 1210 1215 Glu Tyr Arg Gln His Pro Cys Ala Gln Asp Asn Gly Gly Cys Ser 1220 1225 1230 His Ile Cys Leu Val Lys Gly Asp Gly Thr Thr Arg Cys Ser Cys 1235 1240 1245 Pro Met His Leu Val Leu Leu Gln Asp Glu Leu Ser Cys Gly Glu 1250 1255 1260 Pro Pro Thr Cys Ser Pro Gln Gln Phe Thr Cys Phe Thr Gly Glu 1265 1270 1275 Ile Asp Cys Ile Pro Val Ala Trp Arg Cys Asp Gly Phe Thr Glu 1280 1285 1290 Cys Glu Asp His Ser Asp Glu Leu Asn Cys Pro Val Cys Ser Glu 1295 1300 1305 Ser Gln Phe Gln Cys Ala Ser Gly Gln Cys Ile Asp Gly Ala Leu 1310 1315 1320 Arg Cys Asn Gly Asp Ala Asn Cys Gln Asp Lys Ser Asp Glu Lys 1325 1330 1335 Asn Cys Glu Val Leu Cys Leu Ile Asp Gln Phe Arg Cys Ala Asn 1340 1345 1350 Gly Gln Cys Ile Gly Lys His Lys Lys Cys Asp His Asn Val Asp 1355 1360 1365 Cys Ser Asp Lys Ser Asp Glu Leu Asp Cys Tyr Pro Thr Glu Glu 1370 1375 1380 Pro Ala Pro Gln Ala Thr Asn Thr Val Gly Ser Val Ile Gly Val 1385 1390 1395 Ile Val Thr Ile Phe Val Ser Gly Thr Val Tyr Phe Ile Cys Gln 1400 1405 1410 Arg Met Leu Cys Pro Arg Met Lys Gly Asp Gly Glu Thr Met Thr 1415 1420 1425 Asn Asp Tyr Val Val His Gly Pro Ala Ser Val Pro Leu Gly Tyr 1430 1435 1440 Val Pro His Pro Ser Ser Leu Ser Gly Ser Leu Pro Gly Met Ser 1445 1450 1455 Arg Gly Lys Ser Met Ile Ser Ser Leu Ser Ile Met Gly Gly Ser 1460 1465 1470 Ser Gly Pro Pro Tyr Asp Arg Ala His Val Thr Gly Ala Ser Ser 1475 1480 1485 Ser Ser Ser Ser Ser Thr Lys Gly Thr Tyr Phe Pro Ala Ile Leu 1490 1495 1500 Asn Pro Pro Pro Ser Pro Ala Thr Glu Arg Ser His Tyr Thr Met 1505 1510 1515 Glu Phe Gly Tyr Ser Ser Asn Ser Pro Ser Thr His Arg Ser Tyr 1520 1525 1530 Ser Tyr Arg Pro Tyr Ser Tyr Arg His Phe Ala Pro Pro Thr Thr 1535 1540 1545 Pro Cys Ser Thr Asp Val Cys Asp Ser Asp Tyr Ala Pro Ser Arg 1550 1555 1560 Arg Met Thr Ser Val Ala Thr Ala Lys Gly Tyr Thr Ser Asp Leu 1565 1570 1575 Asn Tyr Asp Ser Glu Pro Val Pro Pro Pro Pro Thr Pro Arg Ser 1580 1585 1590 Gln Tyr Leu Ser Ala Glu Glu Asn Tyr Glu Ser Cys Pro Pro Ser 1595 1600 1605 Pro Tyr Thr Glu Arg Ser Tyr Ser His His Leu Tyr Pro Pro Pro 1610 1615 1620 Pro Ser Pro Cys Thr Asp Ser Ser 1625 1630 63069DNAArtificialHA-tagged LRP6, deletion of propeller domains 1 and 2. 6atggagacag acacactcct gctatgggta ctgctgctct gggttccagg ttccactggt 60gacgctagat acccatacga tgttccagat tacgctggag gtggcgctag cgtcccagag 120gctttccttt tgttttcacg gagagcagat atcagacgaa tttctctgga aacaaacaat 180aataatgtgg ctattccact cactggtgtc aaagaagctt ctgctttgga ttttgatgtg 240acagacaacc gaatttattg gactgatata tcactcaaga ccatcagcag agcctttatg 300aatggcagtg cactggaaca tgtggtagaa ttcggcttag attatccaga aggcatggca 360gtagactggc ttgggaagaa cttgtactgg gcagacacag gaacgaatcg aattgaggtg 420tcaaagttgg atgggcagca ccgacaagtt ttggtgtgga aagacctaga tagtcccaga 480gctctcgcgt tggaccctgc cgaaggattt atgtattgga ctgaatgggg tggaaaacct 540aagatagaca gagctgcaat ggatggaagt gaacgtacta ccttagttcc aaatgtgggg 600cgggcaaacg gcctaactat tgattatgct aaaaggaggc tttattggac agacctggac

660accaacttaa tagaatcttc aaatatgctt gggctcaacc gtgaagttat agcagatgac 720ttgcctcatc cttttggctt aactcagtac caagattata tctactggac ggactggagc 780cgacgcagca ttgagcgtgc caacaaaacc agtggccaaa accgcaccat cattcagggc 840catttggatt atgtgatgga catcctcgtc tttcactcat ctcgacagtc agggtggaat 900gaatgtgctt ccagcaatgg gcactgctcc cacctctgct tggctgtgcc agttgggggt 960tttgtttgtg gatgccctgc ccactactct cttaatgctg acaacaggac ttgtagtgct 1020cctacgactt tcctgctctt cagtcaaaag agtgccatca accgcatggt gattgatgaa 1080caacagagcc ccgacatcat ccttcccatc cacagccttc ggaatgtccg ggccattgac 1140tatgacccac tggacaagca actctattgg attgactcac gacaaaacat gatccgaaag 1200gcacaagaag atggcagcca gggctttact gtggttgtga gctcagttcc gagtcagaac 1260ctggaaatac aaccctatga cctcagcatt gatatttaca gccgctacat ctactggact 1320tgtgaggcta ccaatgtcat taatgtgaca agattagatg ggagatcagt tggagtggtg 1380ctgaaaggcg agcaggacag acctcgagcc gttgtggtaa acccagagaa agggtatatg 1440tattttacca atcttcagga aaggtctcct aaaattgaac gggctgcttt ggatgggaca 1500gaacgggagg tcctcttttt cagtggctta agtaaaccaa ttgctttagc ccttgatagc 1560aggctgggca agctcttttg ggctgattca gatctccggc gaattgaaag cagtgatctc 1620tcaggtgcta accggatagt attagaagac tccaatatct tgcagcctgt gggacttact 1680gtgtttgaaa actggctcta ttggattgat aaacagcagc aaatgattga aaaaattgac 1740atgacaggtc gagagggtag aaccaaagtc caagctcgaa ttgcccagct tagtgacatt 1800catgcagtaa aggagctgaa ccttcaagaa tacagacagc acccttgtgc tcaggataat 1860ggtggctgtt cacatatttg tcttgtaaag ggggatggta ctacaaggtg ttcttgcccc 1920atgcacctgg ttctacttca agatgagcta tcatgtggag aacctccaac atgttctcct 1980cagcagttta cttgtttcac gggggaaatt gactgtatcc ctgtggcttg gcggtgcgat 2040gggtttactg aatgtgaaga ccacagtgat gaactcaatt gtcctgtatg ctcagagtcc 2100cagttccagt gtgccagtgg gcagtgtatt gatggtgccc tccgatgcaa tggagatgca 2160aactgccagg acaaatcaga tgagaagaac tgtgaagtgc tttgtttaat tgatcagttc 2220cgctgtgcca atggtcagtg cattggaaag cacaagaagt gtgatcataa tgtggattgc 2280agtgacaagt cagatgaact ggattgttat ccgactgaag aaccagcacc acaggccacc 2340aatacagttg gttctgttat tggcgtaatt gtcaccattt ttgtgtctgg aactgtatac 2400tttatctgcc agaggatgtt gtgtccacgt atgaagggag atggggaaac tatgactaat 2460gactatgtag ttcatggacc agcttctgtg cctcttggtt atgtgccaca cccaagttct 2520ttgtcaggat ctcttccagg aatgtctcga ggtaaatcaa tgatcagctc cctcagtatc 2580atggggggaa gcagtggacc cccctatgac cgagcccatg ttacaggagc atcatcaagt 2640agttcttcaa gcaccaaagg cacttacttc cctgcaattt tgaaccctcc accatcccca 2700gccacagagc gatcacatta cactatggaa tttggatatt cttcaaacag tccttccact 2760cataggtcat acagctacag gccatatagc taccggcact ttgcaccccc caccacaccc 2820tgcagcacag atgtttgtga cagtgactat gctcctagtc ggagaatgac ctcagtggca 2880acagccaagg gctataccag tgacttgaac tatgattcag aacctgtgcc cccacctccc 2940acaccccgaa gccaatactt gtcagcagag gagaactatg aaagctgccc accttctcca 3000tacacagaga ggagctattc tcatcacctc tacccaccgc caccctctcc ctgtacagac 3060tcctcctga 306971022PRTArtificialHA-tagged LRP6, deletion of propeller domains 1 and 2. 7Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Asp Ala Arg Tyr Pro Tyr Asp Val Pro Asp Tyr Ala 20 25 30 Gly Gly Gly Ala Ser Val Pro Glu Ala Phe Leu Leu Phe Ser Arg Arg 35 40 45 Ala Asp Ile Arg Arg Ile Ser Leu Glu Thr Asn Asn Asn Asn Val Ala 50 55 60 Ile Pro Leu Thr Gly Val Lys Glu Ala Ser Ala Leu Asp Phe Asp Val 65 70 75 80 Thr Asp Asn Arg Ile Tyr Trp Thr Asp Ile Ser Leu Lys Thr Ile Ser 85 90 95 Arg Ala Phe Met Asn Gly Ser Ala Leu Glu His Val Val Glu Phe Gly 100 105 110 Leu Asp Tyr Pro Glu Gly Met Ala Val Asp Trp Leu Gly Lys Asn Leu 115 120 125 Tyr Trp Ala Asp Thr Gly Thr Asn Arg Ile Glu Val Ser Lys Leu Asp 130 135 140 Gly Gln His Arg Gln Val Leu Val Trp Lys Asp Leu Asp Ser Pro Arg 145 150 155 160 Ala Leu Ala Leu Asp Pro Ala Glu Gly Phe Met Tyr Trp Thr Glu Trp 165 170 175 Gly Gly Lys Pro Lys Ile Asp Arg Ala Ala Met Asp Gly Ser Glu Arg 180 185 190 Thr Thr Leu Val Pro Asn Val Gly Arg Ala Asn Gly Leu Thr Ile Asp 195 200 205 Tyr Ala Lys Arg Arg Leu Tyr Trp Thr Asp Leu Asp Thr Asn Leu Ile 210 215 220 Glu Ser Ser Asn Met Leu Gly Leu Asn Arg Glu Val Ile Ala Asp Asp 225 230 235 240 Leu Pro His Pro Phe Gly Leu Thr Gln Tyr Gln Asp Tyr Ile Tyr Trp 245 250 255 Thr Asp Trp Ser Arg Arg Ser Ile Glu Arg Ala Asn Lys Thr Ser Gly 260 265 270 Gln Asn Arg Thr Ile Ile Gln Gly His Leu Asp Tyr Val Met Asp Ile 275 280 285 Leu Val Phe His Ser Ser Arg Gln Ser Gly Trp Asn Glu Cys Ala Ser 290 295 300 Ser Asn Gly His Cys Ser His Leu Cys Leu Ala Val Pro Val Gly Gly 305 310 315 320 Phe Val Cys Gly Cys Pro Ala His Tyr Ser Leu Asn Ala Asp Asn Arg 325 330 335 Thr Cys Ser Ala Pro Thr Thr Phe Leu Leu Phe Ser Gln Lys Ser Ala 340 345 350 Ile Asn Arg Met Val Ile Asp Glu Gln Gln Ser Pro Asp Ile Ile Leu 355 360 365 Pro Ile His Ser Leu Arg Asn Val Arg Ala Ile Asp Tyr Asp Pro Leu 370 375 380 Asp Lys Gln Leu Tyr Trp Ile Asp Ser Arg Gln Asn Met Ile Arg Lys 385 390 395 400 Ala Gln Glu Asp Gly Ser Gln Gly Phe Thr Val Val Val Ser Ser Val 405 410 415 Pro Ser Gln Asn Leu Glu Ile Gln Pro Tyr Asp Leu Ser Ile Asp Ile 420 425 430 Tyr Ser Arg Tyr Ile Tyr Trp Thr Cys Glu Ala Thr Asn Val Ile Asn 435 440 445 Val Thr Arg Leu Asp Gly Arg Ser Val Gly Val Val Leu Lys Gly Glu 450 455 460 Gln Asp Arg Pro Arg Ala Val Val Val Asn Pro Glu Lys Gly Tyr Met 465 470 475 480 Tyr Phe Thr Asn Leu Gln Glu Arg Ser Pro Lys Ile Glu Arg Ala Ala 485 490 495 Leu Asp Gly Thr Glu Arg Glu Val Leu Phe Phe Ser Gly Leu Ser Lys 500 505 510 Pro Ile Ala Leu Ala Leu Asp Ser Arg Leu Gly Lys Leu Phe Trp Ala 515 520 525 Asp Ser Asp Leu Arg Arg Ile Glu Ser Ser Asp Leu Ser Gly Ala Asn 530 535 540 Arg Ile Val Leu Glu Asp Ser Asn Ile Leu Gln Pro Val Gly Leu Thr 545 550 555 560 Val Phe Glu Asn Trp Leu Tyr Trp Ile Asp Lys Gln Gln Gln Met Ile 565 570 575 Glu Lys Ile Asp Met Thr Gly Arg Glu Gly Arg Thr Lys Val Gln Ala 580 585 590 Arg Ile Ala Gln Leu Ser Asp Ile His Ala Val Lys Glu Leu Asn Leu 595 600 605 Gln Glu Tyr Arg Gln His Pro Cys Ala Gln Asp Asn Gly Gly Cys Ser 610 615 620 His Ile Cys Leu Val Lys Gly Asp Gly Thr Thr Arg Cys Ser Cys Pro 625 630 635 640 Met His Leu Val Leu Leu Gln Asp Glu Leu Ser Cys Gly Glu Pro Pro 645 650 655 Thr Cys Ser Pro Gln Gln Phe Thr Cys Phe Thr Gly Glu Ile Asp Cys 660 665 670 Ile Pro Val Ala Trp Arg Cys Asp Gly Phe Thr Glu Cys Glu Asp His 675 680 685 Ser Asp Glu Leu Asn Cys Pro Val Cys Ser Glu Ser Gln Phe Gln Cys 690 695 700 Ala Ser Gly Gln Cys Ile Asp Gly Ala Leu Arg Cys Asn Gly Asp Ala 705 710 715 720 Asn Cys Gln Asp Lys Ser Asp Glu Lys Asn Cys Glu Val Leu Cys Leu 725 730 735 Ile Asp Gln Phe Arg Cys Ala Asn Gly Gln Cys Ile Gly Lys His Lys 740 745 750 Lys Cys Asp His Asn Val Asp Cys Ser Asp Lys Ser Asp Glu Leu Asp 755 760 765 Cys Tyr Pro Thr Glu Glu Pro Ala Pro Gln Ala Thr Asn Thr Val Gly 770 775 780 Ser Val Ile Gly Val Ile Val Thr Ile Phe Val Ser Gly Thr Val Tyr 785 790 795 800 Phe Ile Cys Gln Arg Met Leu Cys Pro Arg Met Lys Gly Asp Gly Glu 805 810 815 Thr Met Thr Asn Asp Tyr Val Val His Gly Pro Ala Ser Val Pro Leu 820 825 830 Gly Tyr Val Pro His Pro Ser Ser Leu Ser Gly Ser Leu Pro Gly Met 835 840 845 Ser Arg Gly Lys Ser Met Ile Ser Ser Leu Ser Ile Met Gly Gly Ser 850 855 860 Ser Gly Pro Pro Tyr Asp Arg Ala His Val Thr Gly Ala Ser Ser Ser 865 870 875 880 Ser Ser Ser Ser Thr Lys Gly Thr Tyr Phe Pro Ala Ile Leu Asn Pro 885 890 895 Pro Pro Ser Pro Ala Thr Glu Arg Ser His Tyr Thr Met Glu Phe Gly 900 905 910 Tyr Ser Ser Asn Ser Pro Ser Thr His Arg Ser Tyr Ser Tyr Arg Pro 915 920 925 Tyr Ser Tyr Arg His Phe Ala Pro Pro Thr Thr Pro Cys Ser Thr Asp 930 935 940 Val Cys Asp Ser Asp Tyr Ala Pro Ser Arg Arg Met Thr Ser Val Ala 945 950 955 960 Thr Ala Lys Gly Tyr Thr Ser Asp Leu Asn Tyr Asp Ser Glu Pro Val 965 970 975 Pro Pro Pro Pro Thr Pro Arg Ser Gln Tyr Leu Ser Ala Glu Glu Asn 980 985 990 Tyr Glu Ser Cys Pro Pro Ser Pro Tyr Thr Glu Arg Ser Tyr Ser His 995 1000 1005 His Leu Tyr Pro Pro Pro Pro Ser Pro Cys Thr Asp Ser Ser 1010 1015 1020 82160DNAArtificialHA-tagged LRP6, deletion of propeller domains 1, 2 and 3. 8atggagacag acacactcct gctatgggta ctgctgctct gggttccagg ttccactggt 60gacgctagat acccatacga tgttccagat tacgctggag gtggcgctag ccctacgact 120ttcctgctct tcagtcaaaa gagtgccatc aaccgcatgg tgattgatga acaacagagc 180cccgacatca tccttcccat ccacagcctt cggaatgtcc gggccattga ctatgaccca 240ctggacaagc aactctattg gattgactca cgacaaaaca tgatccgaaa ggcacaagaa 300gatggcagcc agggctttac tgtggttgtg agctcagttc cgagtcagaa cctggaaata 360caaccctatg acctcagcat tgatatttac agccgctaca tctactggac ttgtgaggct 420accaatgtca ttaatgtgac aagattagat gggagatcag ttggagtggt gctgaaaggc 480gagcaggaca gacctcgagc cgttgtggta aacccagaga aagggtatat gtattttacc 540aatcttcagg aaaggtctcc taaaattgaa cgggctgctt tggatgggac agaacgggag 600gtcctctttt tcagtggctt aagtaaacca attgctttag cccttgatag caggctgggc 660aagctctttt gggctgattc agatctccgg cgaattgaaa gcagtgatct ctcaggtgct 720aaccggatag tattagaaga ctccaatatc ttgcagcctg tgggacttac tgtgtttgaa 780aactggctct attggattga taaacagcag caaatgattg aaaaaattga catgacaggt 840cgagagggta gaaccaaagt ccaagctcga attgcccagc ttagtgacat tcatgcagta 900aaggagctga accttcaaga atacagacag cacccttgtg ctcaggataa tggtggctgt 960tcacatattt gtcttgtaaa gggggatggt actacaaggt gttcttgccc catgcacctg 1020gttctacttc aagatgagct atcatgtgga gaacctccaa catgttctcc tcagcagttt 1080acttgtttca cgggggaaat tgactgtatc cctgtggctt ggcggtgcga tgggtttact 1140gaatgtgaag accacagtga tgaactcaat tgtcctgtat gctcagagtc ccagttccag 1200tgtgccagtg ggcagtgtat tgatggtgcc ctccgatgca atggagatgc aaactgccag 1260gacaaatcag atgagaagaa ctgtgaagtg ctttgtttaa ttgatcagtt ccgctgtgcc 1320aatggtcagt gcattggaaa gcacaagaag tgtgatcata atgtggattg cagtgacaag 1380tcagatgaac tggattgtta tccgactgaa gaaccagcac cacaggccac caatacagtt 1440ggttctgtta ttggcgtaat tgtcaccatt tttgtgtctg gaactgtata ctttatctgc 1500cagaggatgt tgtgtccacg tatgaaggga gatggggaaa ctatgactaa tgactatgta 1560gttcatggac cagcttctgt gcctcttggt tatgtgccac acccaagttc tttgtcagga 1620tctcttccag gaatgtctcg aggtaaatca atgatcagct ccctcagtat catgggggga 1680agcagtggac ccccctatga ccgagcccat gttacaggag catcatcaag tagttcttca 1740agcaccaaag gcacttactt ccctgcaatt ttgaaccctc caccatcccc agccacagag 1800cgatcacatt acactatgga atttggatat tcttcaaaca gtccttccac tcataggtca 1860tacagctaca ggccatatag ctaccggcac tttgcacccc ccaccacacc ctgcagcaca 1920gatgtttgtg acagtgacta tgctcctagt cggagaatga cctcagtggc aacagccaag 1980ggctatacca gtgacttgaa ctatgattca gaacctgtgc ccccacctcc cacaccccga 2040agccaatact tgtcagcaga ggagaactat gaaagctgcc caccttctcc atacacagag 2100aggagctatt ctcatcacct ctacccaccg ccaccctctc cctgtacaga ctcctcctga 21609719PRTArtificialHA-tagged LRP6, deletion of propeller domains 1, 2 and 3. 9Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Asp Ala Arg Tyr Pro Tyr Asp Val Pro Asp Tyr Ala 20 25 30 Gly Gly Gly Ala Ser Pro Thr Thr Phe Leu Leu Phe Ser Gln Lys Ser 35 40 45 Ala Ile Asn Arg Met Val Ile Asp Glu Gln Gln Ser Pro Asp Ile Ile 50 55 60 Leu Pro Ile His Ser Leu Arg Asn Val Arg Ala Ile Asp Tyr Asp Pro 65 70 75 80 Leu Asp Lys Gln Leu Tyr Trp Ile Asp Ser Arg Gln Asn Met Ile Arg 85 90 95 Lys Ala Gln Glu Asp Gly Ser Gln Gly Phe Thr Val Val Val Ser Ser 100 105 110 Val Pro Ser Gln Asn Leu Glu Ile Gln Pro Tyr Asp Leu Ser Ile Asp 115 120 125 Ile Tyr Ser Arg Tyr Ile Tyr Trp Thr Cys Glu Ala Thr Asn Val Ile 130 135 140 Asn Val Thr Arg Leu Asp Gly Arg Ser Val Gly Val Val Leu Lys Gly 145 150 155 160 Glu Gln Asp Arg Pro Arg Ala Val Val Val Asn Pro Glu Lys Gly Tyr 165 170 175 Met Tyr Phe Thr Asn Leu Gln Glu Arg Ser Pro Lys Ile Glu Arg Ala 180 185 190 Ala Leu Asp Gly Thr Glu Arg Glu Val Leu Phe Phe Ser Gly Leu Ser 195 200 205 Lys Pro Ile Ala Leu Ala Leu Asp Ser Arg Leu Gly Lys Leu Phe Trp 210 215 220 Ala Asp Ser Asp Leu Arg Arg Ile Glu Ser Ser Asp Leu Ser Gly Ala 225 230 235 240 Asn Arg Ile Val Leu Glu Asp Ser Asn Ile Leu Gln Pro Val Gly Leu 245 250 255 Thr Val Phe Glu Asn Trp Leu Tyr Trp Ile Asp Lys Gln Gln Gln Met 260 265 270 Ile Glu Lys Ile Asp Met Thr Gly Arg Glu Gly Arg Thr Lys Val Gln 275 280 285 Ala Arg Ile Ala Gln Leu Ser Asp Ile His Ala Val Lys Glu Leu Asn 290 295 300 Leu Gln Glu Tyr Arg Gln His Pro Cys Ala Gln Asp Asn Gly Gly Cys 305 310 315 320 Ser His Ile Cys Leu Val Lys Gly Asp Gly Thr Thr Arg Cys Ser Cys 325 330 335 Pro Met His Leu Val Leu Leu Gln Asp Glu Leu Ser Cys Gly Glu Pro 340 345 350 Pro Thr Cys Ser Pro Gln Gln Phe Thr Cys Phe Thr Gly Glu Ile Asp 355 360 365 Cys Ile Pro Val Ala Trp Arg Cys Asp Gly Phe Thr Glu Cys Glu Asp 370 375 380 His Ser Asp Glu Leu Asn Cys Pro Val Cys Ser Glu Ser Gln Phe Gln 385 390 395 400 Cys Ala Ser Gly Gln Cys Ile Asp Gly Ala Leu Arg Cys Asn Gly Asp 405 410 415 Ala Asn Cys Gln Asp Lys Ser Asp Glu Lys Asn Cys Glu Val Leu Cys 420 425 430 Leu Ile Asp Gln Phe Arg Cys Ala Asn Gly Gln Cys Ile Gly Lys His 435 440 445 Lys Lys Cys Asp His Asn Val Asp Cys Ser Asp Lys Ser Asp Glu Leu 450 455 460 Asp Cys Tyr Pro Thr Glu Glu Pro Ala Pro Gln Ala Thr Asn Thr Val 465 470 475 480 Gly Ser Val Ile Gly Val Ile Val Thr Ile Phe Val Ser Gly Thr Val 485 490 495 Tyr Phe Ile Cys Gln Arg Met Leu Cys Pro Arg Met Lys Gly Asp Gly 500 505 510 Glu Thr Met Thr Asn Asp Tyr Val Val His Gly Pro Ala Ser Val Pro 515 520 525 Leu Gly Tyr Val Pro His Pro Ser Ser Leu Ser Gly Ser Leu Pro Gly 530 535

