HK1178175B - Anti-lrp6 antibodies - Google Patents

Description

anti-LRP 6 antibodies

Cross reference to related applications

The present application claims the benefit of U.S. provisional application No.61/317,137 filed on 24/2010 and U.S. provisional application No.61/394,836 filed on 20/10/2010, the complete disclosures of which are incorporated herein by reference.

Technical Field

The invention relates to anti-LRP 6 antibodies and methods of using anti-LRP 6 antibodies to treat cancer or a bone disorder.

Background

Similar to most other morphogen and growth factor signaling pathways, mammalian Wnt signaling is mobilized many times during development and tissue homeostasis via the use of 19 different ligands, 10 receptors, and multiple co-receptors, including LRP5/6, Ror1/2, and Ryk (vanAmerongen and Nusse, 2009). In addition, different secretory antagonists that bind Wnt (such as SFRP1/2/3/4/5 and WIF 1) or LRP5/6 (including DKK1/2/4 and SOST) modulate the interaction between the ligand and the receptor. These membrane and extracellular proteins and their various isoforms provide differential regulation in expression levels and in combination with protein interactions. Most Wnt isoforms were shown to be able to bind to the co-receptor LRP5/6, while the involvement of LRP5/6 refined canonical or β -catenin dependent Wnt signaling. Wnt heterodimerizes LRP5/6 and FZD to mediate LRP5/6 endodomain phosphorylation and axin binding (Tamai et al, 2000; Semenov et al, 2001; Tamai et al, 2004). DVL is brought into the complex by direct binding to both axin and FZD, while DVL oligomerization likely enlarges these protein complexes on the cytoplasmic face of the membrane, sequestering GSK3 and inhibiting its phosphorylation and β -catenin destabilization (Mi et al, 2006; bilc et al, 2007; Schwarz-Romond et al, 2007; Cselenyi et al, 2008; Piao et al, 2008; WilliamsonZeng et al, 2008; Wu et al, 2009).

A very large number of ligand isoforms that exhibit considerable primary sequence divergence that mediate canonical Wnt signaling in mammals are in contrast to highly homologous co-receptor pairs. The extracellular domains of LRP6 and LRP5 consist mainly of four homologous regions, designated from N-terminus to C-terminus as E1 to E4, each containing a YWTD type β -propeller and an EGF-like domain (Jeon et al, 2001). Each repeat at similar positions in LRP6 and LRP5 was highly conserved, while different repeats within the same protein were considerably more divergent. Interestingly, Bourhis et al (2010) demonstrated that Wnt9b binds exclusively within the E1-E2 region in vitro, whereas Wnt3a binds only the fragment containing E3-E4, suggesting that each repeat or the combination of two adjacent repeats binds to a different subset of Wnt isoforms. This arrangement can accommodate the diversity of Wnt proteins, and may also allow differential modulation of them by LRP5/6 antagonistic ligands. In Notch and VEGF receptors whose extracellular regions contain repetitive EGF-like and Ig domains, respectively, binding of multiple ligand isoforms is localized to the same region of one or both repeats, although the presence of other repeats enhances binding (Rebay et al, 1991; Davis-Smyth et al, 1996; Cunningham et al, 1997).

For receptor tyrosine kinases, ligand-induced dimerization initiates stimulation of kinase activity and signaling. Although ligand-induced receptor-co-receptor heterodimerization is necessary for canonical Wnt signaling, the role of LRP5/6 or FZD homodimerization has not been clearly defined. Forced dimerization of different recombinant LRP6 proteins can activate or inhibit Wnt signaling.

β -catenin dependent Wnt signaling is initiated by Wnt isoform binding to both the receptor FZD and the co-receptor LRP5/6, which then assemble multimeric complexes on the cytoplasmic membrane to recruit and inactivate the kinase GSK 3. Whether and how the different interactions between Wnt isoforms and receptors mechanically regulate this process remains to be determined.

Summary of The Invention

One aspect of the invention provides an isolated antibody that binds to LRP6, wherein the antibody inhibits signaling induced by a first Wnt isoform and potentiates signaling induced by a second Wnt isoform. In one embodiment, the first Wnt isoform is selected from the group consisting of: wnt3 and Wnt3 a. In one embodiment, the second Wnt isoform is selected from the group consisting of: wnt1, 2b, 4,6, 7a, 7b, 8a, 9b, 10a and 10 b. In another embodiment, the first Wnt isoform is selected from the group consisting of: wnts1, 2b, 6, 8a, 9b, and 10b and the second Wnt isoform is selected from the group consisting of: wnt3 and Wnt3 a.

One aspect of the invention provides an antibody that binds to the E3-E4 region of LRP6. Another aspect of the invention provides an antibody that binds to the E1-E2 region of LRP6. Yet another aspect provides an antibody that binds to two different regions of LRP6, such as the E1-E2 region and the E3-E4 region of LRP6. In one aspect, these antibodies inhibit Wnt signaling induced by the combination of Wnt1 and Wnt3 a. In one aspect, these antibodies inhibit autocrine Wnt signaling.

One aspect of the invention provides a method of treating an individual having cancer comprising administering to the individual an effective amount of an isolated antibody that binds to LRP6 and inhibits signaling induced by a Wnt isoform selected from the group consisting of Wnt3 and Wnt3a and an isolated antibody that binds to LRP6 and inhibits signaling induced by a Wnt isoform selected from the group consisting of Wnt1, 2b, 6, 8a, 9b, and 10 b.