540 Met Ser Arg Gly Lys Ser Met Ile Ser Ser Leu Ser Ile Met Gly Gly 545 550 555 560 Ser Ser Gly Pro Pro Tyr Asp Arg Ala His Val Thr Gly Ala Ser Ser 565 570 575 Ser Ser Ser Ser Ser Thr Lys Gly Thr Tyr Phe Pro Ala Ile Leu Asn 580 585 590 Pro Pro Pro Ser Pro Ala Thr Glu Arg Ser His Tyr Thr Met Glu Phe 595 600 605 Gly Tyr Ser Ser Asn Ser Pro Ser Thr His Arg Ser Tyr Ser Tyr Arg 610 615 620 Pro Tyr Ser Tyr Arg His Phe Ala Pro Pro Thr Thr Pro Cys Ser Thr 625 630 635 640 Asp Val Cys Asp Ser Asp Tyr Ala Pro Ser Arg Arg Met Thr Ser Val 645 650 655 Ala Thr Ala Lys Gly Tyr Thr Ser Asp Leu Asn Tyr Asp Ser Glu Pro 660 665 670 Val Pro Pro Pro Pro Thr Pro Arg Ser Gln Tyr Leu Ser Ala Glu Glu 675 680 685 Asn Tyr Glu Ser Cys Pro Pro Ser Pro Tyr Thr Glu Arg Ser Tyr Ser 690 695 700 His His Leu Tyr Pro Pro Pro Pro Ser Pro Cys Thr Asp Ser Ser 705 710 715 101224DNAArtificialHA-tagged LRP6, deletion of propeller domains 1-4. 10atggagacag acacactcct gctatgggta ctgctgctct gggttccagg ttccactggt 60gacgctagat acccatacga tgttccagat tacgctggag gtggcgctag cggagaacct 120ccaacatgtt ctcctcagca gtttacttgt ttcacggggg aaattgactg tatccctgtg 180gcttggcggt gcgatgggtt tactgaatgt gaagaccaca gtgatgaact caattgtcct 240gtatgctcag agtcccagtt ccagtgtgcc agtgggcagt gtattgatgg tgccctccga 300tgcaatggag atgcaaactg ccaggacaaa tcagatgaga agaactgtga agtgctttgt 360ttaattgatc agttccgctg tgccaatggt cagtgcattg gaaagcacaa gaagtgtgat 420cataatgtgg attgcagtga caagtcagat gaactggatt gttatccgac tgaagaacca 480gcaccacagg ccaccaatac agttggttct gttattggcg taattgtcac catttttgtg 540tctggaactg tatactttat ctgccagagg atgttgtgtc cacgtatgaa gggagatggg 600gaaactatga ctaatgacta tgtagttcat ggaccagctt ctgtgcctct tggttatgtg 660ccacacccaa gttctttgtc aggatctctt ccaggaatgt ctcgaggtaa atcaatgatc 720agctccctca gtatcatggg gggaagcagt ggacccccct atgaccgagc ccatgttaca 780ggagcatcat caagtagttc ttcaagcacc aaaggcactt acttccctgc aattttgaac 840cctccaccat ccccagccac agagcgatca cattacacta tggaatttgg atattcttca 900aacagtcctt ccactcatag gtcatacagc tacaggccat atagctaccg gcactttgca 960ccccccacca caccctgcag cacagatgtt tgtgacagtg actatgctcc tagtcggaga 1020atgacctcag tggcaacagc caagggctat accagtgact tgaactatga ttcagaacct 1080gtgcccccac ctcccacacc ccgaagccaa tacttgtcag cagaggagaa ctatgaaagc 1140tgcccacctt ctccatacac agagaggagc tattctcatc acctctaccc accgccaccc 1200tctccctgta cagactcctc ctga 122411407PRTArtificialHA-tagged LRP6, deletion of propeller domains 1-4. 11Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Asp Ala Arg Tyr Pro Tyr Asp Val Pro Asp Tyr Ala 20 25 30 Gly Gly Gly Ala Ser Gly Glu Pro Pro Thr Cys Ser Pro Gln Gln Phe 35 40 45 Thr Cys Phe Thr Gly Glu Ile Asp Cys Ile Pro Val Ala Trp Arg Cys 50 55 60 Asp Gly Phe Thr Glu Cys Glu Asp His Ser Asp Glu Leu Asn Cys Pro 65 70 75 80 Val Cys Ser Glu Ser Gln Phe Gln Cys Ala Ser Gly Gln Cys Ile Asp 85 90 95 Gly Ala Leu Arg Cys Asn Gly Asp Ala Asn Cys Gln Asp Lys Ser Asp 100 105 110 Glu Lys Asn Cys Glu Val Leu Cys Leu Ile Asp Gln Phe Arg Cys Ala 115 120 125 Asn Gly Gln Cys Ile Gly Lys His Lys Lys Cys Asp His Asn Val Asp 130 135 140 Cys Ser Asp Lys Ser Asp Glu Leu Asp Cys Tyr Pro Thr Glu Glu Pro 145 150 155 160 Ala Pro Gln Ala Thr Asn Thr Val Gly Ser Val Ile Gly Val Ile Val 165 170 175 Thr Ile Phe Val Ser Gly Thr Val Tyr Phe Ile Cys Gln Arg Met Leu 180 185 190 Cys Pro Arg Met Lys Gly Asp Gly Glu Thr Met Thr Asn Asp Tyr Val 195 200 205 Val His Gly Pro Ala Ser Val Pro Leu Gly Tyr Val Pro His Pro Ser 210 215 220 Ser Leu Ser Gly Ser Leu Pro Gly Met Ser Arg Gly Lys Ser Met Ile 225 230 235 240 Ser Ser Leu Ser Ile Met Gly Gly Ser Ser Gly Pro Pro Tyr Asp Arg 245 250 255 Ala His Val Thr Gly Ala Ser Ser Ser Ser Ser Ser Ser Thr Lys Gly 260 265 270 Thr Tyr Phe Pro Ala Ile Leu Asn Pro Pro Pro Ser Pro Ala Thr Glu 275 280 285 Arg Ser His Tyr Thr Met Glu Phe Gly Tyr Ser Ser Asn Ser Pro Ser 290 295 300 Thr His Arg Ser Tyr Ser Tyr Arg Pro Tyr Ser Tyr Arg His Phe Ala 305 310 315 320 Pro Pro Thr Thr Pro Cys Ser Thr Asp Val Cys Asp Ser Asp Tyr Ala 325 330 335 Pro Ser Arg Arg Met Thr Ser Val Ala Thr Ala Lys Gly Tyr Thr Ser 340 345 350 Asp Leu Asn Tyr Asp Ser Glu Pro Val Pro Pro Pro Pro Thr Pro Arg 355 360 365 Ser Gln Tyr Leu Ser Ala Glu Glu Asn Tyr Glu Ser Cys Pro Pro Ser 370 375 380 Pro Tyr Thr Glu Arg Ser Tyr Ser His His Leu Tyr Pro Pro Pro Pro 385 390 395 400 Ser Pro Cys Thr Asp Ser Ser 405 122148DNAArtificialHA-tagged LRP6, deletion of propeller domains 2-4 12atggagacag acacactcct gctatgggta ctgctgctct gggttccagg ttccactggt 60gacgctagat acccatacga tgttccagat tacgctggag gtggcgctag cgcccctttg 120ttgctttatg caaacagacg ggacttgcga ttggttgatg ctacaaatgg caaagagaat 180gctacgattg tagttggagg cttggaggat gcagctgcgg tggactttgt gtttagtcat 240ggcttgatat actggagtga tgtcagcgaa gaagccatta aacgaacaga atttaacaaa 300actgagagtg tgcagaatgt tgttgtttct ggattattgt cccccgatgg gctggcatgt 360gattggcttg gagaaaaatt gtactggaca gattctgaaa ctaatcggat tgaagtttct 420aatttagatg gatctttacg aaaagtttta ttttggcaag agttggatca acccagagct 480attgccttag atccttcaag tgggttcatg tactggacag actggggaga agtgccaaag 540atagaacgtg ctggaatgga tggttcaagt cgcttcatta taataaacag tgaaatttac 600tggccaaatg gactgacttt ggattatgaa gaacaaaagc tttattgggc agatgcaaaa 660cttaatttca tccacaaatc aaatctggat ggaacaaatc ggcaggcagt ggttaaaggt 720tcccttccac atccttttgc cttgacgtta tttgaggaca tattgtactg gactgactgg 780agcacacact ccattttggc ttgcaacaag tatactggtg agggtctgcg tgaaatccat 840tctgacatct tctctcccat ggatatacat gccttcagcc aacagaggca gccaaatgcc 900acaaatccat gtggaattga caatgggggt tgttcccatt tgtgtttgat gtctccagtc 960aagccttttt atcagtgtgc ttgccccact ggggtcaaac tcctggagaa tggaaaaacc 1020tgcaaagatg gtgccggaga acctccaaca tgttctcctc agcagtttac ttgtttcacg 1080ggggaaattg actgtatccc tgtggcttgg cggtgcgatg ggtttactga atgtgaagac 1140cacagtgatg aactcaattg tcctgtatgc tcagagtccc agttccagtg tgccagtggg 1200cagtgtattg atggtgccct ccgatgcaat ggagatgcaa actgccagga caaatcagat 1260gagaagaact gtgaagtgct ttgtttaatt gatcagttcc gctgtgccaa tggtcagtgc 1320attggaaagc acaagaagtg tgatcataat gtggattgca gtgacaagtc agatgaactg 1380gattgttatc cgactgaaga accagcacca caggccacca atacagttgg ttctgttatt 1440ggcgtaattg tcaccatttt tgtgtctgga actgtatact ttatctgcca gaggatgttg 1500tgtccacgta tgaagggaga tggggaaact atgactaatg actatgtagt tcatggacca 1560gcttctgtgc ctcttggtta tgtgccacac ccaagttctt tgtcaggatc tcttccagga 1620atgtctcgag gtaaatcaat gatcagctcc ctcagtatca tggggggaag cagtggaccc 1680ccctatgacc gagcccatgt tacaggagca tcatcaagta gttcttcaag caccaaaggc 1740acttacttcc ctgcaatttt gaaccctcca ccatccccag ccacagagcg atcacattac 1800actatggaat ttggatattc ttcaaacagt ccttccactc ataggtcata cagctacagg 1860ccatatagct accggcactt tgcacccccc accacaccct gcagcacaga tgtttgtgac 1920agtgactatg ctcctagtcg gagaatgacc tcagtggcaa cagccaaggg ctataccagt 1980gacttgaact atgattcaga acctgtgccc ccacctccca caccccgaag ccaatacttg 2040tcagcagagg agaactatga aagctgccca ccttctccat acacagagag gagctattct 2100catcacctct acccaccgcc accctctccc tgtacagact cctcctga 214813715PRTArtificialHA-tagged LRP6, deletion of propeller domains 2-4 13Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Asp Ala Arg Tyr Pro Tyr Asp Val Pro Asp Tyr Ala 20 25 30 Gly Gly Gly Ala Ser Ala Pro Leu Leu Leu Tyr Ala Asn Arg Arg Asp 35 40 45 Leu Arg Leu Val Asp Ala Thr Asn Gly Lys Glu Asn Ala Thr Ile Val 50 55 60 Val Gly Gly Leu Glu Asp Ala Ala Ala Val Asp Phe Val Phe Ser His 65 70 75 80 Gly Leu Ile Tyr Trp Ser Asp Val Ser Glu Glu Ala Ile Lys Arg Thr 85 90 95 Glu Phe Asn Lys Thr Glu Ser Val Gln Asn Val Val Val Ser Gly Leu 100 105 110 Leu Ser Pro Asp Gly Leu Ala Cys Asp Trp Leu Gly Glu Lys Leu Tyr 115 120 125 Trp Thr Asp Ser Glu Thr Asn Arg Ile Glu Val Ser Asn Leu Asp Gly 130 135 140 Ser Leu Arg Lys Val Leu Phe Trp Gln Glu Leu Asp Gln Pro Arg Ala 145 150 155 160 Ile Ala Leu Asp Pro Ser Ser Gly Phe Met Tyr Trp Thr Asp Trp Gly 165 170 175 Glu Val Pro Lys Ile Glu Arg Ala Gly Met Asp Gly Ser Ser Arg Phe 180 185 190 Ile Ile Ile Asn Ser Glu Ile Tyr Trp Pro Asn Gly Leu Thr Leu Asp 195 200 205 Tyr Glu Glu Gln Lys Leu Tyr Trp Ala Asp Ala Lys Leu Asn Phe Ile 210 215 220 His Lys Ser Asn Leu Asp Gly Thr Asn Arg Gln Ala Val Val Lys Gly 225 230 235 240 Ser Leu Pro His Pro Phe Ala Leu Thr Leu Phe Glu Asp Ile Leu Tyr 245 250 255 Trp Thr Asp Trp Ser Thr His Ser Ile Leu Ala Cys Asn Lys Tyr Thr 260 265 270 Gly Glu Gly Leu Arg Glu Ile His Ser Asp Ile Phe Ser Pro Met Asp 275 280 285 Ile His Ala Phe Ser Gln Gln Arg Gln Pro Asn Ala Thr Asn Pro Cys 290 295 300 Gly Ile Asp Asn Gly Gly Cys Ser His Leu Cys Leu Met Ser Pro Val 305 310 315 320 Lys Pro Phe Tyr Gln Cys Ala Cys Pro Thr Gly Val Lys Leu Leu Glu 325 330 335 Asn Gly Lys Thr Cys Lys Asp Gly Ala Gly Glu Pro Pro Thr Cys Ser 340 345 350 Pro Gln Gln Phe Thr Cys Phe Thr Gly Glu Ile Asp Cys Ile Pro Val 355 360 365 Ala Trp Arg Cys Asp Gly Phe Thr Glu Cys Glu Asp His Ser Asp Glu 370 375 380 Leu Asn Cys Pro Val Cys Ser Glu Ser Gln Phe Gln Cys Ala Ser Gly 385 390 395 400 Gln Cys Ile Asp Gly Ala Leu Arg Cys Asn Gly Asp Ala Asn Cys Gln 405 410 415 Asp Lys Ser Asp Glu Lys Asn Cys Glu Val Leu Cys Leu Ile Asp Gln 420 425 430 Phe Arg Cys Ala Asn Gly Gln Cys Ile Gly Lys His Lys Lys Cys Asp 435 440 445 His Asn Val Asp Cys Ser Asp Lys Ser Asp Glu Leu Asp Cys Tyr Pro 450 455 460 Thr Glu Glu Pro Ala Pro Gln Ala Thr Asn Thr Val Gly Ser Val Ile 465 470 475 480 Gly Val Ile Val Thr Ile Phe Val Ser Gly Thr Val Tyr Phe Ile Cys 485 490 495 Gln Arg Met Leu Cys Pro Arg Met Lys Gly Asp Gly Glu Thr Met Thr 500 505 510 Asn Asp Tyr Val Val His Gly Pro Ala Ser Val Pro Leu Gly Tyr Val 515 520 525 Pro His Pro Ser Ser Leu Ser Gly Ser Leu Pro Gly Met Ser Arg Gly 530 535 540 Lys Ser Met Ile Ser Ser Leu Ser Ile Met Gly Gly Ser Ser Gly Pro 545 550 555 560 Pro Tyr Asp Arg Ala His Val Thr Gly Ala Ser Ser Ser Ser Ser Ser 565 570 575 Ser Thr Lys Gly Thr Tyr Phe Pro Ala Ile Leu Asn Pro Pro Pro Ser 580 585 590 Pro Ala Thr Glu Arg Ser His Tyr Thr Met Glu Phe Gly Tyr Ser Ser 595 600 605 Asn Ser Pro Ser Thr His Arg Ser Tyr Ser Tyr Arg Pro Tyr Ser Tyr 610 615 620 Arg His Phe Ala Pro Pro Thr Thr Pro Cys Ser Thr Asp Val Cys Asp 625 630 635 640 Ser Asp Tyr Ala Pro Ser Arg Arg Met Thr Ser Val Ala Thr Ala Lys 645 650 655 Gly Tyr Thr Ser Asp Leu Asn Tyr Asp Ser Glu Pro Val Pro Pro Pro 660 665 670 Pro Thr Pro Arg Ser Gln Tyr Leu Ser Ala Glu Glu Asn Tyr Glu Ser 675 680 685 Cys Pro Pro Ser Pro Tyr Thr Glu Arg Ser Tyr Ser His His Leu Tyr 690 695 700 Pro Pro Pro Pro Ser Pro Cys Thr Asp Ser Ser 705 710 715 143057DNAArtificialHA-tagged LRP6, deletion of propeller domains 3-4 14atggagacag acacactcct gctatgggta ctgctgctct gggttccagg ttccactggt 60gacgctagat acccatacga tgttccagat tacgctggag gtggcgctag cgcccctttg 120ttgctttatg caaacagacg ggacttgcga ttggttgatg ctacaaatgg caaagagaat 180gctacgattg tagttggagg cttggaggat gcagctgcgg tggactttgt gtttagtcat 240ggcttgatat actggagtga tgtcagcgaa gaagccatta aacgaacaga atttaacaaa 300actgagagtg tgcagaatgt tgttgtttct ggattattgt cccccgatgg gctggcatgt 360gattggcttg gagaaaaatt gtactggaca gattctgaaa ctaatcggat tgaagtttct 420aatttagatg gatctttacg aaaagtttta ttttggcaag agttggatca acccagagct 480attgccttag atccttcaag tgggttcatg tactggacag actggggaga agtgccaaag 540atagaacgtg ctggaatgga tggttcaagt cgcttcatta taataaacag tgaaatttac 600tggccaaatg gactgacttt ggattatgaa gaacaaaagc tttattgggc agatgcaaaa 660cttaatttca tccacaaatc aaatctggat ggaacaaatc ggcaggcagt ggttaaaggt 720tcccttccac atccttttgc cttgacgtta tttgaggaca tattgtactg gactgactgg 780agcacacact ccattttggc ttgcaacaag tatactggtg agggtctgcg tgaaatccat 840tctgacatct tctctcccat ggatatacat gccttcagcc aacagaggca gccaaatgcc 900acaaatccat gtggaattga caatgggggt tgttcccatt tgtgtttgat gtctccagtc 960aagccttttt atcagtgtgc ttgccccact ggggtcaaac tcctggagaa tggaaaaacc 1020tgcaaagatg gtgccacaga attattgctt ttagctcgaa ggacagactt gagacgcatt 1080tctttggata caccagattt tacagacatt gttctgcagt tagaagacat ccgtcatgcc 1140attgccatag attacgatcc tgtggaaggc tacatctact ggactgatga tgaagtgagg 1200gccatacgcc gttcatttat agatggatct ggcagtcagt ttgtggtcac tgctcaaatt 1260gcccatcctg atggtattgc tgtggactgg gttgcacgaa atctttattg gacagacact 1320ggcactgatc gaatagaagt gacaaggctc aatgggacca tgaggaagat cttgatttca 1380gaggacttag aggaaccccg ggctattgtg ttagatccca tggttgggta catgtattgg 1440actgactggg gagaaattcc gaaaattgag cgagcagctc tggatggttc tgaccgtgta 1500gtattggtta acacttctct tggttggcca aatggtttag ccttggatta tgatgaaggc 1560aaaatatact ggggagatgc caaaacagac aagattgagg ttatgaatac tgatggcact 1620gggagacgag tactagtgga agacaaaatt cctcacatat ttggatttac tttgttgggt 1680gactatgttt actggactga ctggcagagg cgtagcattg aaagagttca taaacgaagt 1740gcagagaggg aagtgatcat agatcagctg cctgacctca tgggcctaaa ggctacaaat 1800gttcatcgag tgattggttc caacccctgt gctgaggaaa acgggggatg tagccatctc 1860tgcctctata gacctcaggg ccttcgctgt gcttgcccta ttggctttga actcatcagt 1920gacatgaaga cctgcattgt cccaggagaa cctccaacat gttctcctca gcagtttact 1980tgtttcacgg gggaaattga ctgtatccct gtggcttggc ggtgcgatgg gtttactgaa 2040tgtgaagacc acagtgatga actcaattgt cctgtatgct cagagtccca gttccagtgt 2100gccagtgggc agtgtattga tggtgccctc cgatgcaatg gagatgcaaa ctgccaggac 2160aaatcagatg agaagaactg tgaagtgctt tgtttaattg atcagttccg ctgtgccaat 2220ggtcagtgca ttggaaagca caagaagtgt gatcataatg tggattgcag tgacaagtca 2280gatgaactgg attgttatcc gactgaagaa ccagcaccac aggccaccaa tacagttggt 2340tctgttattg gcgtaattgt caccattttt gtgtctggaa ctgtatactt tatctgccag 2400aggatgttgt gtccacgtat gaagggagat ggggaaacta tgactaatga ctatgtagtt 2460catggaccag cttctgtgcc tcttggttat gtgccacacc caagttcttt gtcaggatct 2520cttccaggaa tgtctcgagg taaatcaatg atcagctccc tcagtatcat ggggggaagc 2580agtggacccc cctatgaccg agcccatgtt acaggagcat catcaagtag ttcttcaagc 2640accaaaggca cttacttccc tgcaattttg aaccctccac catccccagc cacagagcga 2700tcacattaca ctatggaatt tggatattct tcaaacagtc cttccactca taggtcatac 2760agctacaggc catatagcta ccggcacttt gcacccccca ccacaccctg cagcacagat

2820gtttgtgaca gtgactatgc tcctagtcgg agaatgacct cagtggcaac agccaagggc 2880tataccagtg acttgaacta tgattcagaa cctgtgcccc cacctcccac accccgaagc 2940caatacttgt cagcagagga gaactatgaa agctgcccac cttctccata cacagagagg 3000agctattctc atcacctcta cccaccgcca ccctctccct gtacagactc ctcctga 3057151018PRTArtificialHA-tagged LRP6, deletion of propeller domains 3-4 15Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Asp Ala Arg Tyr Pro Tyr Asp Val Pro Asp Tyr Ala 20 25 30 Gly Gly Gly Ala Ser Ala Pro Leu Leu Leu Tyr Ala Asn Arg Arg Asp 35 40 45 Leu Arg Leu Val Asp Ala Thr Asn Gly Lys Glu Asn Ala Thr Ile Val 50 55 60 Val Gly Gly Leu Glu Asp Ala Ala Ala Val Asp Phe Val Phe Ser His 65 70 75 80 Gly Leu Ile Tyr Trp Ser Asp Val Ser Glu Glu Ala Ile Lys Arg Thr 85 90 95 Glu Phe Asn Lys Thr Glu Ser Val Gln Asn Val Val Val Ser Gly Leu 100 105 110 Leu Ser Pro Asp Gly Leu Ala Cys Asp Trp Leu Gly Glu Lys Leu Tyr 115 120 125 Trp Thr Asp Ser Glu Thr Asn Arg Ile Glu Val Ser Asn Leu Asp Gly 130 135 140 Ser Leu Arg Lys Val Leu Phe Trp Gln Glu Leu Asp Gln Pro Arg Ala 145 150 155 160 Ile Ala Leu Asp Pro Ser Ser Gly Phe Met Tyr Trp Thr Asp Trp Gly 165 170 175 Glu Val Pro Lys Ile Glu Arg Ala Gly Met Asp Gly Ser Ser Arg Phe 180 185 190 Ile Ile Ile Asn Ser Glu Ile Tyr Trp Pro Asn Gly Leu Thr Leu Asp 195 200 205 Tyr Glu Glu Gln Lys Leu Tyr Trp Ala Asp Ala Lys Leu Asn Phe Ile 210 215 220 His Lys Ser Asn Leu Asp Gly Thr Asn Arg Gln Ala Val Val Lys Gly 225 230 235 240 Ser Leu Pro His Pro Phe Ala Leu Thr Leu Phe Glu Asp Ile Leu Tyr 245 250 255 Trp Thr Asp Trp Ser Thr His Ser Ile Leu Ala Cys Asn Lys Tyr Thr 260 265 270 Gly Glu Gly Leu Arg Glu Ile His Ser Asp Ile Phe Ser Pro Met Asp 275 280 285 Ile His Ala Phe Ser Gln Gln Arg Gln Pro Asn Ala Thr Asn Pro Cys 290 295 300 Gly Ile Asp Asn Gly Gly Cys Ser His Leu Cys Leu Met Ser Pro Val 305 310 315 320 Lys Pro Phe Tyr Gln Cys Ala Cys Pro Thr Gly Val Lys Leu Leu Glu 325 330 335 Asn Gly Lys Thr Cys Lys Asp Gly Ala Thr Glu Leu Leu Leu Leu Ala 340 345 350 Arg Arg Thr Asp Leu Arg Arg Ile Ser Leu Asp Thr Pro Asp Phe Thr 355 360 365 Asp Ile Val Leu Gln Leu Glu Asp Ile Arg His Ala Ile Ala Ile Asp 370 375 380 Tyr Asp Pro Val Glu Gly Tyr Ile Tyr Trp Thr Asp Asp Glu Val Arg 385 390 395 400 Ala Ile Arg Arg Ser Phe Ile Asp Gly Ser Gly Ser Gln Phe Val Val 405 410 415 Thr Ala Gln Ile Ala His Pro Asp Gly Ile Ala Val Asp Trp Val Ala 420 425 430 Arg Asn Leu Tyr Trp Thr Asp Thr Gly Thr Asp Arg Ile Glu Val Thr 435 440 445 Arg Leu Asn Gly Thr Met Arg Lys Ile Leu Ile Ser Glu Asp Leu Glu 450 455 460 Glu Pro Arg Ala Ile Val Leu Asp Pro Met Val Gly Tyr Met Tyr Trp 465 470 475 480 Thr Asp Trp Gly Glu Ile Pro Lys Ile Glu Arg Ala Ala Leu Asp Gly 485 490 495 Ser Asp Arg Val Val Leu Val Asn Thr Ser Leu Gly Trp Pro Asn Gly 500 505 510 Leu Ala Leu Asp Tyr Asp Glu Gly Lys Ile Tyr Trp Gly Asp Ala Lys 515 520 525 Thr Asp Lys Ile Glu Val Met Asn Thr Asp Gly Thr Gly Arg Arg Val 530 535 540 Leu Val Glu Asp Lys Ile Pro His Ile Phe Gly Phe Thr Leu Leu Gly 545 550 555 560 Asp Tyr Val Tyr Trp Thr Asp Trp Gln Arg Arg Ser Ile Glu Arg Val 565 570 575 His Lys Arg Ser Ala Glu Arg Glu Val Ile Ile Asp Gln Leu Pro Asp 580 585 590 Leu Met Gly Leu Lys Ala Thr Asn Val His Arg Val Ile Gly Ser Asn 595 600 605 Pro Cys Ala Glu Glu Asn Gly Gly Cys Ser His Leu Cys Leu Tyr Arg 610 615 620 Pro Gln Gly Leu Arg Cys Ala Cys Pro Ile Gly Phe Glu Leu Ile Ser 625 630 635 640 Asp Met Lys Thr Cys Ile Val Pro Gly Glu Pro Pro Thr Cys Ser Pro 645 650 655 Gln Gln Phe Thr Cys Phe Thr Gly Glu Ile Asp Cys Ile Pro Val Ala 660 665 670 Trp Arg Cys Asp Gly Phe Thr Glu Cys Glu Asp His Ser Asp Glu Leu 675 680 685 Asn Cys Pro Val Cys Ser Glu Ser Gln Phe Gln Cys Ala Ser Gly Gln 690 695 700 Cys Ile Asp Gly Ala Leu Arg Cys Asn Gly Asp Ala Asn Cys Gln Asp 705 710 715 720 Lys Ser Asp Glu Lys Asn Cys Glu Val Leu Cys Leu Ile Asp Gln Phe 725 730 735 Arg Cys Ala Asn Gly Gln Cys Ile Gly Lys His Lys Lys Cys Asp His 740 745 750 Asn Val Asp Cys Ser Asp Lys Ser Asp Glu Leu Asp Cys Tyr Pro Thr 755 760 765 Glu Glu Pro Ala Pro Gln Ala Thr Asn Thr Val Gly Ser Val Ile Gly 770 775 780 Val Ile Val Thr Ile Phe Val Ser Gly Thr Val Tyr Phe Ile Cys Gln 785 790 795 800 Arg Met Leu Cys Pro Arg Met Lys Gly Asp Gly Glu Thr Met Thr Asn 805 810 815 Asp Tyr Val Val His Gly Pro Ala Ser Val Pro Leu Gly Tyr Val Pro 820 825 830 His Pro Ser Ser Leu Ser Gly Ser Leu Pro Gly Met Ser Arg Gly Lys 835 840 845 Ser Met Ile Ser Ser Leu Ser Ile Met Gly Gly Ser Ser Gly Pro Pro 850 855 860 Tyr Asp Arg Ala His Val Thr Gly Ala Ser Ser Ser Ser Ser Ser Ser 865 870 875 880 Thr Lys Gly Thr Tyr Phe Pro Ala Ile Leu Asn Pro Pro Pro Ser Pro 885 890 895 Ala Thr Glu Arg Ser His Tyr Thr Met Glu Phe Gly Tyr Ser Ser Asn 900 905 910 Ser Pro Ser Thr His Arg Ser Tyr Ser Tyr Arg Pro Tyr Ser Tyr Arg 915 920 925 His Phe Ala Pro Pro Thr Thr Pro Cys Ser Thr Asp Val Cys Asp Ser 930 935 940 Asp Tyr Ala Pro Ser Arg Arg Met Thr Ser Val Ala Thr Ala Lys Gly 945 950 955 960 Tyr Thr Ser Asp Leu Asn Tyr Asp Ser Glu Pro Val Pro Pro Pro Pro 965 970 975 Thr Pro Arg Ser Gln Tyr Leu Ser Ala Glu Glu Asn Tyr Glu Ser Cys 980 985 990 Pro Pro Ser Pro Tyr Thr Glu Arg Ser Tyr Ser His His Leu Tyr Pro 995 1000 1005 Pro Pro Pro Ser Pro Cys Thr Asp Ser Ser 1010 1015 16282PRTHomo sapiens 16Ala Thr Ile Val Val Gly Gly Leu Glu Asp Ala Ala Ala Val Asp Phe 1 5 10 15 Val Phe Ser His Gly Leu Ile Tyr Trp Ser Asp Val Ser Glu Glu Ala 20 25 30 Ile Lys Arg Thr Glu Phe Asn Lys Thr Glu Ser Val Gln Asn Val Val 35 40 45 Val Ser Gly Leu Leu Ser Pro Asp Gly Leu Ala Cys Asp Trp Leu Gly 50 55 60 Glu Lys Leu Tyr Trp Thr Asp Ser Glu Thr Asn Arg Ile Glu Val Ser 65 70 75 80 Asn Leu Asp Gly Ser Leu Arg Lys Val Leu Phe Trp Gln Glu Leu Asp 85 90 95 Gln Pro Arg Ala Ile Ala Leu Asp Pro Ser Ser Gly Phe Met Tyr Trp 100 105 110 Thr Asp Trp Gly Glu Val Pro Lys Ile Glu Arg Ala Gly Met Asp Gly 115 120 125 Ser Ser Arg Phe Ile Ile Ile Asn Ser Glu Ile Tyr Trp Pro Asn Gly 130 135 140 Leu Thr Leu Asp Tyr Glu Glu Gln Lys Leu Tyr Trp Ala Asp Ala Lys 145 150 155 160 Leu Asn Phe Ile His Lys Ser Asn Leu Asp Gly Thr Asn Arg Gln Ala 165 170 175 Val Val Lys Gly Ser Leu Pro His Pro Phe Ala Leu Thr Leu Phe Glu 180 185 190 Asp Ile Leu Tyr Trp Thr Asp Trp Ser Thr His Ser Ile Leu Ala Cys 195 200 205 Asn Lys Tyr Thr Gly Glu Gly Leu Arg Glu Ile His Ser Asp Ile Phe 210 215 220 Ser Pro Met Asp Ile His Ala Phe Ser Gln Gln Arg Gln Pro Asn Ala 225 230 235 240 Thr Asn Pro Cys Gly Ile Asp Asn Gly Gly Cys Ser His Leu Cys Leu 245 250 255 Met Ser Pro Val Lys Pro Phe Tyr Gln Cys Ala Cys Pro Thr Gly Val 260 265 270 Lys Leu Leu Glu Asn Gly Lys Thr Cys Lys 275 280 17441DNAMus musculus 17atggaaaggc actggatctt tctcttcctg ttttcagtaa ctgcaggtgt ccactcccag 60gtccaacttc agcagtctgg ggctgaactg gcaaaacctg gggcctcagt gaagatgtcc 120tgcaaggctt ctggctacac ctttactagc tactggatgc actgggtaaa acagaggcct 180ggacagggtc tggaatggat tggatacatt aatcctagca ctggttatac tgagtacaat 240cagaacttca gggacaaggc cacattgact gcagacaaat cctccagcac agccaacatg 300caactgagca gcctgacatc tgaggactct gcagtctatt actgtgcaag aactgcgcaa 360tactacggta gtcctagggg ttactatgct atggactcct ggggtcaagg aacctcagtc 420accgtctcct cagccaaaac a 44118147PRTMus musculus 18Met Glu Arg His Trp Ile Phe Leu Phe Leu Phe Ser Val Thr Ala Gly 1 5 10 15 Val His Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Lys 20 25 30 Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 Thr Ser Tyr Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu 50 55 60 Glu Trp Ile Gly Tyr Ile Asn Pro Ser Thr Gly Tyr Thr Glu Tyr Asn 65 70 75 80 Gln Asn Phe Arg Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser 85 90 95 Thr Ala Asn Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Thr Ala Gln Tyr Tyr Gly Ser Pro Arg Gly Tyr 115 120 125 Tyr Ala Met Asp Ser Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser 130 135 140 Ala Lys Thr 145 19393DNAMus musculus 19atgaggttct ctgctcagct tctggggctg cttgtgctct ggatccctgg atccactgca 60gatattgtga tgacgcaggc tgcattctcc aatccagtca ctcttggaac atcagcttcc 120atctcctgca ggtctagtaa gagtctccta catagtaacg gcatcactta tttgtgttgg 180tttctgcaga agccaggcca gtctcctcag ctcctgattt atctgatgtc caaccttgcc 240tcaggagtcc cagacaggtt cagtagcagt gggtcaggaa ctgatttcac actgagaatc 300agcagagtgg aggctgagga tgtgggtgtt tatttctgtg ctcaaaatct agatcttccg 360tggacgttcg gtgggggcac caagctggaa atc 39320131PRTMus musculus 20Met Arg Phe Ser Ala Gln Leu Leu Gly Leu Leu Val Leu Trp Ile Pro 1 5 10 15 Gly Ser Thr Ala Asp Ile Val Met Thr Gln Ala Ala Phe Ser Asn Pro 20 25 30 Val Thr Leu Gly Thr Ser Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser 35 40 45 Leu Leu His Ser Asn Gly Ile Thr Tyr Leu Cys Trp Phe Leu Gln Lys 50 55 60 Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Leu Met Ser Asn Leu Ala 65 70 75 80 Ser Gly Val Pro Asp Arg Phe Ser Ser Ser Gly Ser Gly Thr Asp Phe 85 90 95 Thr Leu Arg Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Phe 100 105 110 Cys Ala Gln Asn Leu Asp Leu Pro Trp Thr Phe Gly Gly Gly Thr Lys 115 120 125 Leu Glu Ile 130 21449DNAMus musculus 21atggcgtgga tctctatcat cctcttccta gtggcaacag ctttaggtgt ccactcccag 60gttcaactgc agcagtctgg ggctgagctg gtgaagcctg gggcctcagt gaagatgtcc 120tgcaaggctt ttggctacac cttcacttcc tatccaatag agtggatgaa acagaatcat 180gggaagagcc tagagtggat tggaaatttt catccttaca atgataatac taagtacaat 240gaaaaattca agggcaaggc caaattgact gttgaaaaat cctctagcac agtctacttg 300gagctcagcc gatcaacatc tgatgactct gctgtttatt actgtgcaag ggggtactct 360ggtaactact tctctgctat ggactactgg ggtcaaggaa cctcagtcac cgtctcctca 420gccaaaacaa cagccccatc ggtctatcc 44922150PRTMus musculus 22Met Ala Trp Ile Ser Ile Ile Leu Phe Leu Val Ala Thr Ala Leu Gly 1 5 10 15 Val His Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys 20 25 30 Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Phe Gly Tyr Thr Phe 35 40 45 Thr Ser Tyr Pro Ile Glu Trp Met Lys Gln Asn His Gly Lys Ser Leu 50 55 60 Glu Trp Ile Gly Asn Phe His Pro Tyr Asn Asp Asn Thr Lys Tyr Asn 65 70 75 80 Glu Lys Phe Lys Gly Lys Ala Lys Leu Thr Val Glu Lys Ser Ser Ser 85 90 95 Thr Val Tyr Leu Glu Leu Ser Arg Ser Thr Ser Asp Asp Ser Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Gly Tyr Ser Gly Asn Tyr Phe Ser Ala Met Asp 115 120 125 Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Lys Thr Thr 130 135 140 Ala Pro Ser Val Tyr Pro 145 150 23411DNAMus musculus 23atgggcatca agatggagtt tcagacccag gtctttgtat tcgtgttgct ctggttgtct 60ggtgttgatg gagacattgt gatgacccag tctcaaaaat tcatgtccac atcagtagga 120gacagggtca gcatcacctg caaggccagt cagaatgttc gtaatgatgt agcctggtat 180caacagaaac cagggcagtc tcctaaatca ctgatttact tggcatccaa ccggcacact 240ggagtccctg atcgcttcac aggcagtgga tctgggacag atttcactct caccattagc 300aatgtgcaat ctgaagacct ggcagattat ttctgtctgc aacattggaa ttatccgtac 360acgttcggag gggggaccaa gctggaaata aaacgggctg atgctgcacc g 41124137PRTMus musculus 24Met Gly Ile Lys Met Glu Phe Gln Thr Gln Val Phe Val Phe Val Leu 1 5 10 15 Leu Trp Leu Ser Gly Val Asp Gly Asp Ile Val Met Thr Gln Ser Gln 20 25 30 Lys Phe Met Ser Thr Ser Val Gly Asp Arg Val Ser Ile Thr Cys Lys 35 40 45 Ala Ser Gln Asn Val Arg Asn Asp Val Ala Trp Tyr Gln Gln Lys Pro 50 55 60 Gly Gln Ser Pro Lys Ser Leu Ile Tyr Leu Ala Ser Asn Arg His Thr 65 70 75 80 Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr 85 90 95 Leu Thr Ile Ser Asn Val Gln Ser Glu Asp Leu Ala Asp Tyr Phe Cys 100 105 110 Leu Gln His Trp Asn Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu 115 120 125 Glu Ile Lys Arg Ala Asp Ala Ala Pro 130 135 25423DNAMus musculus 25atgaacttcg ggctcagatt gattttcctt gtccttgttt taaaaggtgt cctgtgtgac 60gtgaagctcg tggagtctgg gggaggctta gtgaaggttg gcgggtccct gaaactctcc 120tgtgcagcct ctggattcac tttcagtagt tattacatgt cttgggttcg ccagactcca 180gagaagaggc tggagttggt cgcagtcatt aatactaatg gtggtagcac ctactattca 240gacactgtga agggccgatt caccatctcc agagacaatg ccaagaacac cctgtacctg 300caaatgagca gtctgaagtc tgaggacaca gccttgtatt actgttcaag acaaccctat 360tatggtaacc cttttgacta ctggggccaa ggcaccactc tcacagtctc ctcagccaaa 420aca 42326141PRTMus musculus 26Met Asn Phe Gly Leu Arg Leu Ile Phe Leu Val Leu Val Leu Lys Gly 1 5

10 15 Val Leu Cys Asp Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys 20 25 30 Val Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35 40 45 Ser Ser Tyr Tyr Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu 50 55 60 Glu Leu Val Ala Val Ile Asn Thr Asn Gly Gly Ser Thr Tyr Tyr Ser 65 70 75 80 Asp Thr Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn 85 90 95 Thr Leu Tyr Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Leu 100 105 110 Tyr Tyr Cys Ser Arg Gln Pro Tyr Tyr Gly Asn Pro Phe Asp Tyr Trp 115 120 125 Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Ala Lys Thr 130 135 140 27378DNAMus musculus 27atggttttca cacctcagat acttggactt atgctttttt ggatttcagc ctccagaagt 60gatattgtgc taactcagtc tccagccacc ctgtctgtga ctccaggaga tagcgtcagt 120ctttcctgca gggccagcca aagtattagc aacaacctac actggtatca acaaaaatca 180catgagtctc caaggcttct catcaagtat gtttcccagt ccatctctgg gatcccctcc 240aggttcagtg gcagtggatc agggacagat ttcactctca gtatcaacag tgtggagact 300gaagattttg gaatgtattt ctgtcaacag agtaacaact ggccgctcac gttcggtgct 360gggaccaagc tggagctg 37828126PRTMus musculus 28Met Val Phe Thr Pro Gln Ile Leu Gly Leu Met Leu Phe Trp Ile Ser 1 5 10 15 Ala Ser Arg Ser Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser 20 25 30 Val Thr Pro Gly Asp Ser Val Ser Leu Ser Cys Arg Ala Ser Gln Ser 35 40 45 Ile Ser Asn Asn Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro 50 55 60 Arg Leu Leu Ile Lys Tyr Val Ser Gln Ser Ile Ser Gly Ile Pro Ser 65 70 75 80 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn 85 90 95 Ser Val Glu Thr Glu Asp Phe Gly Met Tyr Phe Cys Gln Gln Ser Asn 100 105 110 Asn Trp Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu 115 120 125 29426DNAMus musculus 29atgaacttcg ggctcagatt gattttcctt gtccttgttt taaaaggtgt cctgtgtgac 60gtgaagctcg tggagtctgg gggaggctta gtgaagcttg gagggtccct aaaactctcc 120tgtgcagcct ctggattcac tttcagtagc tattacatgt cttgggttcg ccagactcca 180gagaagaggc tggagttggt cgcagccatt aatagtaatg gtggtagtac ctactatgca 240gacactatga agggccgatt caccatctcc agagacaatg ccaagaacac cctttacctg 300caaatgatca gtctgaagtc tgaggacaca gccttctatt actgtgcaag cgagttggcc 360gggtatggta ccccgtttgc ttactggggc cacgggactc tggtcactgt ctctgcagcc 420aaaacg 42630142PRTMus musculus 30Met Asn Phe Gly Leu Arg Leu Ile Phe Leu Val Leu Val Leu Lys Gly 1 5 10 15 Val Leu Cys Asp Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys 20 25 30 Leu Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35 40 45 Ser Ser Tyr Tyr Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu 50 55 60 Glu Leu Val Ala Ala Ile Asn Ser Asn Gly Gly Ser Thr Tyr Tyr Ala 65 70 75 80 Asp Thr Met Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn 85 90 95 Thr Leu Tyr Leu Gln Met Ile Ser Leu Lys Ser Glu Asp Thr Ala Phe 100 105 110 Tyr Tyr Cys Ala Ser Glu Leu Ala Gly Tyr Gly Thr Pro Phe Ala Tyr 115 120 125 Trp Gly His Gly Thr Leu Val Thr Val Ser Ala Ala Lys Thr 130 135 140 31378DNAMus musculus 31atggttttca cacctcagat acttggactt atgctttttt ggatttcagc ctccagaggt 60gatattgtgc tcactcagtt tccagccacc ctgtctgtga ctccaggaga tagcgtcagt 120ctttcctgca gggccagcca aagtattagc agcaacctac actggtatca acaaacatca 180catgagtctc caaggcttct catcaagtat gcttcccagt ccatctctgg catcccctcc 240aggttcagtg gcactggatc agggacagat ttcactctca gtatcaacag tgtggagact 300gaagattttg gaatgtattt ctgtcaacag agtaacacct ggccgctcac gttcggtgct 360gggaccaagc tggagctg 37832126PRTMus musculus 32Met Val Phe Thr Pro Gln Ile Leu Gly Leu Met Leu Phe Trp Ile Ser 1 5 10 15 Ala Ser Arg Gly Asp Ile Val Leu Thr Gln Phe Pro Ala Thr Leu Ser 20 25 30 Val Thr Pro Gly Asp Ser Val Ser Leu Ser Cys Arg Ala Ser Gln Ser 35 40 45 Ile Ser Ser Asn Leu His Trp Tyr Gln Gln Thr Ser His Glu Ser Pro 50 55 60 Arg Leu Leu Ile Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser 65 70 75 80 Arg Phe Ser Gly Thr Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn 85 90 95 Ser Val Glu Thr Glu Asp Phe Gly Met Tyr Phe Cys Gln Gln Ser Asn 100 105 110 Thr Trp Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu 115 120 125 33468DNAMus musculus 33atgtcctctc cacagtccct gcagacactg accctaagca tggaatggag atggatcttt 60ctcttcctcc tgtcaggaac tacaggtgtc cactctgaga tccagctgca gcagtctgga 120cctgagctgg tgaagcctgg ggcttcagtg aaggtatcct gcaaggcttc tggttatgca 180ttcactagct acaacatgta ctgggtgaaa cagagccatg gaaagggcct tgagtggatt 240ggatatattg atccttacaa tggcggtact gactccaacc agaacttcaa gggcaaggcc 300acattgactg ttgacaagtc ctccagcaca gccttcatgc atctcaacag cctgacatct 360gaggactctg cagtctatta ctgtgcaaga ggggggatgg gattacgacg ggaccacttt 420gactactggg gccaaggcac cagtctcacg gtctcctcag ccaaaaca 46834156PRTMus musculus 34Met Ser Ser Pro Gln Ser Leu Gln Thr Leu Thr Leu Ser Met Glu Trp 1 5 10 15 Arg Trp Ile Phe Leu Phe Leu Leu Ser Gly Thr Thr Gly Val His Ser 20 25 30 Glu Ile Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 35 40 45 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Ser Tyr 50 55 60 Asn Met Tyr Trp Val Lys Gln Ser His Gly Lys Gly Leu Glu Trp Ile 65 70 75 80 Gly Tyr Ile Asp Pro Tyr Asn Gly Gly Thr Asp Ser Asn Gln Asn Phe 85 90 95 Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Phe 100 105 110 Met His Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 115 120 125 Ala Arg Gly Gly Met Gly Leu Arg Arg Asp His Phe Asp Tyr Trp Gly 130 135 140 Gln Gly Thr Ser Leu Thr Val Ser Ser Ala Lys Thr 145 150 155 35381DNAMus musculus 35atggattttc aggtgcagat tttcagcttc ctgctaatca gtgcctcagt cataatgtcc 60agaggacaaa ttgttctcac ccagtctcca gcaatcatgt ctgcatctcc aggggagaag 120gtcaccatat cctgcagtgc caactcaagt gtacgttaca tgttctggta ccagcagaag 180ccaggatcct tccccaaacc ctggacttat cgcacatcca acctggcttc tggagtccct 240gctcgcttca gtggcagtgg gtctgggacc tcttactctc tcacaatcag cagtatggag 300gctgaagatg ttgccactta ttactgccag cagtatcata gttacccgtg gacgttcggt 360ggaggcacca agctggaaat c 38136127PRTMus musculus 36Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser 1 5 10 15 Val Ile Met Ser Arg Gly Gln Ile Val Leu Thr Gln Ser Pro Ala Ile 20 25 30 Met Ser Ala Ser Pro Gly Glu Lys Val Thr Ile Ser Cys Ser Ala Asn 35 40 45 Ser Ser Val Arg Tyr Met Phe Trp Tyr Gln Gln Lys Pro Gly Ser Phe 50 55 60 Pro Lys Pro Trp Thr Tyr Arg Thr Ser Asn Leu Ala Ser Gly Val Pro 65 70 75 80 Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile 85 90 95 Ser Ser Met Glu Ala Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Tyr 100 105 110 His Ser Tyr Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 115 120 125 37420DNAMus musculus 37atgggatgga gccggatctt tctcttcctc ctgtcaataa ttgcaagtgt ccattgccag 60gtccagctgc agcagtctgg acctgagctg gtgaagcctg gggcttcagt gaggatatcc 120tgcaaggctt ctggctacac cttcacaacc tactatatac actggttgaa acagaggcct 180ggacagggac ttgagtggat tggatggatt tttcctggaa atgttaatac taagtacaat 240gcgaagttca agggcaaggc cacactgact gcagacaagt cctccagcac agcctacatg 300cagctcagca gcctgacctc tgaggactct gcggtctatt tctgtgcaag agaggaatta 360cagtactact ttgactactg gggccaaggc tccactctca cagtctcctc agccaaaaca 4203880PRTMus musculus 38Met Gly Trp Ser Arg Ile Phe Leu Phe Leu Leu Ser Ile Ile Ala Ser 1 5 10 15 Val His Cys Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys 20 25 30 Pro Gly Ala Ser Val Arg Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 Thr Thr Tyr Tyr Ile His Trp Leu Lys Gln Arg Pro Gly Gln Gly Leu 50 55 60 Glu Trp Ile Gly Trp Ile Phe Pro Gly Asn Val Asn Thr Lys Tyr Asn 65 70 75 80 39327DNAMus musculus 39atgtccagag gacaaattgt tctcacccag tctccagcaa tcatgtctgc atctccaggg 60gagaaggtca ccatatcctg cagtgccaac tcaagtgtac gtttcatgtt ctggtaccag 120cagaagccag gatcctcccc caaacccttg atttatcgca catccaacct ggcctctgga 180gtccctgctc gcttcagtgg ctgtgggtct gggacctctt actctctcac aatcagcagc 240atggaggctg aagatgccgc cacttattac tgccagcagt atcatagtta cccgtggacg 300ttcggtggag gcaccaagtt ggaaatc 32740109PRTMus musculus 40Met Ser Arg Gly Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser 1 5 10 15 Ala Ser Pro Gly Glu Lys Val Thr Ile Ser Cys Ser Ala Asn Ser Ser 20 25 30 Val Arg Phe Met Phe Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys 35 40 45 Pro Leu Ile Tyr Arg Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg 50 55 60 Phe Ser Gly Cys Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser 65 70 75 80 Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Tyr His Ser 85 90 95 Tyr Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 100 105 41423DNAMus musculus 41atgaacttcg ggctcagatt gattttcctt gtccttgttt taaaaggtgt cctgtgtgac 60gtgaagctcg tggagtctgg gggaggctta gtgaggcttg gagggtccct gaaactctcc 120tgtgcagcct ctggattcac tttcagtacc tattacatgt cttgggttcg ccagactcca 180gagaagaggc tggagttggt cgcaaccatt aatactaatg gtggtagcac ctactatcca 240gacactttga agggccgatt caccatctcc agagacaatg ccaagaacac cctgtacctg 300caaatgagca gtctgaggtc tgaggacaca gccttgtatt actgtgcgag acagcgtaac 360tacggagtgg ctgtggactc ctggggtcaa ggaacctcag tcaccgtctc ctcagccaaa 420acg 42342141PRTMus musculus 42Met Asn Phe Gly Leu Arg Leu Ile Phe Leu Val Leu Val Leu Lys Gly 1 5 10 15 Val Leu Cys Asp Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Arg 20 25 30 Leu Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35 40 45 Ser Thr Tyr Tyr Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu 50 55 60 Glu Leu Val Ala Thr Ile Asn Thr Asn Gly Gly Ser Thr Tyr Tyr Pro 65 70 75 80 Asp Thr Leu Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn 85 90 95 Thr Leu Tyr Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Leu 100 105 110 Tyr Tyr Cys Ala Arg Gln Arg Asn Tyr Gly Val Ala Val Asp Ser Trp 115 120 125 Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Lys Thr 130 135 140 43378DNAMus musculus 43atggttttca cacctcagat acttggactt atgctttttt ggatttcagc ctccagaggt 60gatattgtgc tgactcagtc tccagccacc ctgtctgtga ctccaggaga tagcgtcagt 120ctttcctgca gggccagcca aagtattagc aacaacctac actggtatca acaaaaatca 180catgagtctc caaggcttct catcaagtat gcttcccagt ccatctctgg gatcccctcc 240aggttcagtg gcagtggatc agggacagat ttcactctca gtatcaacag tgtggagact 300gaagattttg gaatgtattt ctgtcaacag actaacaact ggcctctcac gttcggtgct 360gggaccaagc tggagcgg 37844126PRTMus musculus 44Met Val Phe Thr Pro Gln Ile Leu Gly Leu Met Leu Phe Trp Ile Ser 1 5 10 15 Ala Ser Arg Gly Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser 20 25 30 Val Thr Pro Gly Asp Ser Val Ser Leu Ser Cys Arg Ala Ser Gln Ser 35 40 45 Ile Ser Asn Asn Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro 50 55 60 Arg Leu Leu Ile Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser 65 70 75 80 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn 85 90 95 Ser Val Glu Thr Glu Asp Phe Gly Met Tyr Phe Cys Gln Gln Thr Asn 100 105 110 Asn Trp Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Arg 115 120 125 45423DNAMus musculus 45atgaaagtgt tgagtctgtt gtacctgttg acagccattc ctggtttcct gtctgatgta 60cagcttcagg agtcaggacc tggcctcgtg aaaccttctc agtctctgtc tctcacctgc 120tctgtcactg gctactccat caccagtggt tattactgga actggatccg gcaatttcca 180ggagacaaac tggaatggat gggccacata aactacgacg gtagagataa ctacaaccca 240tctctcaaaa atcgaatctc catcactcgt gacacatcta agaaccagtt tttcctgaag 300ttgaattctg tgactactga ggacacagct acatattact gtgcaagaga gtttggtaac 360ttcccttact actttgacta ctggggccaa ggcaccactc tcacagtctc ctcagccaaa 420aca 42346141PRTMus musculus 46Met Lys Val Leu Ser Leu Leu Tyr Leu Leu Thr Ala Ile Pro Gly Phe 1 5 10 15 Leu Ser Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro 20 25 30 Ser Gln Ser Leu Ser Leu Thr Cys Ser Val Thr Gly Tyr Ser Ile Thr 35 40 45 Ser Gly Tyr Tyr Trp Asn Trp Ile Arg Gln Phe Pro Gly Asp Lys Leu 50 55 60 Glu Trp Met Gly His Ile Asn Tyr Asp Gly Arg Asp Asn Tyr Asn Pro 65 70 75 80 Ser Leu Lys Asn Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln 85 90 95 Phe Phe Leu Lys Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr 100 105 110 Tyr Cys Ala Arg Glu Phe Gly Asn Phe Pro Tyr Tyr Phe Asp Tyr Trp 115 120 125 Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Ala Lys Thr 130 135 140 47381DNAMus musculus 47atggattttc aggtgcagat tctcagcatc ctgctaatca gtgcctcagt cataatgtcc 60agaggacaaa ttgttctcac ccagtctcca gcaatcatgt ctgcatctcc aggggagaag 120gtcaccatat cctgcagtgc ctactcaagt gtacgtttca tgttctggta ccagcagaag 180ccaggatcct cccccaaacc cttgatttat cgcacatcca acctggcttc tggagtctct 240gctcgcttca gtggcagtgg gtctgggacc tcttactctc tcacaatcag cagcatggag 300gctgaagatg ctgccactta ttactgccag cagtatcata gttacccgtg gacgttcggt 360ggaggcacca agttggaaat c 38148127PRTMus musculus 48Met Asp Phe Gln Val Gln Ile Leu Ser Ile Leu Leu Ile Ser Ala Ser 1 5 10 15 Val Ile Met Ser Arg Gly Gln Ile Val Leu Thr Gln Ser Pro Ala Ile 20 25 30 Met Ser Ala Ser Pro Gly Glu Lys Val Thr Ile Ser Cys Ser Ala Tyr 35 40 45 Ser Ser Val Arg Phe Met Phe Trp Tyr Gln Gln Lys Pro Gly Ser Ser 50 55 60 Pro Lys Pro Leu Ile Tyr Arg Thr Ser Asn Leu Ala Ser Gly Val Ser 65 70 75 80 Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile 85 90

95 Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Tyr 100 105 110 His Ser Tyr Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 115 120 125 49423DNAMus musculus 49atgaacttcg ggctcagatt gactttcctt gtccttgttt taaaaggtgt cctgtgtgac 60gtgaagctcg tggagtcggg gggaggctta gtgaaggttg gagggtccct gaaactctcc 120tgtgcagcct ctggattcac tttcagtagt tattacatgt cttgggttcg ccagactcca 180gagaagaggc tggagttggt cgcagtcatt aatagtaatg gtggtagcac ctactattca 240gagactgtga agggccgatt caccatctcc agagacaatg ccaagaacac cctgtacctg 300caaatgagca gtctgaagtc tgaggacaca gccttgtatt actgttcaag acaaccctat 360tatggtaacc cttttgacta ctggggccaa ggcaccactc tcacagtctc ctcagccaaa 420aca 42350141PRTMus musculus 50Met Asn Phe Gly Leu Arg Leu Thr Phe Leu Val Leu Val Leu Lys Gly 1 5 10 15 Val Leu Cys Asp Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys 20 25 30 Val Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35 40 45 Ser Ser Tyr Tyr Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu 50 55 60 Glu Leu Val Ala Val Ile Asn Ser Asn Gly Gly Ser Thr Tyr Tyr Ser 65 70 75 80 Glu Thr Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn 85 90 95 Thr Leu Tyr Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Leu 100 105 110 Tyr Tyr Cys Ser Arg Gln Pro Tyr Tyr Gly Asn Pro Phe Asp Tyr Trp 115 120 125 Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Ala Lys Thr 130 135 140 51378DNAMus musculus 51atggttttca cacctcagat acttggactt atgctttttt ggatttcagc ctccagaggt 60gatattgtgc taactcagtc tccagccacc ctgtctgtga ctccaggaga tagcgtcagt 120ctttcctgca gggccagcca aagtattagc aacaacctac actggtatca acaaaaatca 180catgagtctc caaggcttct catcaagtat gtttcccagt ccatctctgg gatcccctcc 240aggttcagtg gcagtggatc agggacagat ttcactctca gtatcaacag tgtggagact 300gaagattttg gaatgtattt ctgtcaacag agtaacaact ggccgctcac gttcggtgct 360gggaccaagc tggagctg 37852126PRTMus musculus 52Met Val Phe Thr Pro Gln Ile Leu Gly Leu Met Leu Phe Trp Ile Ser 1 5 10 15 Ala Ser Arg Gly Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser 20 25 30 Val Thr Pro Gly Asp Ser Val Ser Leu Ser Cys Arg Ala Ser Gln Ser 35 40 45 Ile Ser Asn Asn Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro 50 55 60 Arg Leu Leu Ile Lys Tyr Val Ser Gln Ser Ile Ser Gly Ile Pro Ser 65 70 75 80 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn 85 90 95 Ser Val Glu Thr Glu Asp Phe Gly Met Tyr Phe Cys Gln Gln Ser Asn 100 105 110 Asn Trp Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu 115 120 125 53487DNAMus musculus 53atgtcctctc cacagtccct gcagacactg accctaagca tggaatggag atggatcttt 60ctcttcctcc tgtcaggaac tacaggtgtc cactctgaga tccagctgca gcagtctgga 120cctgagctgg tgaagcctgg ggcttcagtg aaggtatcct gcaaggcttc tggttatgca 180ttcactagct acaacatgta ctgggtgaag cagagccatg gaaagagcct tgagtggatt 240ggatatattg atccttacaa tggtggtact aactacaacc agaagttcaa gggcaaggcc 300acattgactg ttgacaagtc ctccagcaca gcctacatgc atctcaacag cctgacatct 360gaggactctg cagtctatta ctgtgcaaga ggggggatgg gattacgacg ggactacttt 420gacttctggg gccaaggcac cactctcaca gtctcctcag ccaaaacgac acccccatct 480gtctatc 48754162PRTMus musculus 54Met Ser Ser Pro Gln Ser Leu Gln Thr Leu Thr Leu Ser Met Glu Trp 1 5 10 15 Arg Trp Ile Phe Leu Phe Leu Leu Ser Gly Thr Thr Gly Val His Ser 20 25 30 Glu Ile Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 35 40 45 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Ser Tyr 50 55 60 Asn Met Tyr Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile 65 70 75 80 Gly Tyr Ile Asp Pro Tyr Asn Gly Gly Thr Asn Tyr Asn Gln Lys Phe 85 90 95 Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 100 105 110 Met His Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 115 120 125 Ala Arg Gly Gly Met Gly Leu Arg Arg Asp Tyr Phe Asp Phe Trp Gly 130 135 140 Gln Gly Thr Thr Leu Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser 145 150 155 160 Val Tyr 55396DNAMus musculus 55atggattcac aggcccaggt tcttatgtta ctgctgctat gggtatctgg tacctgtggg 60gacattgtga tgtcacagtc tccatcctcc ctagctgtgt cagttggaga gaaggttact 120atgagctgca agtccagtca gagcctttta tatagtagca atcaaaagaa ctacttggcc 180tggtaccagc agaaaccagg gcagtctcct aaagtgctga tttactgggc atccactagg 240gaatctgggg tccctgatcg cttcacaggc agtggatctg ggacagattt cactctcacc 300atcagcagtg tgaaggctga agacctggca gtttattact gtcagcaata ttatagctat 360ccgtacacgt tcggaggggg gaccaagctg gaaata 39656132PRTMus musculus 56Met Asp Ser Gln Ala Gln Val Leu Met Leu Leu Leu Leu Trp Val Ser 1 5 10 15 Gly Thr Cys Gly Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala 20 25 30 Val Ser Val Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser 35 40 45 Leu Leu Tyr Ser Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln 50 55 60 Lys Pro Gly Gln Ser Pro Lys Val Leu Ile Tyr Trp Ala Ser Thr Arg 65 70 75 80 Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp 85 90 95 Phe Thr Leu Thr Ile Ser Ser Val Lys Ala Glu Asp Leu Ala Val Tyr 100 105 110 Tyr Cys Gln Gln Tyr Tyr Ser Tyr Pro Tyr Thr Phe Gly Gly Gly Thr 115 120 125 Lys Leu Glu Ile 130 57420DNAMus musculus 57atgggatgga gccggatctt tctcttcctc ctgtcaataa ttgcaagtgt ccattgccag 60gtccagctgc agcagtctgg acctgagctg gtgaagcctg gggcttcagt gaggatatcc 120tgcaaggctt ctggctacac cttcacaacc tactatatac actggttgaa acagaggcct 180ggacagggac ttgagtggat tggatggatt tttcctggaa atgttaatac taagtacaat 240gcgaagttca agggcaaggc cacactgact gcagacaaat cctccagcac agcctacatg 300cagctcagca gcctgacctc tgaggactct gcggtctatt tctgtgcaag agagggatta 360cagtactact ttgactactg gggccaaggc accactctca cagtctcctc agccaaaaca 42058140PRTMus musculus 58Met Gly Trp Ser Arg Ile Phe Leu Phe Leu Leu Ser Ile Ile Ala Ser 1 5 10 15 Val His Cys Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys 20 25 30 Pro Gly Ala Ser Val Arg Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 Thr Thr Tyr Tyr Ile His Trp Leu Lys Gln Arg Pro Gly Gln Gly Leu 50 55 60 Glu Trp Ile Gly Trp Ile Phe Pro Gly Asn Val Asn Thr Lys Tyr Asn 65 70 75 80 Ala Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser 85 90 95 Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val 100 105 110 Tyr Phe Cys Ala Arg Glu Gly Leu Gln Tyr Tyr Phe Asp Tyr Trp Gly 115 120 125 Gln Gly Thr Thr Leu Thr Val Ser Ser Ala Lys Thr 130 135 140 59327DNAMus musculus 59atgtccagag gacaaattgt tctcacccag tctccagcaa tcatgtctgc atctccaggg 60gagaaggtca ccatatcctg cagtgccaac tcaagtgtac gtttcatgtt ctggtaccag 120cagaagccag gatcctcccc caaacccttg atttatcgca catccaacct ggcttctgga 180gtccctgctc gcttcagtgg cagtgggtct gggacctctt actctctcac aatcagcagc 240atggaggctg aagatgctgc cacttattac tgccagcagt atcatagtta cccgtggacg 300ttcggtggag gcaccaagtt ggaaatc 32760109PRTMus musculus 60Met Ser Arg Gly Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser 1 5 10 15 Ala Ser Pro Gly Glu Lys Val Thr Ile Ser Cys Ser Ala Asn Ser Ser 20 25 30 Val Arg Phe Met Phe Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys 35 40 45 Pro Leu Ile Tyr Arg Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg 50 55 60 Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser 65 70 75 80 Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Tyr His Ser 85 90 95 Tyr Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 100 105 61426DNAMus musculus 61atgaacttcg ggctcagatt gattttcctt gtccttgttt taaaaggtgt cctgtgtgac 60gtgaaactcg tggagtctgg gggaggctta gtgaaggttg gagggtccct gaaactctcc 120tgtgcagcct ctggattcac tttcagtagc tattatatgt cttgggttcg ccagactcca 180gagaagaggc tggagttggt cgcagccatt aatattaatg gtggtagcac ctactatcca 240gacactgtga agggccgatt caccatctcc agagacaatg ccaagaacac cctgtacctg 300caaatgagca gtctgaagtc tgaggacaca gccttctatt actgtgcaag cgagttggcc 360ggctatggta ccccgtttgc ttactggggc caagggactc tggtcactgt ctctgcagcc 420aaaacg 42662142PRTMus musculus 62Met Asn Phe Gly Leu Arg Leu Ile Phe Leu Val Leu Val Leu Lys Gly 1 5 10 15 Val Leu Cys Asp Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys 20 25 30 Val Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35 40 45 Ser Ser Tyr Tyr Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu 50 55 60 Glu Leu Val Ala Ala Ile Asn Ile Asn Gly Gly Ser Thr Tyr Tyr Pro 65 70 75 80 Asp Thr Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn 85 90 95 Thr Leu Tyr Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Phe 100 105 110 Tyr Tyr Cys Ala Ser Glu Leu Ala Gly Tyr Gly Thr Pro Phe Ala Tyr 115 120 125 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala Ala Lys Thr 130 135 140 63378DNAMus musculus 63atggttttca cacctcagat acttggactt atgctttttt ggatttcagc ctccagaggt 60gatattgtgc taactcagtc tccagccacc ctgtctgtga ctccaggaga tagcgtcagt 120ctttcctgca gggccagcca aagtattagc aacaacctac actggtatca acaaaaatca 180catgagtctc caaggcttct catcaagtat gcttcccagt ccatctctgg gatcccctcc 240aggttcagtg gcagtggatc agggacagat ttcactctca gtatcaacag tgtggagact 300gaagattttg gaatgtattt ctgtcaacag agtaacaact ggccgctcac gttcggtgct 360gggaccaagc tggagctg 37864126PRTMus musculus 64Met Val Phe Thr Pro Gln Ile Leu Gly Leu Met Leu Phe Trp Ile Ser 1 5 10 15 Ala Ser Arg Gly Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser 20 25 30 Val Thr Pro Gly Asp Ser Val Ser Leu Ser Cys Arg Ala Ser Gln Ser 35 40 45 Ile Ser Asn Asn Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro 50 55 60 Arg Leu Leu Ile Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser 65 70 75 80 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn 85 90 95 Ser Val Glu Thr Glu Asp Phe Gly Met Tyr Phe Cys Gln Gln Ser Asn 100 105 110 Asn Trp Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu 115 120 125 65423DNAMus musculus 65atgaacttcg ggctcagatt gattttcctt gtccttgttt taaaaggtgt cctgtgtgac 60gtgaacctcg tggagtctgg gggaggctta gtgaagcttg gagggtccct gaaactctcc 120tgtgcagcct ctggattcac tttcagtagc ttttacatgt cttgggttcg ccagactcca 180gagaagaggc tggacttggt cgcaaccatt aatactaatg gtggtagcac ctactattca 240gacactgtga agggccgatt caccatctcc agagacaatg ccaaaaacac cctgtacctg 300caaatgaaca gtctgaagtc tgaggacaca gccttgtttt attgtgtaag acagccttac 360tacggaggga ctatggacta ctggggtcaa ggaacctcag tcaccgtctc ctcagccaaa 420acg 42366141PRTMus musculus 66Met Asn Phe Gly Leu Arg Leu Ile Phe Leu Val Leu Val Leu Lys Gly 1 5 10 15 Val Leu Cys Asp Val Asn Leu Val Glu Ser Gly Gly Gly Leu Val Lys 20 25 30 Leu Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35 40 45 Ser Ser Phe Tyr Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu 50 55 60 Asp Leu Val Ala Thr Ile Asn Thr Asn Gly Gly Ser Thr Tyr Tyr Ser 65 70 75 80 Asp Thr Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn 85 90 95 Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Leu 100 105 110 Phe Tyr Cys Val Arg Gln Pro Tyr Tyr Gly Gly Thr Met Asp Tyr Trp 115 120 125 Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Lys Thr 130 135 140 67378DNAMus musculus 67atggttttca cacctcagat acttggactt atgctttttt ggatttcagc ctccagaggt 60gatattgtgc tgactcagtc tccagccacc ctgtctgtga ctccgggaga tcgcgtcagt 120ctttcctgca gggccagcca aagtattaac aacaatctac actggtatca acaaaagtca 180catgagtctc caaggcttct catcaaatat gcttcccagt ccatctctgg gatcccctcc 240aggttcagtg gcagtggatc agggacagat ttcactctca gtatcaacag tgtggagact 300gaagattttg gaatgtattt ctgtcaacag actaacaact ggcctctcac gttcggtgct 360gggaccaagc tggagctg 37868126PRTMus musculus 68Met Val Phe Thr Pro Gln Ile Leu Gly Leu Met Leu Phe Trp Ile Ser 1 5 10 15 Ala Ser Arg Gly Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser 20 25 30 Val Thr Pro Gly Asp Arg Val Ser Leu Ser Cys Arg Ala Ser Gln Ser 35 40 45 Ile Asn Asn Asn Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro 50 55 60 Arg Leu Leu Ile Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser 65 70 75 80 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn 85 90 95 Ser Val Glu Thr Glu Asp Phe Gly Met Tyr Phe Cys Gln Gln Thr Asn 100 105 110 Asn Trp Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu 115 120 125 69423DNAMus musculus 69atgaaagtgt tgagtctgtt gtacctgttg acagccattc ctggaatcct gtctgatgta 60caggttcagg agtcaggacc tggcctcgtg aaaccttctc agtctctgtc tctcacctgc 120tctgtcactg gctactccat caccagtggt tattactgga actggatccg gcagtttcca 180ggaaacaaac tggaatggat gggctacata agctacgacg gtagaaataa ctacaaccca 240tctctcaaaa atcgaatctc catcactcgt gacacatcta agaaccagtt tttcctgaag 300ttgaattctg tgactactga ggacacagct acatattact gtgcaagaga aaatagtaac 360tacccttact actatgacta ctggggccaa ggcaccactc tcacagtctc ctcagccaaa 420acg 42370141PRTMus musculus 70Met Lys Val Leu Ser Leu Leu Tyr Leu Leu Thr Ala Ile Pro Gly Ile 1 5 10 15 Leu Ser Asp Val Gln Val Gln Glu Ser Gly Pro Gly Leu Val Lys Pro 20 25 30 Ser Gln Ser Leu Ser Leu Thr Cys Ser Val Thr Gly Tyr Ser Ile Thr 35 40 45 Ser Gly Tyr Tyr Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu 50 55 60 Glu Trp Met Gly Tyr Ile Ser Tyr Asp Gly Arg Asn Asn Tyr Asn Pro 65 70 75 80 Ser Leu Lys Asn Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln 85 90 95 Phe Phe Leu Lys Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr 100

105 110 Tyr Cys Ala Arg Glu Asn Ser Asn Tyr Pro Tyr Tyr Tyr Asp Tyr Trp 115 120 125 Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Ala Lys Thr 130 135 140 71426DNAMus musculus 71aggatgaaag tgttgagtct gttgtacctg ttgacagcca ttcctggaat cctgtctgat 60gtacaggttc aggagtcagg acctggcctc gtgaaacctt ctcagtctct gtctctcacc 120tgctctgtca ctggctactc catcaccagt ggttattact ggaactggat ccggcagttt 180ccaggaaaca aactggaatg gatgggctac ataagctacg acggtagaaa taactacaac 240ccatctctca aaaatcgaat ctccatcact cgtgacacat ctaagaacca gtttttcctg 300aagttgaatt ctgtgactac tgaggacaca gctacatatt actgtgcaag agaaaatagt 360aactaccctt actactatga ctactggggc caaggcacca ctctcacagt ctcctcagcc 420aaaacg 42672142PRTMus musculus 72Arg Met Lys Val Leu Ser Leu Leu Tyr Leu Leu Thr Ala Ile Pro Gly 1 5 10 15 Ile Leu Ser Asp Val Gln Val Gln Glu Ser Gly Pro Gly Leu Val Lys 20 25 30 Pro Ser Gln Ser Leu Ser Leu Thr Cys Ser Val Thr Gly Tyr Ser Ile 35 40 45 Thr Ser Gly Tyr Tyr Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys 50 55 60 Leu Glu Trp Met Gly Tyr Ile Ser Tyr Asp Gly Arg Asn Asn Tyr Asn 65 70 75 80 Pro Ser Leu Lys Asn Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn 85 90 95 Gln Phe Phe Leu Lys Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr 100 105 110 Tyr Tyr Cys Ala Arg Glu Asn Ser Asn Tyr Pro Tyr Tyr Tyr Asp Tyr 115 120 125 Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Ala Lys Thr 130 135 140 73426DNAMus musculus 73atgaacttcg ggctcagatt gattttcctt gtccttgttt taaaaggtgt cctgtgtgac 60gtgaagctcg tggagtctgg gggaggctta gtgaagcttg gagggtccct gagactctcc 120tgtgcagcct ctggattcag tttcagtacc tcttacatgt cttgggttcg ccagactcca 180gagaagaggc tggagttggt cgcagccatt aatcttaatg gtggtagtac ctactattca 240gacactgtga agggccgatt caccatctcc agagacaatg ccaagaacac cctgtacctg 300caaatgagca gtctgaagtc tgaggacaca gccttctatt actgtgcaag cgagttggcc 360gggtatggta ccccgtttgc ttactggggc caagggactc tggtcactgt ctctgcagcc 420aaaacg 42674142PRTMus musculus 74Met Asn Phe Gly Leu Arg Leu Ile Phe Leu Val Leu Val Leu Lys Gly 1 5 10 15 Val Leu Cys Asp Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys 20 25 30 Leu Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe 35 40 45 Ser Thr Ser Tyr Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu 50 55 60 Glu Leu Val Ala Ala Ile Asn Leu Asn Gly Gly Ser Thr Tyr Tyr Ser 65 70 75 80 Asp Thr Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn 85 90 95 Thr Leu Tyr Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Phe 100 105 110 Tyr Tyr Cys Ala Ser Glu Leu Ala Gly Tyr Gly Thr Pro Phe Ala Tyr 115 120 125 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala Ala Lys Thr 130 135 140 75378DNAMus musculus 75atggttttca cacctcagat acttggactt atgctttttt ggatttcagc ctccagaggt 60gatattgtgc taactcagtc tccagccacc ctgtctgtga ctccaggaga tagcgtcagt 120ctttcctgta aggccagcca aagtattagc aacaacctac actggtatca acaaaaatca 180catgagtctc caaggcttct catcaagtat acttcccagt ccatctctgg gatcccctcc 240aggttcagtg gcagtggatc agggacagat ttcactctca ctatcaacaa tgtggagact 300gaagattttg gaatgtattt ctgtcaacag agtaacagtt ggccgctcac gttcggtgct 360gggaccaagc tggaggtg 37876126PRTMus musculus 76Met Val Phe Thr Pro Gln Ile Leu Gly Leu Met Leu Phe Trp Ile Ser 1 5 10 15 Ala Ser Arg Gly Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser 20 25 30 Val Thr Pro Gly Asp Ser Val Ser Leu Ser Cys Lys Ala Ser Gln Ser 35 40 45 Ile Ser Asn Asn Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro 50 55 60 Arg Leu Leu Ile Lys Tyr Thr Ser Gln Ser Ile Ser Gly Ile Pro Ser 65 70 75 80 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn 85 90 95 Asn Val Glu Thr Glu Asp Phe Gly Met Tyr Phe Cys Gln Gln Ser Asn 100 105 110 Ser Trp Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Val 115 120 125 77423DNAMus musculus 77atgaacttcg ggctcagatt gattttcctt gtccttgttt taaaaggtgt cctgtgtgac 60gtgaacctcg tggagtctgg gggaggctta gtgaagcttg gagggtccct gaaactctcc 120tgtgcagcct ctggattcac tttcagtagc ttttacatgt cttgggttcg ccagactcca 180gagaagaggc tggacttggt cgcaaccatt aatactaatg gtggtagcac ctactattca 240gacactgtga agggccgatt caccatctcc agagacaatg ccaaaaacac cctgtacctg 300caaatgaaca gtctgaagtc tgaggacaca gccttgtttt attgtgtaag acagccttac 360tacggaggga ctatggacta ctggggtcaa ggaacctcag tcaccgtctc ctcagccaaa 420acg 42378141PRTMus musculus 78Met Asn Phe Gly Leu Arg Leu Ile Phe Leu Val Leu Val Leu Lys Gly 1 5 10 15 Val Leu Cys Asp Val Asn Leu Val Glu Ser Gly Gly Gly Leu Val Lys 20 25 30 Leu Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35 40 45 Ser Ser Phe Tyr Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu 50 55 60 Asp Leu Val Ala Thr Ile Asn Thr Asn Gly Gly Ser Thr Tyr Tyr Ser 65 70 75 80 Asp Thr Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn 85 90 95 Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Leu 100 105 110 Phe Tyr Cys Val Arg Gln Pro Tyr Tyr Gly Gly Thr Met Asp Tyr Trp 115 120 125 Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Lys Thr 130 135 140 79378DNAMus musculus 79atggttttca cacctcagat acttggactt atgctttttt ggatttcagc ctccagaggt 60gatattgtgc tgactcagtc tccagccacc ctgtctgtga ctccgggaga tcgcgtcagt 120ctttcctgca gggccagcca aagtattaac aacaatctac actggtatca acaaaagtca 180catgagtctc caaggcttct catcaaatat gcttcccagt ccatctctgg gatcccctcc 240aggttcagtg gcagtggatc agggacagat ttcactctca gtatcaacag tgtggagact 300gaagattttg gaatgtattt ctgtcaacag actaacaact ggcctctcac gttcggtgct 360gggaccaagc tggagctg 37880126PRTMus musculus 80Met Val Phe Thr Pro Gln Ile Leu Gly Leu Met Leu Phe Trp Ile Ser 1 5 10 15 Ala Ser Arg Gly Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser 20 25 30 Val Thr Pro Gly Asp Arg Val Ser Leu Ser Cys Arg Ala Ser Gln Ser 35 40 45 Ile Asn Asn Asn Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro 50 55 60 Arg Leu Leu Ile Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser 65 70 75 80 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn 85 90 95 Ser Val Glu Thr Glu Asp Phe Gly Met Tyr Phe Cys Gln Gln Thr Asn 100 105 110 Asn Trp Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu 115 120 125 81468DNAMus musculus 81atgatcagtg tcctctctac acagtccctg acaacactga ctctaaccat gggatggagc 60cggatctttc tcttcctcct gtcaataatt gcaggtgtcc attgccaggt ccagctgcag 120cagtctggac ctgagctggt gatgcctggg gcttcagtga ggatatcctg caaggcttct 180ggctacacct tcacaaacta ctatttacac tgggttaagc agaggcctgg acagggactt 240gaatggattg gatggattta tcctggaaat gttaatacta agtacaatga gaagttcaag 300ggcaaggcct cactgactgc agacaaatcc tccagcacag cctacatgca gctcagcagc 360ctgacctctg aggactctgc ggtctatttc tgtgcaagag agggattaca gtactacttt 420gactactggg cccaaggcac cactctcaca gtctcctcag ccaaaaca 46882156PRTMus musculus 82Met Ile Ser Val Leu Ser Thr Gln Ser Leu Thr Thr Leu Thr Leu Thr 1 5 10 15 Met Gly Trp Ser Arg Ile Phe Leu Phe Leu Leu Ser Ile Ile Ala Gly 20 25 30 Val His Cys Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Met 35 40 45 Pro Gly Ala Ser Val Arg Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe 50 55 60 Thr Asn Tyr Tyr Leu His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu 65 70 75 80 Glu Trp Ile Gly Trp Ile Tyr Pro Gly Asn Val Asn Thr Lys Tyr Asn 85 90 95 Glu Lys Phe Lys Gly Lys Ala Ser Leu Thr Ala Asp Lys Ser Ser Ser 100 105 110 Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val 115 120 125 Tyr Phe Cys Ala Arg Glu Gly Leu Gln Tyr Tyr Phe Asp Tyr Trp Ala 130 135 140 Gln Gly Thr Thr Leu Thr Val Ser Ser Ala Lys Thr 145 150 155 83381DNAMus musculus 83atggattttc aggtgcagat tttcagcttc ctgctaatca gtgcctcagt cataatgtcc 60agaggacaaa ttgttctcac ccagtctcca gcaatcatgt ctgcatctcc aggggagaag 120gtcaccatat cctgcagtgc caactcaagt gtacgttaca tgttctggta ccagcagaag 180ccaggatcct tccccaaacc ctggatttat cgcacatcca acctggcttc tggagtccct 240gctcgcttca gtggcagtgg gtctgggacc tcttactctc tcacaatcag cagtatggag 300gctgaagatg ttgccactta ttactgccag cagtatcata gttacccgtg gacgttcggt 360ggaggcacca agctggaaat c 38184127PRTMus musculus 84Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser 1 5 10 15 Val Ile Met Ser Arg Gly Gln Ile Val Leu Thr Gln Ser Pro Ala Ile 20 25 30 Met Ser Ala Ser Pro Gly Glu Lys Val Thr Ile Ser Cys Ser Ala Asn 35 40 45 Ser Ser Val Arg Tyr Met Phe Trp Tyr Gln Gln Lys Pro Gly Ser Phe 50 55 60 Pro Lys Pro Trp Ile Tyr Arg Thr Ser Asn Leu Ala Ser Gly Val Pro 65 70 75 80 Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile 85 90 95 Ser Ser Met Glu Ala Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Tyr 100 105 110 His Ser Tyr Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 115 120 125 85441DNAMus musculus 85atggaaaggc actggatctt tctcttcctg ttttcagtaa ctgcaggtgt ccactcccag 60gtccaacttc agcaatctgg ggctgaactg gcaaaacctg gggcctcagt gaaaatctcc 120tgcaaggctt ctggctacac ctttactagc tactggatgc actgggtaaa acagaggcct 180ggacagggtc tggaatggat tggatacatt aatcctaaca ctggttatag tgagtacaat 240caaaagttca gggacaaggc cacattgact gcaaacaaat cctccagcac agccaacatg 300caactgagca gcctgacatc tgacgactct gcagtctatt actgtgcaag aactgcacaa 360tactacggta gtcctagggg ttactatgct atggactcct ggggtcaagg aacctcagtc 420accgtctcct cagccaaaac g 44186147PRTMus musculus 86Met Glu Arg His Trp Ile Phe Leu Phe Leu Phe Ser Val Thr Ala Gly 1 5 10 15 Val His Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Lys 20 25 30 Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 Thr Ser Tyr Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu 50 55 60 Glu Trp Ile Gly Tyr Ile Asn Pro Asn Thr Gly Tyr Ser Glu Tyr Asn 65 70 75 80 Gln Lys Phe Arg Asp Lys Ala Thr Leu Thr Ala Asn Lys Ser Ser Ser 85 90 95 Thr Ala Asn Met Gln Leu Ser Ser Leu Thr Ser Asp Asp Ser Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Thr Ala Gln Tyr Tyr Gly Ser Pro Arg Gly Tyr 115 120 125 Tyr Ala Met Asp Ser Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser 130 135 140 Ala Lys Thr 145 87393DNAMus musculus 87atgaggttct ctgctcagct tctggggctg cttgtgctct ggatccctgg atccactgca 60gatattgtga tgacgcaggc tgcattctcc aatccagtca ctcttggaac atcagcttcc 120atctcctgca ggtctagtaa gagtctccta catagtaacg gcatcactta tttgtgttgg 180tttctgcaga agccaggcca gtctcctcag ctcctgattt atcagatgtc caaccttgcc 240tcaggagtcc cagacaggtt cagtagcagt gggtcaggaa ctgatttcac actgagaatc 300agcagagtgg aggctgagga tgtgggtgtt tatttctgtg ctcaaaatct agatctgccg 360tggacgttcg gtgggggcac caagctggaa atc 39388131PRTMus musculus 88Met Arg Phe Ser Ala Gln Leu Leu Gly Leu Leu Val Leu Trp Ile Pro 1 5 10 15 Gly Ser Thr Ala Asp Ile Val Met Thr Gln Ala Ala Phe Ser Asn Pro 20 25 30 Val Thr Leu Gly Thr Ser Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser 35 40 45 Leu Leu His Ser Asn Gly Ile Thr Tyr Leu Cys Trp Phe Leu Gln Lys 50 55 60 Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Ala 65 70 75 80 Ser Gly Val Pro Asp Arg Phe Ser Ser Ser Gly Ser Gly Thr Asp Phe 85 90 95 Thr Leu Arg Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Phe 100 105 110 Cys Ala Gln Asn Leu Asp Leu Pro Trp Thr Phe Gly Gly Gly Thr Lys 115 120 125 Leu Glu Ile 130 89468DNAMus musculus 89atgatcagtg tcctctctac acagtccctg acaacactga ctctaaccat gggatggagc 60cggatctttc tcttcctcct gtcaataatt gcaagtgtcc attgccaggt ccagctgcag 120cagtctggac ctgaactggt gaagcctggg gcttcagtga ggatatcctg caaggcttct 180ggctacacct tcacaaccta ctatatacac tggttgaaac agaggcctgg acagggactt 240gagtggattg gatggatttt tcctggaaat gttaatacta agtacaatgc gaagttcaag 300ggcaaggcca cactgactgc agacaaatcc tccagcacag cctacatgca gctcagcagc 360ctgacctctg aggactctgc ggtctatttc tgtgcaagag aggaattaca gtactacttt 420gactactggg gccaaggctc cgctctcaca gtctcctcag ccaaaaca 46890156PRTMus musculus 90Met Ile Ser Val Leu Ser Thr Gln Ser Leu Thr Thr Leu Thr Leu Thr 1 5 10 15 Met Gly Trp Ser Arg Ile Phe Leu Phe Leu Leu Ser Ile Ile Ala Ser 20 25 30 Val His Cys Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys 35 40 45 Pro Gly Ala Ser Val Arg Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe 50 55 60 Thr Thr Tyr Tyr Ile His Trp Leu Lys Gln Arg Pro Gly Gln Gly Leu 65 70 75 80 Glu Trp Ile Gly Trp Ile Phe Pro Gly Asn Val Asn Thr Lys Tyr Asn 85 90 95 Ala Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser 100 105 110 Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val 115 120 125 Tyr Phe Cys Ala Arg Glu Glu Leu Gln Tyr Tyr Phe Asp Tyr Trp Gly 130 135 140 Gln Gly Ser Ala Leu Thr Val Ser Ser Ala Lys Thr 145 150 155 91381DNAMus musculus 91atggattttc aggtgcagat tctcagcatc ctgctaatca gtgcctcagt cataatgtcc 60agaggacaaa ttgttctcac ccagtctcca gcaatcatgt ctgcatctcc aggggagaag 120gtcaccatat cctgcagtgc caactcaagt gtacgtttca tgttctggta ccagcagaag 180ccaggatcct cccccaaacc cttgatttat cgcacatcca acctggcttc tggagtccct 240gctcgcttca gtggcagtgg gtctgggacc tcttactctc tcacaatcag cagcatggag 300gctgaagatg ctgccactta ttactgccag cagtatcata gttacccgtg gacgttcggt 360ggaggcacca agttggaaat c 38192127PRTMus musculus 92Met Asp Phe Gln Val Gln Ile Leu Ser Ile Leu Leu Ile Ser Ala Ser 1 5 10 15 Val Ile Met Ser Arg Gly Gln Ile Val Leu Thr Gln Ser Pro Ala Ile 20 25 30 Met Ser Ala Ser Pro Gly Glu Lys Val Thr Ile Ser Cys Ser Ala Asn 35

40 45 Ser Ser Val Arg Phe Met Phe Trp Tyr Gln Gln Lys Pro Gly Ser Ser 50 55 60 Pro Lys Pro Leu Ile Tyr Arg Thr Ser Asn Leu Ala Ser Gly Val Pro 65 70 75 80 Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile 85 90 95 Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Tyr 100 105 110 His Ser Tyr Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 115 120 125 93441DNAMus musculus 93atggaaaggc actggatctt tctcttcctg ttttcagtaa ctgcaggtgt ccactcccag 60gtccaacttc agcagtctgg ggctgaactg gcaaaacctg gggcctcagt gaagatgtcc 120tgcaaggctt ctggctacac ctttactagc tactggatgc actgggtaaa acagaggcct 180ggacagggtc tggaatggat tggatacatt aatcctagta ctggttatac tgagtacaat 240cagaagttca gggacaaggc cacattgact gcagacaaat cctccagcac agccaacatg 300caactgagca gcctgacatc tgaggactct gcagtctatt actgtacaag aactgcgcaa 360tactacggta gtcctagggg ttactatgct atggactcct ggggtcaagg aacctcagtc 420accgtctcct cggccaaaac g 44194147PRTMus musculus 94Met Glu Arg His Trp Ile Phe Leu Phe Leu Phe Ser Val Thr Ala Gly 1 5 10 15 Val His Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Lys 20 25 30 Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 Thr Ser Tyr Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu 50 55 60 Glu Trp Ile Gly Tyr Ile Asn Pro Ser Thr Gly Tyr Thr Glu Tyr Asn 65 70 75 80 Gln Lys Phe Arg Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser 85 90 95 Thr Ala Asn Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val 100 105 110 Tyr Tyr Cys Thr Arg Thr Ala Gln Tyr Tyr Gly Ser Pro Arg Gly Tyr 115 120 125 Tyr Ala Met Asp Ser Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser 130 135 140 Ala Lys Thr 145 95324DNAMus musculus 95atgacgcagg ctgcattctc caatccagtc actcttggaa catcagcttc catctcctgc 60aggtctagta agagtctcct acatagtaac ggcatcactt atttgtgttg gtttctgcag 120aagccaggcc agtctcctca gctcctgatt tatctgatgt ccaaccttgc ctcaggagtc 180ccagacaggt tcagtagcag tgggtcagga actgatttta cactgagaat cagcagagtg 240gaggctgagg atgtgggtgt ttatttctgt gctcaaaatc tagatcttcc gtggacgttc 300ggtgggggca ccaagctgga aatc 32496108PRTMus musculus 96Met Thr Gln Ala Ala Phe Ser Asn Pro Val Thr Leu Gly Thr Ser Ala 1 5 10 15 Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser Asn Gly Ile 20 25 30 Thr Tyr Leu Cys Trp Phe Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu 35 40 45 Leu Ile Tyr Leu Met Ser Asn Leu Ala Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Ser Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile Ser Arg Val 65 70 75 80 Glu Ala Glu Asp Val Gly Val Tyr Phe Cys Ala Gln Asn Leu Asp Leu 85 90 95 Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 100 105 97423DNAMus musculus 97atgaacttcg ggctcagatt gattttcctt gtccttgttt taaaaggtgt cctgtgtgac 60gtgaacctcg tggagtctgg gggaggctta gtgaagcttg gagggtccct gaaactctcc 120tgtgcagcct ctggattcac tttcagtagc ttttacatgt cttgggttcg ccagactcca 180gagaagaggc tggacttggt cgcagccatt aatactaatg gtggtagcac ctactattca 240gacactgtga agggccgatt caccatctcc agagacaatg ccaagaacac cctgtacctg 300caaatgagca gtctgaagtc tgaggacaca gccttgtttt actgtgcaag acagccttac 360tacggagggc ctatggactt ctggggtcaa ggaacctcag tcaccgtctc ctcagccaaa 420acg 42398141PRTMus musculus 98Met Asn Phe Gly Leu Arg Leu Ile Phe Leu Val Leu Val Leu Lys Gly 1 5 10 15 Val Leu Cys Asp Val Asn Leu Val Glu Ser Gly Gly Gly Leu Val Lys 20 25 30 Leu Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35 40 45 Ser Ser Phe Tyr Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu 50 55 60 Asp Leu Val Ala Ala Ile Asn Thr Asn Gly Gly Ser Thr Tyr Tyr Ser 65 70 75 80 Asp Thr Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn 85 90 95 Thr Leu Tyr Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Leu 100 105 110 Phe Tyr Cys Ala Arg Gln Pro Tyr Tyr Gly Gly Pro Met Asp Phe Trp 115 120 125 Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Lys Thr 130 135 140 99378DNAMus musculus 99atggttttca cacctcagat acttggactt atgctttttt ggatttcagc ctccagaggt 60gatattgtgc taactcagtc tccagccacc ctgtctgtga ctccaggaga tagcgtcagt 120ctttcctgca gggccagcca aagtattaac aacaacctac actggtatca acaaaaatca 180catgagtctc caaggcttct catcaagtat gcttcccagt ccatctctgg gatcccctcc 240aggttcagtg gcagtggatc agggacagat ttcactctca gtatcaacag tgtggagact 300gaagattttg gaatgtattt ctgtcaacag agtaacaact ggcctctcac gttcggtgct 360gggaccaagc tggagctg 378100126PRTMus musculus 100Met Val Phe Thr Pro Gln Ile Leu Gly Leu Met Leu Phe Trp Ile Ser 1 5 10 15 Ala Ser Arg Gly Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser 20 25 30 Val Thr Pro Gly Asp Ser Val Ser Leu Ser Cys Arg Ala Ser Gln Ser 35 40 45 Ile Asn Asn Asn Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro 50 55 60 Arg Leu Leu Ile Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser 65 70 75 80 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn 85 90 95 Ser Val Glu Thr Glu Asp Phe Gly Met Tyr Phe Cys Gln Gln Ser Asn 100 105 110 Asn Trp Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu 115 120 125 101423DNAMus musculus 101atgaacttcg ggctcagatt gattttcctt gtccttgttt taaaaggtgt cctgtgtgac 60gtgaacctcg tggagtctgg gggaggctta gtgaagcttg gagggtccct gaaactctcc 120tgtgcagcct ctggattcac tttcagtaga ttttacatgt cttgggttcg ccagactcca 180gagaagaggc tggacttggt cgcagccatt aatactaatg gtggtagcac ctattattca 240gacactgtga agggccgatt caccatctcc agagacaatg ccaagaacac cctgtacctg 300caaatgagca gtctgaagtc tgaggacaca gccttgtttt actgtgcaag acagccttac 360tacggagggc ctatggagtt ctggggtcaa ggaacctcag tcaccgtctc ctcagccaaa 420acg 423102141PRTMus musculus 102Met Asn Phe Gly Leu Arg Leu Ile Phe Leu Val Leu Val Leu Lys Gly 1 5 10 15 Val Leu Cys Asp Val Asn Leu Val Glu Ser Gly Gly Gly Leu Val Lys 20 25 30 Leu Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35 40 45 Ser Arg Phe Tyr Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu 50 55 60 Asp Leu Val Ala Ala Ile Asn Thr Asn Gly Gly Ser Thr Tyr Tyr Ser 65 70 75 80 Asp Thr Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn 85 90 95 Thr Leu Tyr Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Leu 100 105 110 Phe Tyr Cys Ala Arg Gln Pro Tyr Tyr Gly Gly Pro Met Glu Phe Trp 115 120 125 Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Lys Thr 130 135 140 103390DNAMus musculus 103atgggcatca agatggagtc acagactcag gtctttgtat acatgttgct gtggttgtct 60ggtgttgatg gagacattgt gatgacccag tctcaaaaat tcatgtccac atcagtagga 120gacagggtca gcgtcacctg caaggccagt cagaatgtgg gtactaatgt agcctggtat 180caacagaaac cagggcaatc tcctaaagca ctgatttact cggcatccta ccggtacagt 240ggagtccctg atcgcttcac aggcagtgga tctgggtcag atttcactct caccatcagc 300aatgtgcagt ctgaagactt ggcagagtat ttctgtcagc aatataacag ctatcctctc 360acgttcggtg ctgggaccaa gctggagctg 390104130PRTMus musculus 104Met Gly Ile Lys Met Glu Ser Gln Thr Gln Val Phe Val Tyr Met Leu 1 5 10 15 Leu Trp Leu Ser Gly Val Asp Gly Asp Ile Val Met Thr Gln Ser Gln 20 25 30 Lys Phe Met Ser Thr Ser Val Gly Asp Arg Val Ser Val Thr Cys Lys 35 40 45 Ala Ser Gln Asn Val Gly Thr Asn Val Ala Trp Tyr Gln Gln Lys Pro 50 55 60 Gly Gln Ser Pro Lys Ala Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr Ser 65 70 75 80 Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Ser Asp Phe Thr 85 90 95 Leu Thr Ile Ser Asn Val Gln Ser Glu Asp Leu Ala Glu Tyr Phe Cys 100 105 110 Gln Gln Tyr Asn Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu 115 120 125 Glu Leu 130 10531DNAMus musculus 105ggctacacct ttactagcta ctggatgcac t 3110652DNAMus musculus 106tacattaatc ctagcactgg ttatactgag tacaatcaga acttcaggga ca 5210749DNAMus musculus 107actgcgcaat actacggtag tcctaggggt tactatgcta tggactcct 4910810PRTMus musculus 108Gly Tyr Thr Phe Thr Ser Tyr Trp Met His 1 5 10 10917PRTMus musculus 109Tyr Ile Asn Pro Ser Thr Gly Tyr Thr Glu Tyr Asn Gln Asn Phe Arg 1 5 10 15 Asp 11016PRTMus musculus 110Thr Ala Gln Tyr Tyr Gly Ser Pro Arg Gly Tyr Tyr Ala Met Asp Ser 1 5 10 15 11149DNAMus musculus 111aggtctagta agagtctcct acatagtaac ggcatcactt atttgtgtt 4911222DNAMus musculus 112ctgatgtcca accttgcctc ag 2211328DNAMus musculus 113gctcaaaatc tagatcttcc gtggacgt 2811416PRTMus musculus 114Arg Ser Ser Lys Ser Leu Leu His Ser Asn Gly Ile Thr Tyr Leu Cys 1 5 10 15 1157PRTMus musculus 115Leu Met Ser Asn Leu Ala Ser 1 5 1169PRTMus musculus 116Ala Gln Asn Leu Asp Leu Pro Trp Thr 1 5 11730DNAMus musculus 117ggctacacct tcacttccta tccaatagag 3011851DNAMus musculus 118aattttcatc cttacaatga taatactaag tacaatgaaa aattcaaggg c 5111936DNAMus musculus 119gggtactctg gtaactactt ctctgctatg gactac 3612010PRTMus musculus 120Gly Tyr Thr Phe Thr Ser Tyr Pro Ile Glu 1 5 10 12117PRTMus musculus 121Asn Phe His Pro Tyr Asn Asp Asn Thr Lys Tyr Asn Glu Lys Phe Lys 1 5 10 15 Gly 12212PRTMus musculus 122Gly Tyr Ser Gly Asn Tyr Phe Ser Ala Met Asp Tyr 1 5 10 12333DNAMus musculus 123aaggccagtc agaatgttcg taatgatgta gcc 3312421DNAMus musculus 124ttggcatcca accggcacac t 2112527DNAMus musculus 125ctgcaacatt ggaattatcc gtacacg 2712611PRTMus musculus 126Lys Ala Ser Gln Asn Val Arg Asn Asp Val Ala 1 5 10 1277PRTMus musculus 127Leu Ala Ser Asn Arg His Thr 1 5 1289PRTMus musculus 128Leu Gln His Trp Asn Tyr Pro Tyr Thr 1 5 12931DNAMus musculus 129ggattcactt tcagtagtta ttacatgtct t 3113051DNAMus musculus 130gtcattaata ctaatggtgg tagcacctac tattcagaca ctgtgaaggg c 5113131DNAMus musculus 131caaccctatt atggtaaccc ttttgactac t 3113210PRTMus musculus 132Gly Phe Thr Phe Ser Ser Tyr Tyr Met Ser 1 5 10 13317PRTMus musculus 133Val Ile Asn Thr Asn Gly Gly Ser Thr Tyr Tyr Ser Asp Thr Val Lys 1 5 10 15 Gly 13410PRTMus musculus 134Gln Pro Tyr Tyr Gly Asn Pro Phe Asp Tyr 1 5 10 13534DNAMus musculus 135agggccagcc aaagtattag caacaaccta cact 3413622DNAMus musculus 136tatgtttccc agtccatctc tg 2213728DNAMus musculus 137caacagagta acaactggcc gctcacgt 2813811PRTMus musculus 138Arg Ala Ser Gln Ser Ile Ser Asn Asn Leu His 1 5 10 1397PRTMus musculus 139Tyr Val Ser Gln Ser Ile Ser 1 5 1409PRTMus musculus 140Gln Gln Ser Asn Asn Trp Pro Leu Thr 1 5 14131DNAMus musculus 141ggattcactt tcagtagcta ttacatgtct t 3114252DNAMus musculus 142gccattaata gtaatggtgg tagtacctac tatgcagaca ctatgaaggg cc 5214334DNAMus musculus 143gagttggccg ggtatggtac cccgtttgct tact 3414410PRTMus musculus 144Gly Phe Thr Phe Ser Ser Tyr Tyr Met Ser 1 5 10 14517PRTMus musculus 145Ala Ile Asn Ser Asn Gly Gly Ser Thr Tyr Tyr Ala Asp Thr Met Lys 1 5 10 15 Gly 14611PRTMus musculus 146Glu Leu Ala Gly Tyr Gly Thr Pro Phe Ala Tyr 1 5 10 14734DNAMus musculus 147agggccagcc aaagtattag cagcaaccta cact 3414822DNAMus musculus 148tatgcttccc agtccatctc tg 2214928DNAMus musculus 149caacagagta acacctggcc gctcacgt 2815011PRTMus musculus 150Arg Ala Ser Gln Ser Ile Ser Ser Asn Leu His 1 5 10 1517PRTMus musculus 151Tyr Ala Ser Gln Ser Ile Ser 1 5 1529PRTMus musculus 152Gln Gln Ser Asn Thr Trp Pro Leu Thr 1 5 15331DNAMus musculus 153ggttatgcat tcactagcta caacatgtac t 3115452DNAMus musculus 154tatattgatc cttacaatgg cggtactgac tccaaccaga acttcaaggg ca 5215537DNAMus musculus 155ggggggatgg gattacgacg ggaccacttt gactact 3715610PRTMus musculus 156Gly Tyr Ala Phe Thr Ser Tyr Asn Met Tyr 1 5 10 15717PRTMus musculus 157Tyr Ile Asp Pro Tyr Asn Gly Gly Thr Asp Ser Asn Gln Asn Phe Lys 1 5 10 15 Gly 15812PRTMus musculus 158Gly Gly Met Gly Leu Arg Arg Asp His Phe Asp Tyr 1 5 10 15930DNAMus musculus 159agtgccaact caagtgtacg ttacatgttc 3016021DNAMus musculus 160cgcacatcca acctggcttc t 2116127DNAMus musculus 161cagcagtatc atagttaccc gtggacg 2716210PRTMus musculus 162Ser Ala Asn Ser Ser Val Arg Tyr Met Phe 1 5 10 1637PRTMus musculus 163Arg Thr Ser Asn Leu Ala Ser 1 5 1649PRTMus musculus 164Gln Gln Tyr His Ser Tyr Pro Trp Thr 1 5 16531DNAMus musculus 165ggctacacct tcacaaccta ctatatacac t 3116652DNAMus musculus 166tggatttttc ctggaaatgt taatactaag tacaatgcga agttcaaggg ca 5216728DNAMus musculus 167gaggaattac agtactactt tgactact 2816810PRTMus musculus 168Gly Tyr Thr Phe Thr Thr Tyr Tyr Ile His 1 5 10 16917PRTMus musculus 169Trp Ile Phe Pro Gly Asn Val Asn Thr Lys Tyr Asn Ala Lys Phe Lys 1 5 10 15 Gly 1709PRTMus musculus 170Glu Glu Leu Gln Tyr Tyr Phe Asp Tyr 1 5 17131DNAMus musculus 171agtgccaact caagtgtacg tttcatgttc t 3117222DNAMus musculus 172cgcacatcca acctggcctc tg 2217328DNAMus musculus 173cagcagtatc atagttaccc gtggacgt 2817410PRTMus musculus 174Ser Ala Asn Ser Ser Val Arg Phe Met Phe 1 5 10 1757PRTMus musculus 175Arg Thr Ser Asn Leu Ala Ser 1 5 1769PRTMus musculus 176Gln Gln Tyr His Ser Tyr Pro Trp Thr 1 5 17731DNAMus musculus 177ggattcactt tcagtaccta ttacatgtct t 3117852DNAMus musculus 178accattaata ctaatggtgg tagcacctac tatccagaca ctttgaaggg cc 5217931DNAMus musculus

179cagcgtaact acggagtggc tgtggactcc t 3118010PRTMus musculus 180Gly Phe Thr Phe Ser Thr Tyr Tyr Met Ser 1 5 10 18117PRTMus musculus 181Thr Ile Asn Thr Asn Gly Gly Ser Thr Tyr Tyr Pro Asp Thr Leu Lys 1 5 10 15 Gly 18210PRTMus musculus 182Gln Arg Asn Tyr Gly Val Ala Val Asp Ser 1 5 10 18334DNAMus musculus 183agggccagcc aaagtattag caacaaccta cact 3418422DNAMus musculus 184tatgcttccc agtccatctc tg 2218528DNAMus musculus 185caacagacta acaactggcc tctcacgt 2818611PRTMus musculus 186Arg Ala Ser Gln Ser Ile Ser Asn Asn Leu His 1 5 10 1877PRTMus musculus 187Tyr Ala Ser Gln Ser Ile Ser 1 5 1889PRTMus musculus 188Gln Gln Thr Asn Asn Trp Pro Leu Thr 1 5 18934DNAMus musculus 189ggctactcca tcaccagtgg ttattactgg aact 3419049DNAMus musculus 190cacataaact acgacggtag agataactac aacccatctc tcaaaaatc 4919134DNAMus musculus 191gagtttggta acttccctta ctactttgac tact 3419211PRTMus musculus 192Gly Tyr Ser Ile Thr Ser Gly Tyr Tyr Trp Asn 1 5 10 19316PRTMus musculus 193His Ile Asn Tyr Asp Gly Arg Asp Asn Tyr Asn Pro Ser Leu Lys Asn 1 5 10 15 19411PRTMus musculus 194Glu Phe Gly Asn Phe Pro Tyr Tyr Phe Asp Tyr 1 5 10 19531DNAMus musculus 195agtgcctact caagtgtacg tttcatgttc t 3119622DNAMus musculus 196cgcacatcca acctggcttc tg 2219728DNAMus musculus 197cagcagtatc atagttaccc gtggacgt 2819810PRTMus musculus 198Ser Ala Tyr Ser Ser Val Arg Phe Met Phe 1 5 10 1997PRTMus musculus 199Arg Thr Ser Asn Leu Ala Ser 1 5 2009PRTMus musculus 200Gln Gln Tyr His Ser Tyr Pro Trp Thr 1 5 20131DNAMus musculus 201ggattcactt tcagtagtta ttacatgtct t 3120252DNAMus musculus 202gtcattaata gtaatggtgg tagcacctac tattcagaga ctgtgaaggg cc 5220331DNAMus musculus 203caaccctatt atggtaaccc ttttgactac t 3120410PRTMus musculus 204Gly Phe Thr Phe Ser Ser Tyr Tyr Met Ser 1 5 10 20517PRTMus musculus 205Val Ile Asn Ser Asn Gly Gly Ser Thr Tyr Tyr Ser Glu Thr Val Lys 1 5 10 15 Gly 20610PRTMus musculus 206Gln Pro Tyr Tyr Gly Asn Pro Phe Asp Tyr 1 5 10 20734DNAMus musculus 207agggccagcc aaagtattag caacaaccta cact 3420822DNAMus musculus 208tatgtttccc agtccatctc tg 2220928DNAMus musculus 209caacagagta acaactggcc gctcacgt 2821011PRTMus musculus 210Arg Ala Ser Gln Ser Ile Ser Asn Asn Leu His 1 5 10 2117PRTMus musculus 211Tyr Val Ser Gln Ser Ile Ser 1 5 2129PRTMus musculus 212Gln Gln Ser Asn Asn Trp Pro Leu Thr 1 5 21331DNAMus musculus 213ggttatgcat tcactagcta caacatgtac t 3121452DNAMus musculus 214tatattgatc cttacaatgg tggtactaac tacaaccaga agttcaaggg ca 5221537DNAMus musculus 215ggggggatgg gattacgacg ggactacttt gacttct 3721610PRTMus musculus 216Gly Tyr Ala Phe Thr Ser Tyr Asn Met Tyr 1 5 10 21717PRTMus musculus 217Tyr Ile Asp Pro Tyr Asn Gly Gly Thr Asn Tyr Asn Gln Lys Phe Lys 1 5 10 15 Gly 21812PRTMus musculus 218Gly Gly Met Gly Leu Arg Arg Asp Tyr Phe Asp Phe 1 5 10 21952DNAMus musculus 219aagtccagtc agagcctttt atatagtagc aatcaaaaga actacttggc ct 5222022DNAMus musculus 220tgggcatcca ctagggaatc tg 2222128DNAMus musculus 221cagcaatatt atagctatcc gtacacgt 2822217PRTMus musculus 222Lys Ser Ser Gln Ser Leu Leu Tyr Ser Ser Asn Gln Lys Asn Tyr Leu 1 5 10 15 Ala 2237PRTMus musculus 223Trp Ala Ser Thr Arg Glu Ser 1 5 2249PRTMus musculus 224Gln Gln Tyr Tyr Ser Tyr Pro Tyr Thr 1 5 22531DNAMus musculus 225ggctacacct tcacaaccta ctatatacac t 3122652DNAMus musculus 226tggatttttc ctggaaatgt taatactaag tacaatgcga agttcaaggg ca 5222728DNAMus musculus 227gagggattac agtactactt tgactact 2822810PRTMus musculus 228Gly Tyr Thr Phe Thr Thr Tyr Tyr Ile His 1 5 10 22917PRTMus musculus 229Trp Ile Phe Pro Gly Asn Val Asn Thr Lys Tyr Asn Ala Lys Phe Lys 1 5 10 15 Gly 2309PRTMus musculus 230Glu Gly Leu Gln Tyr Tyr Phe Asp Tyr 1 5 23131DNAMus musculus 231agtgccaact caagtgtacg tttcatgttc t 3123222DNAMus musculus 232cgcacatcca acctggcttc tg 2223328DNAMus musculus 233cagcagtatc atagttaccc gtggacgt 2823410PRTMus musculus 234Ser Ala Asn Ser Ser Val Arg Phe Met Phe 1 5 10 2357PRTMus musculus 235Arg Thr Ser Asn Leu Ala Ser 1 5 2369PRTMus musculus 236Gln Gln Tyr His Ser Tyr Pro Trp Thr 1 5 23731DNAMus musculus 237ggattcactt tcagtagcta ttatatgtct t 3123852PRTMus musculus 238Gly Cys Cys Ala Thr Thr Ala Ala Thr Ala Thr Thr Ala Ala Thr Gly 1 5 10 15 Gly Thr Gly Gly Thr Ala Gly Cys Ala Cys Cys Thr Ala Cys Thr Ala 20 25 30 Thr Cys Cys Ala Gly Ala Cys Ala Cys Thr Gly Thr Gly Ala Ala Gly 35 40 45 Gly Gly Cys Cys 50 23934DNAMus musculus 239gagttggccg gctatggtac cccgtttgct tact 3424010PRTMus musculus 240Gly Phe Thr Phe Ser Ser Tyr Tyr Met Ser 1 5 10 24117PRTMus musculus 241Ala Ile Asn Ile Asn Gly Gly Ser Thr Tyr Tyr Pro Asp Thr Val Lys 1 5 10 15 Gly 24211PRTMus musculus 242Glu Leu Ala Gly Tyr Gly Thr Pro Phe Ala Tyr 1 5 10 24334DNAMus musculus 243agggccagcc aaagtattag caacaaccta cact 3424422DNAMus musculus 244tatgcttccc agtccatctc tg 2224528DNAMus musculus 245caacagagta acaactggcc gctcacgt 2824611PRTMus musculus 246Arg Ala Ser Gln Ser Ile Ser Asn Asn Leu His 1 5 10 2477PRTMus musculus 247Tyr Ala Ser Gln Ser Ile Ser 1 5 2489PRTMus musculus 248Gln Gln Ser Asn Asn Trp Pro Leu Thr 1 5 24931DNAMus musculus 249ggattcactt tcagtagctt ttacatgtct t 3125052DNAMus musculus 250accattaata ctaatggtgg tagcacctac tattcagaca ctgtgaaggg cc 5225131DNAMus musculus 251cagccttact acggagggac tatggactac t 3125210PRTMus musculus 252Gly Phe Thr Phe Ser Ser Phe Tyr Met Ser 1 5 10 25317PRTMus musculus 253Thr Ile Asn Thr Asn Gly Gly Ser Thr Tyr Tyr Ser Asp Thr Val Lys 1 5 10 15 Gly 25410PRTMus musculus 254Gln Pro Tyr Tyr Gly Gly Thr Met Asp Tyr 1 5 10 25534DNAMus musculus 255agggccagcc aaagtattaa caacaatcta cact 3425622DNAMus musculus 256tatgcttccc agtccatctc tg 2225728DNAMus musculus 257caacagacta acaactggcc tctcacgt 2825811PRTMus musculus 258Arg Ala Ser Gln Ser Ile Asn Asn Asn Leu His 1 5 10 2597PRTMus musculus 259Tyr Ala Ser Gln Ser Ile Ser 1 5 2609PRTMus musculus 260Gln Gln Thr Asn Asn Trp Pro Leu Thr 1 5 26134DNAMus musculus 261ggctactcca tcaccagtgg ttattactgg aact 3426249DNAMus musculus 262tacataagct acgacggtag aaataactac aacccatctc tcaaaaatc 4926334DNAMus musculus 263gaaaatagta actaccctta ctactatgac tact 3426411PRTMus musculus 264Gly Tyr Ser Ile Thr Ser Gly Tyr Tyr Trp Asn 1 5 10 26516PRTMus musculus 265Tyr Ile Ser Tyr Asp Gly Arg Asn Asn Tyr Asn Pro Ser Leu Lys Asn 1 5 10 15 26611PRTMus musculus 266Glu Asn Ser Asn Tyr Pro Tyr Tyr Tyr Asp Tyr 1 5 10 26734DNAMus musculus 267ggctactcca tcaccagtgg ttattactgg aact 3426849DNAMus musculus 268tacataagct acgacggtag aaataactac aacccatctc tcaaaaatc 4926934DNAMus musculus 269gaaaatagta actaccctta ctactatgac tact 3427011PRTMus musculus 270Gly Tyr Ser Ile Thr Ser Gly Tyr Tyr Trp Asn 1 5 10 27116PRTMus musculus 271Tyr Ile Ser Tyr Asp Gly Arg Asn Asn Tyr Asn Pro Ser Leu Lys Asn 1 5 10 15 27211PRTMus musculus 272Glu Asn Ser Asn Tyr Pro Tyr Tyr Tyr Asp Tyr 1 5 10 27331DNAMus musculus 273ggattcagtt tcagtacctc ttacatgtct t 3127452DNAMus musculus 274gccattaatc ttaatggtgg tagtacctac tattcagaca ctgtgaaggg cc 5227534DNAMus musculus 275gagttggccg ggtatggtac cccgtttgct tact 3427610PRTMus musculus 276Gly Phe Ser Phe Ser Thr Ser Tyr Met Ser 1 5 10 27717PRTMus musculus 277Ala Ile Asn Leu Asn Gly Gly Ser Thr Tyr Tyr Ser Asp Thr Val Lys 1 5 10 15 Gly 27811PRTMus musculus 278Glu Leu Ala Gly Tyr Gly Thr Pro Phe Ala Tyr 1 5 10 27934DNAMus musculus 279aaggccagcc aaagtattag caacaaccta cact 3428022DNAMus musculus 280tatacttccc agtccatctc tg 2228128DNAMus musculus 281caacagagta acagttggcc gctcacgt 2828211PRTMus musculus 282Lys Ala Ser Gln Ser Ile Ser Asn Asn Leu His 1 5 10 2837PRTMus musculus 283Tyr Thr Ser Gln Ser Ile Ser 1 5 2849PRTMus musculus 284Gln Gln Ser Asn Ser Trp Pro Leu Thr 1 5 28531DNAMus musculus 285ggattcactt tcagtagctt ttacatgtct t 3128652DNAMus musculus 286accattaata ctaatggtgg tagcacctac tattcagaca ctgtgaaggg cc 5228731DNAMus musculus 287cagccttact acggagggac tatggactac t 3128810PRTMus musculus 288Gly Phe Thr Phe Ser Ser Phe Tyr Met Ser 1 5 10 28917PRTMus musculus 289Thr Ile Asn Thr Asn Gly Gly Ser Thr Tyr Tyr Ser Asp Thr Val Lys 1 5 10 15 Gly 29010PRTMus musculus 290Gln Pro Tyr Tyr Gly Gly Thr Met Asp Tyr 1 5 10 29134DNAMus musculus 291agggccagcc aaagtattaa caacaatcta cact 3429222DNAMus musculus 292tatgcttccc agtccatctc tg 2229328DNAMus musculus 293caacagacta acaactggcc tctcacgt 2829411PRTMus musculus 294Arg Ala Ser Gln Ser Ile Asn Asn Asn Leu His 1 5 10 2957PRTMus musculus 295Tyr Ala Ser Gln Ser Ile Ser 1 5 2969PRTMus musculus 296Gln Gln Thr Asn Asn Trp Pro Leu Thr 1 5 29730DNAMus musculus 297ggctacacct tcacaaacta ctatttacac 3029851DNAMus musculus 298tggatttatc ctggaaatgt taatactaag tacaatgaga agttcaaggg c 5129927DNAMus musculus 299gagggattac agtactactt tgactac 2730010PRTMus musculus 300Gly Tyr Thr Phe Thr Asn Tyr Tyr Leu His 1 5 10 30117PRTMus musculus 301Trp Ile Tyr Pro Gly Asn Val Asn Thr Lys Tyr Asn Glu Lys Phe Lys 1 5 10 15 Gly 3029PRTMus musculus 302Glu Gly Leu Gln Tyr Tyr Phe Asp Tyr 1 5 30331DNAMus musculus 303agtgccaact caagtgtacg ttacatgttc t 3130422DNAMus musculus 304cgcacatcca acctggcttc tg 2230528DNAMus musculus 305cagcagtatc atagttaccc gtggacgt 2830610PRTMus musculus 306Ser Ala Asn Ser Ser Val Arg Tyr Met Phe 1 5 10 3077PRTMus musculus 307Arg Thr Ser Asn Leu Ala Ser 1 5 3089PRTMus musculus 308Gln Gln Tyr His Ser Tyr Pro Trp Thr 1 5 30931DNAMus musculus 309ggctacacct ttactagcta ctggatgcac t 3131052DNAMus musculus 310tacattaatc ctaacactgg ttatagtgag tacaatcaaa agttcaggga ca 5231149DNAMus musculus 311actgcacaat actacggtag tcctaggggt tactatgcta tggactcct 4931210PRTMus musculus 312Gly Tyr Thr Phe Thr Ser Tyr Trp Met His 1 5 10 31317PRTMus musculus 313Tyr Ile Asn Pro Asn Thr Gly Tyr Ser Glu Tyr Asn Gln Lys Phe Arg 1 5 10 15 Asp 31416PRTMus musculus 314Thr Ala Gln Tyr Tyr Gly Ser Pro Arg Gly Tyr Tyr Ala Met Asp Ser 1 5 10 15 31549DNAMus musculus 315aggtctagta agagtctcct acatagtaac ggcatcactt atttgtgtt 4931622DNAMus musculus 316cagatgtcca accttgcctc ag 2231728DNAMus musculus 317gctcaaaatc tagatctgcc gtggacgt 2831816PRTMus musculus 318Arg Ser Ser Lys Ser Leu Leu His Ser Asn Gly Ile Thr Tyr Leu Cys 1 5 10 15 3197PRTMus musculus 319Gln Met Ser Asn Leu Ala Ser 1 5 3209PRTMus musculus 320Ala Gln Asn Leu Asp Leu Pro Trp Thr 1 5 32131DNAMus musculus 321ggctacacct tcacaaccta ctatatacac t 3132252DNAMus musculus 322tggatttttc ctggaaatgt taatactaag tacaatgcga agttcaaggg ca 5232328DNAMus musculus 323gaggaattac agtactactt tgactact 2832410PRTMus musculus 324Gly Tyr Thr Phe Thr Thr Tyr Tyr Ile His 1 5 10 32517PRTMus musculus 325Trp Ile Phe Pro Gly Asn Val Asn Thr Lys Tyr Asn Ala Lys Phe Lys 1 5 10 15 Gly 3269PRTMus musculus 326Glu Glu Leu Gln Tyr Tyr Phe Asp Tyr 1 5 32731DNAMus musculus 327agtgccaact caagtgtacg tttcatgttc t 3132822DNAMus musculus 328cgcacatcca acctggcttc tg 2232928DNAMus musculus 329cagcagtatc atagttaccc gtggacgt 2833010PRTMus musculus 330Ser Ala Asn Ser Ser Val Arg Phe Met Phe 1 5 10 3317PRTMus musculus 331Arg Thr Ser Asn Leu Ala Ser 1 5 3329PRTMus musculus 332Gln Gln Tyr His Ser Tyr Pro Trp Thr 1 5 33331DNAMus musculus 333ggctacacct ttactagcta ctggatgcac t 3133452DNAMus musculus 334tacattaatc ctagtactgg ttatactgag tacaatcaga agttcaggga ca 5233549DNAMus musculus 335actgcgcaat actacggtag tcctaggggt tactatgcta tggactcct 4933610PRTMus musculus 336Gly Tyr Thr Phe Thr Ser Tyr Trp Met His 1 5 10 33717PRTMus musculus 337Tyr Ile Asn Pro Ser Thr Gly Tyr Thr Glu Tyr Asn Gln Lys Phe Arg 1 5 10 15 Asp 33816PRTMus musculus 338Thr Ala Gln Tyr Tyr Gly Ser Pro Arg Gly Tyr Tyr Ala Met Asp Ser 1 5 10 15 33949DNAMus musculus 339aggtctagta agagtctcct acatagtaac ggcatcactt atttgtgtt 4934022DNAMus musculus 340ctgatgtcca accttgcctc ag 2234128DNAMus musculus 341gctcaaaatc tagatcttcc gtggacgt 2834216PRTMus musculus 342Arg Ser Ser Lys Ser Leu Leu His Ser Asn Gly Ile Thr Tyr Leu Cys 1 5 10 15 3437PRTMus musculus 343Leu Met Ser Asn Leu Ala Ser 1 5 3449PRTMus musculus 344Ala Gln Asn Leu Asp Leu Pro Trp Thr 1 5 34531DNAMus musculus 345ggattcactt tcagtagctt ttacatgtct t 3134652DNAMus musculus 346gccattaata ctaatggtgg tagcacctac tattcagaca ctgtgaaggg cc 5234731DNAMus musculus 347cagccttact acggagggcc tatggacttc t 3134810PRTMus musculus 348Gly Phe Thr Phe Ser Ser Phe Tyr Met Ser 1 5 10 34917PRTMus musculus 349Ala Ile Asn Thr Asn Gly Gly Ser Thr Tyr

Tyr Ser Asp Thr Val Lys 1 5 10 15 Gly 35010PRTMus musculus 350Gln Pro Tyr Tyr Gly Gly Pro Met Asp Phe 1 5 10 35134DNAMus musculus 351agggccagcc aaagtattaa caacaaccta cact 3435222DNAMus musculus 352tatgcttccc agtccatctc tg 2235328DNAMus musculus 353caacagagta acaactggcc tctcacgt 2835411PRTMus musculus 354Arg Ala Ser Gln Ser Ile Asn Asn Asn Leu His 1 5 10 3557PRTMus musculus 355Tyr Ala Ser Gln Ser Ile Ser 1 5 3569PRTMus musculus 356Gln Gln Ser Asn Asn Trp Pro Leu Thr 1 5 35730DNAMus musculus 357ggattcactt tcagtagatt ttacatgtct 3035851DNAMus musculus 358gccattaata ctaatggtgg tagcacctat tattcagaca ctgtgaaggg c 5135930DNAMus musculus 359cagccttact acggagggcc tatggagttc 3036010PRTMus musculus 360Gly Phe Thr Phe Ser Arg Phe Tyr Met Ser 1 5 10 36117PRTMus musculus 361Ala Ile Asn Thr Asn Gly Gly Ser Thr Tyr Tyr Ser Asp Thr Val Lys 1 5 10 15 Gly 36210PRTMus musculus 362Gln Pro Tyr Tyr Gly Gly Pro Met Glu Phe 1 5 10 36334DNAMus musculus 363aaggccagtc agaatgtggg tactaatgta gcct 3436422DNAMus musculus 364tcggcatcct accggtacag tg 2236528DNAMus musculus 365cagcaatata acagctatcc tctcacgt 2836611PRTMus musculus 366Lys Ala Ser Gln Asn Val Gly Thr Asn Val Ala 1 5 10 3677PRTMus musculus 367Ser Ala Ser Tyr Arg Tyr Ser 1 5 3689PRTMus musculus 368Gln Gln Tyr Asn Ser Tyr Pro Leu Thr 1 5 36910DNAArtificialTCF Consensus sequence 369agatcaaagg 10370276PRTHomo sapiens 370Ile Val Leu Gln Leu Glu Asp Ile Arg His Ala Ile Ala Ile Asp Tyr 1 5 10 15 Asp Pro Val Glu Gly Tyr Ile Tyr Trp Thr Asp Asp Glu Val Arg Ala 20 25 30 Ile Arg Arg Ser Phe Ile Asp Gly Ser Gly Ser Gln Phe Val Val Thr 35 40 45 Ala Gln Ile Ala His Pro Asp Gly Ile Ala Val Asp Trp Val Ala Arg 50 55 60 Asn Leu Tyr Trp Thr Asp Thr Gly Thr Asp Arg Ile Glu Val Thr Arg 65 70 75 80 Leu Asn Gly Thr Met Arg Lys Ile Leu Ile Ser Glu Asp Leu Glu Glu 85 90 95 Pro Arg Ala Ile Val Leu Asp Pro Met Val Gly Tyr Met Tyr Trp Thr 100 105 110 Asp Trp Gly Glu Ile Pro Lys Ile Glu Arg Ala Ala Leu Asp Gly Ser 115 120 125 Asp Arg Val Val Leu Val Asn Thr Ser Leu Gly Trp Pro Asn Gly Leu 130 135 140 Ala Leu Asp Tyr Asp Glu Gly Lys Ile Tyr Trp Gly Asp Ala Lys Thr 145 150 155 160 Asp Lys Ile Glu Val Met Asn Thr Asp Gly Thr Gly Arg Arg Val Leu 165 170 175 Val Glu Asp Lys Ile Pro His Ile Phe Gly Phe Thr Leu Leu Gly Asp 180 185 190 Tyr Val Tyr Trp Thr Asp Trp Gln Arg Arg Ser Ile Glu Arg Val His 195 200 205 Lys Arg Ser Ala Glu Arg Glu Val Ile Ile Asp Gln Leu Pro Asp Leu 210 215 220 Met Gly Leu Lys Ala Thr Asn Val His Arg Val Ile Gly Ser Asn Pro 225 230 235 240 Cys Ala Glu Glu Asn Gly Gly Cys Ser His Leu Cys Leu Tyr Arg Pro 245 250 255 Gln Gly Leu Arg Cys Ala Cys Pro Ile Gly Phe Glu Leu Ile Ser Asp 260 265 270 Met Lys Thr Cys 275 37148PRTHomo sapiens 371Ile Leu Tyr Trp Thr Asp Trp Ser Thr His Ser Ile Leu Ala Cys Asn 1 5 10 15 Lys Tyr Thr Gly Glu Gly Leu Arg Glu Ile His Ser Asp Ile Phe Ser 20 25 30 Pro Met Asp Ile His Ala Phe Ser Gln Gln Arg Gln Pro Asn Ala Thr 35 40 45 37248PRTMus musculus 372Thr Leu Tyr Trp Thr Asp Trp Asn Thr His Ser Ile Leu Ala Cys Asn 1 5 10 15 Lys Tyr Thr Gly Glu Gly Leu Arg Glu Ile His Ser Asn Ile Phe Ser 20 25 30 Pro Met Asp Ile His Ala Phe Ser Gln Gln Arg Gln Pro Asn Ala Thr 35 40 45 3731613PRTMus musculus 373Met Gly Ala Val Leu Arg Ser Leu Leu Ala Cys Ser Phe Cys Val Leu 1 5 10 15 Leu Arg Ala Ala Pro Leu Leu Leu Tyr Ala Asn Arg Arg Asp Leu Arg 20 25 30 Leu Val Asp Ala Thr Asn Gly Lys Glu Asn Ala Thr Ile Val Val Gly 35 40 45 Gly Leu Glu Asp Ala Ala Ala Val Asp Phe Val Phe Gly His Gly Leu 50 55 60 Ile Tyr Trp Ser Asp Val Ser Glu Glu Ala Ile Lys Arg Thr Glu Phe 65 70 75 80 Asn Lys Thr Glu Ser Val Gln Asn Val Val Val Ser Gly Leu Leu Ser 85 90 95 Pro Asp Gly Leu Ala Cys Asp Trp Leu Gly Glu Lys Leu Tyr Trp Thr 100 105 110 Asp Ser Glu Thr Asn Arg Ile Glu Val Ser Asn Leu Asp Gly Ser Leu 115 120 125 Arg Lys Val Leu Phe Trp Gln Glu Leu Asp Gln Pro Arg Ala Ile Ala 130 135 140 Leu Asp Pro Ser Ser Gly Phe Met Tyr Trp Thr Asp Trp Gly Glu Val 145 150 155 160 Pro Lys Ile Glu Arg Ala Gly Met Asp Gly Ser Ser Arg Phe Val Ile 165 170 175 Ile Asn Thr Glu Ile Tyr Trp Pro Asn Gly Leu Thr Leu Asp Tyr Gln 180 185 190 Glu Arg Lys Leu Tyr Trp Ala Asp Ala Lys Leu Asn Phe Ile His Lys 195 200 205 Ser Asn Leu Asp Gly Thr Asn Arg Gln Ala Val Val Lys Gly Ser Leu 210 215 220 Pro His Pro Phe Ala Leu Thr Leu Phe Glu Asp Thr Leu Tyr Trp Thr 225 230 235 240 Asp Trp Asn Thr His Ser Ile Leu Ala Cys Asn Lys Tyr Thr Gly Glu 245 250 255 Gly Leu Arg Glu Ile His Ser Asn Ile Phe Ser Pro Met Asp Ile His 260 265 270 Ala Phe Ser Gln Gln Arg Gln Pro Asn Ala Thr Asn Pro Cys Gly Ile 275 280 285 Asp Asn Gly Gly Cys Ser His Leu Cys Leu Met Ser Pro Val Lys Pro 290 295 300 Phe Tyr Gln Cys Ala Cys Pro Thr Gly Val Lys Leu Leu Glu Asn Gly 305 310 315 320 Lys Thr Cys Lys Asp Gly Ala Thr Glu Leu Leu Leu Leu Ala Arg Arg 325 330 335 Thr Asp Leu Arg Arg Ile Ser Leu Asp Thr Pro Asp Phe Thr Asp Ile 340 345 350 Val Leu Gln Leu Glu Asp Ile Arg His Ala Ile Ala Ile Asp Tyr Asp 355 360 365 Pro Val Glu Gly Tyr Ile Tyr Trp Thr Asp Asp Glu Val Arg Ala Ile 370 375 380 Arg Arg Ser Phe Ile Asp Gly Ser Gly Ser Gln Phe Val Val Thr Ala 385 390 395 400 Gln Ile Ala His Pro Asp Gly Ile Ala Val Asp Trp Val Ala Arg Asn 405 410 415 Leu Tyr Trp Thr Asp Thr Gly Thr Asp Arg Ile Glu Val Thr Arg Leu 420 425 430 Asn Gly Thr Met Arg Lys Ile Leu Ile Ser Glu Asp Leu Glu Glu Pro 435 440 445 Arg Ala Ile Val Leu Asp Pro Met Val Gly Tyr Met Tyr Trp Thr Asp 450 455 460 Trp Gly Glu Ile Pro Lys Ile Glu Arg Ala Ala Leu Asp Gly Ser Asp 465 470 475 480 Arg Val Val Leu Val Asn Thr Ser Leu Gly Trp Pro Asn Gly Leu Ala 485 490 495 Leu Asp Tyr Asp Glu Gly Thr Ile Tyr Trp Gly Asp Ala Lys Thr Asp 500 505 510 Lys Ile Glu Val Met Asn Thr Asp Gly Thr Gly Arg Arg Val Leu Val 515 520 525 Glu Asp Lys Ile Pro His Ile Phe Gly Phe Thr Leu Leu Gly Asp Tyr 530 535 540 Val Tyr Trp Thr Asp Trp Gln Arg Arg Ser Ile Glu Arg Val His Lys 545 550 555 560 Arg Ser Ala Glu Arg Glu Val Ile Ile Asp Gln Leu Pro Asp Leu Met 565 570 575 Gly Leu Lys Ala Thr Ser Val His Arg Ile Ile Gly Ser Asn Pro Cys 580 585 590 Ala Glu Asp Asn Gly Gly Cys Ser His Leu Cys Leu Tyr Arg Pro Gln 595 600 605 Gly Leu Arg Cys Ala Cys Pro Ile Gly Phe Glu Leu Ile Ser Asp Met 610 615 620 Lys Thr Cys Ile Val Pro Glu Ala Phe Leu Leu Phe Ser Arg Arg Ala 625 630 635 640 Asp Ile Arg Arg Ile Ser Leu Glu Thr Asn Asn Asn Asn Val Ala Ile 645 650 655 Pro Leu Thr Gly Val Lys Glu Ala Ser Ala Leu Asp Phe Asp Val Thr 660 665 670 Asp Asn Arg Ile Tyr Trp Thr Asp Ile Ser Leu Lys Thr Ile Ser Arg 675 680 685 Ala Phe Met Asn Gly Ser Ala Leu Glu His Val Val Glu Phe Gly Leu 690 695 700 Asp Tyr Pro Glu Gly Met Ala Val Asp Trp Leu Gly Lys Asn Leu Tyr 705 710 715 720 Trp Ala Asp Thr Gly Thr Asn Arg Ile Glu Val Ser Lys Leu Asp Gly 725 730 735 Gln His Arg Gln Val Leu Val Trp Lys Asp Leu Asp Ser Pro Arg Ala 740 745 750 Leu Ala Leu Asp Pro Ala Glu Gly Phe Met Tyr Trp Thr Glu Trp Gly 755 760 765 Gly Lys Pro Lys Ile Asp Arg Ala Ala Met Asp Gly Ser Glu Arg Thr 770 775 780 Thr Leu Val Pro Asn Val Gly Arg Ala Asn Gly Leu Thr Ile Asp Tyr 785 790 795 800 Ala Lys Arg Arg Leu Tyr Trp Thr Asp Leu Asp Thr Asn Leu Ile Glu 805 810 815 Ser Ser Asp Met Leu Gly Leu Asn Arg Glu Val Ile Ala Asp Asp Leu 820 825 830 Pro His Pro Phe Gly Leu Thr Gln Tyr Gln Asp Tyr Ile Tyr Trp Thr 835 840 845 Asp Trp Ser Arg Arg Ser Ile Glu Arg Ala Asn Lys Thr Ser Gly Gln 850 855 860 Asn Arg Thr Ile Ile Gln Gly His Leu Asp Tyr Val Met Asp Ile Leu 865 870 875 880 Val Phe His Ser Ser Arg Gln Ala Gly Trp Asn Glu Cys Ala Ser Ser 885 890 895 Asn Gly His Cys Ser His Leu Cys Leu Ala Val Pro Val Gly Gly Phe 900 905 910 Val Cys Gly Cys Pro Ala His Tyr Ser Leu Asn Ala Asp Asn Arg Thr 915 920 925 Cys Ser Ala Pro Thr Thr Phe Leu Leu Phe Ser Gln Lys Ser Ala Ile 930 935 940 Asn Arg Met Val Ile Asp Glu Gln Gln Ser Pro Asp Ile Ile Leu Pro 945 950 955 960 Ile His Ser Leu Arg Asn Val Arg Ala Ile Asp Tyr Asp Pro Leu Asp 965 970 975 Lys Gln Leu Tyr Trp Ile Asp Ser Arg Gln Asn Ser Ile Arg Lys Ala 980 985 990 His Glu Asp Gly Gly Gln Gly Phe Asn Val Val Ala Asn Ser Val Ala 995 1000 1005 Asn Gln Asn Leu Glu Ile Gln Pro Tyr Asp Leu Ser Ile Asp Ile 1010 1015 1020 Tyr Ser Arg Tyr Ile Tyr Trp Thr Cys Glu Ala Thr Asn Val Ile 1025 1030 1035 Asp Val Thr Arg Leu Asp Gly Arg Ser Val Gly Val Val Leu Lys 1040 1045 1050 Gly Glu Gln Asp Arg Pro Arg Ala Ile Val Val Asn Pro Glu Lys 1055 1060 1065 Gly Tyr Met Tyr Phe Thr Asn Leu Gln Glu Arg Ser Pro Lys Ile 1070 1075 1080 Glu Arg Ala Ala Leu Asp Gly Thr Glu Arg Glu Val Leu Phe Phe 1085 1090 1095 Ser Gly Leu Ser Lys Pro Ile Ala Leu Ala Leu Asp Ser Lys Leu 1100 1105 1110 Gly Lys Leu Phe Trp Ala Asp Ser Asp Leu Arg Arg Ile Glu Ser 1115 1120 1125 Ser Asp Leu Ser Gly Ala Asn Arg Ile Val Leu Glu Asp Ser Asn 1130 1135 1140 Ile Leu Gln Pro Val Gly Leu Thr Val Phe Glu Asn Trp Leu Tyr 1145 1150 1155 Trp Ile Asp Lys Gln Gln Gln Met Ile Glu Lys Ile Asp Met Thr 1160 1165 1170 Gly Arg Glu Gly Arg Thr Lys Val Gln Ala Arg Ile Ala Gln Leu 1175 1180 1185 Ser Asp Ile His Ala Val Lys Glu Leu Asn Leu Gln Glu Tyr Arg 1190 1195 1200 Gln His Pro Cys Ala Gln Asp Asn Gly Gly Cys Ser His Ile Cys 1205 1210 1215 Leu Val Lys Gly Asp Gly Thr Thr Arg Cys Ser Cys Pro Met His 1220 1225 1230 Leu Val Leu Leu Gln Asp Glu Leu Ser Cys Gly Glu Pro Pro Thr 1235 1240 1245 Cys Ser Pro Gln Gln Phe Thr Cys Phe Thr Gly Asp Ile Asp Cys 1250 1255 1260 Ile Pro Val Ala Trp Arg Cys Asp Gly Phe Thr Glu Cys Glu Asp 1265 1270 1275 His Ser Asp Glu Leu Asn Cys Pro Val Cys Ser Glu Ser Gln Phe 1280 1285 1290 Gln Cys Ala Ser Gly Gln Cys Ile Asp Gly Ala Leu Arg Cys Asn 1295 1300 1305 Gly Asp Ala Asn Cys Gln Asp Lys Ser Asp Glu Lys Asn Cys Glu 1310 1315 1320 Val Leu Cys Leu Ile Asp Gln Phe Arg Cys Ala Asn Gly Gln Cys 1325 1330 1335 Val Gly Lys His Lys Lys Cys Asp His Ser Val Asp Cys Ser Asp 1340 1345 1350 Arg Ser Asp Glu Leu Asp Cys Tyr Pro Thr Glu Glu Pro Ala Pro 1355 1360 1365 Gln Ala Thr Asn Thr Val Gly Ser Val Ile Gly Val Ile Val Thr 1370 1375 1380 Ile Phe Val Ser Gly Thr Ile Tyr Phe Ile Cys Gln Arg Met Leu 1385 1390 1395 Cys Pro Arg Met Lys Gly Asp Gly Glu Thr Met Thr Asn Asp Tyr 1400 1405 1410 Val Val His Ser Pro Ala Ser Val Pro Leu Gly Tyr Val Pro His 1415 1420 1425 Pro Ser Ser Leu Ser Gly Ser Leu Pro Gly Met Ser Arg Gly Lys 1430 1435 1440 Ser Met Ile Ser Ser Leu Ser Ile Met Gly Gly Ser Ser Gly Pro 1445 1450 1455 Pro Tyr Asp Arg Ala His Val Thr Gly Ala Ser Ser Ser Ser Ser 1460 1465 1470 Ser Ser Thr Lys Gly Thr Tyr Phe Pro Ala Ile Leu Asn Pro Pro 1475 1480 1485 Pro Ser Pro Ala Thr Glu Arg Ser His Tyr Thr Met Glu Phe Gly 1490 1495 1500 Tyr Ser Ser Asn Ser Pro Ser Thr His Arg Ser Tyr Ser Tyr Arg 1505 1510 1515 Pro Tyr Ser Tyr Arg His Phe Ala Pro Pro Thr Thr Pro Cys Ser 1520 1525 1530 Thr Asp Val Cys Asp Ser Asp Tyr Ala Pro Ser Arg Arg Met Thr 1535 1540 1545 Ser Val Ala Thr Ala Lys Gly Tyr Thr Ser Asp Val Asn Tyr Asp 1550 1555 1560 Ser Glu Pro Val Pro Pro Pro Pro Thr Pro Arg Ser Gln Tyr Leu 1565 1570 1575 Ser Ala Glu Glu Asn Tyr Glu Ser Cys Pro Pro Ser Pro Tyr Thr 1580 1585 1590 Glu Arg Ser Tyr Ser His His Leu Tyr Pro Pro Pro Pro Ser Pro 1595 1600 1605 Cys Thr Asp Ser Ser 1610

Claims

1. An isolated antibody or antigen-binding fragment thereof, comprising: a) a heavy chain, comprising a first variable region, comprising an amino acid sequence as set forth in SEQ ID NO: 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 72, 74, 78, 82, 86, 90, 94, 98, or 102; and b) a light chain comprising a second variable region, comprising an amino acid sequence as set forth in SEQ ID NO: 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 76, 80, 84, 88, 92, 96, 100, or 104.

2. An isolated antibody or antigen-binding fragment thereof, comprising: a) a heavy chain, comprising a first variable region, comprising a sequence that has at least 90% identity to an amino acid sequence as set forth in SEQ ID NO: 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 72, 74, 78, 82, 86, 90, 94, 98, or 102; and b) a light chain comprising a second variable region, comprising a sequence that has at least 90% identity to an amino acid sequence as set forth in SEQ ID NO: 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 76, 80, 84, 88, 92, 96, 100, or 104, wherein the antibody binds LRP6 with high affinity.

3. An isolated antibody or antigen-binding fragment thereof, comprising: a) a heavy chain, comprising a first variable region, comprising a sequence that has at least 95% identity to an amino acid sequence as set forth in SEQ ID NO: 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 72, 74, 78, 82, 86, 90, 94, 98, or 102; and b) a light chain comprising a second variable region, comprising a sequence that has at least 95% identity to an amino acid sequence as set forth in SEQ ID NO: 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 76, 80, 84, 88, 92, 96, 100, or 104, wherein the antibody binds LRP6 with high affinity.

4. An isolated antibody or antigen-binding fragment thereof, comprising: a) a heavy chain, comprising a first variable region, comprising a sequence that has at least 99% identity to an amino acid sequence as set forth in SEQ ID NO: 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 72, 74, 78, 82, 86, 90, 94, 98, or 102; and b) a light chain, comprising a second variable region, comprising a sequence that has at least 99% identity to an amino acid sequence as set forth in SEQ ID NO: 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 76, 80, 84, 88, 92, 96, 100, or 104, wherein the antibody binds to LRP6 with high affinity.

5. The antibody of any one of claims 1-4, wherein the antibody stimulates Wnt activity.

6. The antibody of any one of claims 1-4, wherein the antibody antagonizes Dkk1 activity.

7. The antibody of claim 1, wherein the heavy chain and the light chain are connected by a flexible linker to form a single chain antibody.

8. The antibody of claim 7, which is a single-Fv antibody.

9. The antibody of claim 1, which is selected from the group consisting of an Fab antibody, an Fab' antibody, an (Fab')2 antibody, a fully human antibody, a humanized antibody, and a chimeric antibody.

10. A nucleic acid molecule encoding the antibody of any one of claims 1-6.

11. A host cell transformed with the nucleic acid of claim 10.

12. A pharmaceutical composition comprising an antibody of claim 1, further comprising a suitable carrier.

13. A pharmaceutical composition comprising an antibody of claim 1, further comprising at least one therapeutic agent.

14. A method of treating a bone disorder in a patient comprising administering the pharmaceutical agent of claim 12 or 13.

15. The method of claim 14, wherein the bone disorder is selected from the group consisting of rheumatoid arthritis, osteoarthritis, ankylosing spondylosis, osteoporosis, and osteolytic lesions associated with multiple myeloma.

16. A method of healing wounds in a patient comprising administering the pharmaceutical agent of claim 12 or 13.

17. A method of treating gastrointestinal diseases in a patient comprising administering the pharmaceutical agent of claim 12 or 13.

18. The method of claim 17, wherein the gastrointestinal disease is inflammatory bowel disease.

19. A method of detecting the level of LRP6 in a biological sample, comprising contacting the sample with the antibody or antigen-binding fragment of claim 1.

20. A selective binding agent comprising any of SEQ ID NO: 108-110, 114-116, 120-122, 126-128, 132-134, 138-140, 144-146, 150-152, 156-158, 162-164, 168-170, 174-176, 180-182, 186-188, 192-194, 198-200, 204-206, 210-212, 216-218, 222-224, 228-230, 234-236, 240-242, 246-248, 252-254, 258-260, 264-266, 270-272, 276-278, 282-284, 288-290, 294-296, 300-302, 306-308, 312-314, 318-320, 324-326, 330-332, 336-338, 342-344, 348-350, 354-356, 360-362, or 366-368.

21. A nucleic acid molecule encoding the selective binding agent of claim 20.

22. A host cell transformed with the nucleic acid molecule of claim 21.

23. The selective binding agent of claim 20, wherein the heavy chain and the light chain are connected by a flexible linker to form a single chain antibody.

24. The selective binding agent of claim 23, which is a single-Fv antibody.

25. The selective binding agent of claim 20, which is selected from the group consisting of an Fab antibody, an (Fab')2 antibody, a fully human antibody, a humanized antibody, and a chimeric antibody.

26. A pharmaceutical composition comprising the selective binding agent of claim 20, further comprising a suitable carrier.

27. A pharmaceutical composition comprising the selective binding agent of claim 20, further comprising at least one therapeutic agent.

28. A method of treating a bone disorder in a patient, comprising administering the pharmaceutical composition of claim 26 or 27.

29. The method of claim 28, wherein the bone disorder is selected from the group consisting of rheumatoid arthritis, osteoarthritis, ankylosing spondylosis, osteoporosis, and osteolytic lesions associated with multiple myeloma.

30. A method of detecting the level of LRP6 in a biological sample, comprising contacting the sample with the selective binding agent of claim 20.

31. An antibody or antigen-binding fragment thereof according to any of claims 1-6, wherein the antibody or antigen-binding fragment has at least one property selected from the group consisting of: a) competes for binding to LRP6 with antibody selected from the group consisting of 77.2, 135.16, 213.7, 240.8, 413.1, 421.1, 498.3, 537.2, 606.4, 620.1, 856.6, 923.3, 931.1, 993.9, 995.5, 1115.3, 1213.2, 1253.12, 1281.1, 1293.11, 1433.8, 1470.2, and 1903.1; b) binds to the same epitope of LRP6 as an antibody selected from 77.2, 135.16, 213.7, 240.8, 413.1, 421.1, 498.3, 537.2, 606.4, 620.1, 856.6, 923.3, 931.1, 993.9, 995.5, 1115.3, 1213.2, 1253.12, 1281.1, 1293.11, 1433.8, 1470.2, and 1903.1; or c) binds to the same antigen as that bound by an antibody selected from the group consisting of 77.2, 135.16, 213.7, 240.8, 413.1, 421.1, 498.3, 537.2, 606.4, 620.1, 856.6, 923.3, 931.1, 993.9, 995.5, 1115.3, 1213.2, 1253.12, 1281.1, 1293.11, 1433.8, 1470.2, and 1903.1.

32. A selective binding agent according to any of claim 35, 36, or 37, wherein the selective binding agent has at least one property selected from the group consisting of: a) competes for binding to LRP6 with an antibody selected from 77.2, 135.16, 213.7, 240.8, 413.1, 421.1, 498.3, 537.2, 606.4, 620.1, 856.6, 923.3, 931.1, 993.9, 995.5, 1115.3, 1213.2, 1253.12, 1281.1, 1293.11, 1433.8, 1470.2, and 1903.1; b) binds to the same epitope of LRP6 as an antibody selected from the group consisting of 77.2, 135.16, 213.7, 240.8, 413.1, 421.1, 498.3, 537.2, 606.4, 620.1, 856.6, 923.3, 931.1, 993.9, 995.5, 1115.3, 1213.2, 1253.12, 1281.1, 1293.11, 1433.8, 1470.2, and 1903.1; or c) binds to the same antigen as that bound by an antibody selected from the group consisting of 77.2, 135.16, 213.7, 240.8, 413.1, 421.1, 498.3, 537.2, 606.4, 620.1, 856.6, 923.3, 931.1, 993.9, 995.5, 1115.3, 1213.2, 1253.12, 1281.1, 1293.11, 1433.8, 1470.2, and 1903.1.

33. An isolated antibody or antigen-binding fragment thereof that binds to an epitope within residues 263-283 of an LRP6 polypeptide comprising SEQ ID NO: 2.

34. The antibody of claim 33, wherein the antibody binds to the LRP6 polypeptide of SEQ ID NO: 2 with a KD of less than or equal to 10<9>M.

35. An isolated cell line that produces an antibody according to any one of claims 1-6.

36. An isolated cell line that produces a specific binding agent according to claim 20.

37. An isolated cell line that produces an antibody selected from the group of 77.2, 135.16, 213.7, 240.8, 413.1, 421.1, 498.3, 537.2, 606.4, 620.1, 856.6, 923.3, 931.1, 993.9, 995.5, 1115.3, 1213.2, 1253.12, 1281.1, 1293.11, 1433.8, 1470.2, and 1903.1.

38. The antibody of claim 1, wherein said antibody is murine anti-LRP6 monoclonal antibody 77.2.

39. The antibody of claim 38, wherein said antibody is a humanized or chimeric form thereof.

40. The antibody of claim 1, wherein said antibody is murine anti-LRP6 monoclonal antibody 135.16.

41. The antibody of claim 40, wherein said antibody is a humanized or chimeric form thereof.

42. The antibody of claim 1, wherein said antibody is murine anti-LRP6 monoclonal antibody 213.7.

43. The antibody of claim 42, wherein said antibody is a humanized or chimeric form thereof.

44. The antibody of claim 1, wherein said antibody is murine anti-LRP6 monoclonal antibody 240.8.

45. The antibody of claim 44, wherein said antibody is a humanized or chimeric form thereof.

46. The antibody of claim 1, wherein said antibody is murine anti-LRP6 monoclonal antibody 413.1.

47. The antibody of claim 46, wherein said antibody is a humanized or chimeric form thereof.

48. The antibody of claim 1, wherein said antibody is murine anti-LRP6 monoclonal antibody 421.1.

49. The antibody of claim 48, wherein said antibody is a humanized or chimeric form thereof.

50. The antibody of claim 1, wherein said antibody is murine anti-LRP6 monoclonal antibody 498.3.

51. The antibody of claim 50, wherein said antibody is a humanized or chimeric form thereof.

52. The antibody of claim 1, wherein said antibody is murine anti-LRP6 monoclonal antibody 537.2.

53. The antibody of claim 52, wherein said antibody is a humanized or chimeric form thereof.

54. The antibody of claim 1, wherein said antibody is murine anti-LRP6 monoclonal antibody 606.4.

55. The antibody of claim 54, wherein said antibody is a humanized or chimeric form thereof.

56. The antibody of claim 1, wherein said antibody is murine anti-LRP6 monoclonal antibody 620.1.

57. The antibody of claim 56, wherein said antibody is a humanized or chimeric form thereof.

58. The antibody of claim 1, wherein said antibody is murine anti-LRP6 monoclonal antibody 856.6.

59. The antibody of claim 58, wherein said antibody is a humanized or chimeric form thereof.

60. The antibody of claim 1, wherein said antibody is murine anti-LRP6 monoclonal antibody 923.3.

61. The antibody of claim 60, wherein said antibody is a humanized or chimeric form thereof.

62. The antibody of claim 1, wherein said antibody is murine anti-LRP6 monoclonal antibody 931.1.

63. The antibody of claim 62, wherein said antibody is a humanized or chimeric form thereof.

64. The antibody of claim 1, wherein said antibody is murine anti-LRP6 monoclonal antibody 993.9.

65. The antibody of claim 64, wherein said antibody is a humanized or chimeric form thereof.

66. The antibody of claim 1, wherein said antibody is murine anti-LRP6 monoclonal antibody 995.5.

67. The antibody of claim 66, wherein said antibody is a humanized or chimeric form thereof.

68. The antibody of claim 1, wherein said antibody is murine anti-LRP6 monoclonal antibody 1115.3.

69. The antibody of claim 68, wherein said antibody is a humanized or chimeric form thereof.

70. The antibody of claim 1, wherein said antibody is murine anti-LRP6 monoclonal antibody 1213.2.

71. The antibody of claim 70, wherein said antibody is a humanized or chimeric form thereof.

72. The antibody of claim 1, wherein said antibody is murine anti-LRP6 monoclonal antibody 1253.12.

73. The antibody of claim 72, wherein said antibody is a humanized or chimeric form thereof.

74. The antibody of claim 1, wherein said antibody is murine anti-LRP6 monoclonal antibody 1281.1.

75. The antibody of claim 74, wherein said antibody is a humanized or chimeric form thereof.

76. The antibody of claim 1, wherein said antibody is murine anti-LRP6 monoclonal antibody 1293.11.

77. The antibody of claim 76, wherein said antibody is a humanized or chimeric form thereof.

78. The antibody of claim 1, wherein said antibody is murine anti-LRP6 monoclonal antibody 1433.8.

79. The antibody of claim 78, wherein said antibody is a humanized or chimeric form thereof.

80. The antibody of claim 1, wherein said antibody is murine anti-LRP6 monoclonal antibody 1470.2.

81. The antibody of claim 80, wherein said antibody is a humanized or chimeric form thereof.

82. The antibody of claim 1, wherein said antibody is murine anti-LRP6 monoclonal antibody 1903.1.

83. The antibody of claim 82, wherein said antibody is a humanized or chimeric form thereof.