Another aspect of the invention provides a method of treating an individual having cancer comprising administering to the individual an effective amount of an isolated antibody that binds to LRP6 and inhibits signaling induced by Wnt3 and Wnt3a and an isolated antibody that binds to LRP6 and inhibits signaling induced by Wnt1, 2b, 6, 8a, 9b, and 10 b.

Another aspect of the invention provides a method of treating an individual having cancer comprising administering to the individual an effective amount of an isolated antibody that binds to LRP6 and inhibits signaling induced by Wnt3 and Wnt3a and an isolated antibody that binds to LRP6 and inhibits signaling induced by Wnt1, 2b, 4,6, 7a, 7b, 8a, 9b, 10a, and 10 b.

One aspect of the invention provides a method of treating an individual having a bone disorder, such as osteoporosis, osteoarthritis, a bone fracture, and bone injury, comprising administering to the individual an effective amount of an anti-LRP 6 antibody described herein.

Another aspect of the invention provides a method of potentiating Wnt signaling induced by a Wnt isoform in an individual, comprising administering to the individual an effective amount of an anti-LRP 6 antibody described herein and the Wnt isoform to potentiate Wnt signaling induced by the Wnt isoform.

Specific anti-LRP 6 antibodies, including bispecific anti-LRP 6 antibodies, are also provided. In one embodiment, an isolated antibody that binds LRP6 comprises a VH comprising an amino acid sequence selected from the group consisting of seq id nos: SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO. 13 and SEQ ID NO. 15. In one embodiment, the antibody further comprises a VL comprising an amino acid sequence selected from the group consisting of seq id nos: SEQ ID NO. 10 and SEQ ID NO. 12. In one embodiment, an isolated antibody that binds to LRP6 comprises a VH comprising an amino acid sequence having at least 90% homology with the amino acid sequences of seq id No. 9, seq id No. 11, seq id No. 13, and seq id No. 15. In one embodiment, the isolated antibody that binds LRP6 further comprises a VL comprising an amino acid sequence having at least 90% homology to an amino acid sequence selected from the group consisting of seq id nos: SEQ ID NO. 10 and SEQ ID NO. 12.

In one embodiment, the antibody is an isolated bispecific antibody that binds two different regions of LRP6, wherein the antibody comprises a VH comprising an amino acid sequence selected from the group consisting of seq id nos: SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO. 13 and SEQ ID NO. 15. In one embodiment, the bispecific antibody comprises a first VH comprising the amino acid sequence of seq id No. 15 and a second VH comprising an amino acid sequence selected from the group consisting of seq id no: SEQ ID NO. 9, SEQ ID NO. 11 and SEQ ID NO. 13. In one embodiment, the bispecific antibody further comprises a VL comprising an amino acid sequence selected from the group consisting of seq id no: SEQ ID NO. 10 and SEQ ID NO. 12.

In one embodiment, a bispecific antibody that binds to two different regions of LRP6 comprises a VH comprising an amino acid sequence having at least 90% homology to an amino acid sequence selected from the group consisting of seq id nos: SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO. 13, or SEQ ID NO. 15. In one embodiment, a bispecific antibody that binds two different regions of LRP6 comprises a first VH comprising an amino acid sequence having at least 90% homology with the amino acid sequence of seq id No. 15 and a second VH comprising an amino acid sequence having at least 90% homology with an amino acid sequence selected from the group consisting of seq id nos: SEQ ID NO. 9, SEQ ID NO. 11 and SEQ ID NO. 13. In one embodiment, the bispecific antibody further comprises a VL comprising an amino acid sequence having at least 90% homology to an amino acid sequence selected from the group consisting of seq id nos: SEQ ID NO. 10 and SEQ ID NO. 12.

In one embodiment, an isolated bispecific antibody that binds two different regions of LRP6 comprises a first VH domain comprising at least one, at least two, or all three VHHVR sequences selected from (a) HVR-H1 comprising the amino acid sequence of seq id no: 17; (b) HVR-H2 comprising amino acid sequence SEQ ID NO. 18; and (c) HVR-H3 comprising the amino acid sequence of seq id no:19, the second VH domain comprising at least one, at least two, or all three VHHVR sequences selected from: (d) HVR-H1 comprising amino acid sequence SEQ ID NO: 22; (e) HVR-H2 comprising amino acid sequence SEQ ID NO: 23; and (f) HVR-H3 comprising amino acid sequence SEQ ID NO: 24. In one embodiment, an isolated bispecific antibody that binds two different regions of LRP6 comprises a first VH domain comprising all three VHHVR sequences from: (a) HVR-H1 comprising amino acid sequence SEQ ID NO 17; (b) HVR-H2 comprising amino acid sequence SEQ ID NO. 18; (c) HVR-H3 comprising the amino acid sequence seq id No. 19, the second VH domain comprising all three VHHVR sequences from: (d) HVR-H1 comprising amino acid sequence SEQ ID NO: 22; (e) HVR-H2 comprising amino acid sequence SEQ ID NO: 23; and (f) HVR-H3 comprising amino acid sequence SEQ ID NO: 24.

In one embodiment, an isolated bispecific antibody that binds two different regions of LRP6 comprises a first VH domain comprising at least one, at least two, or all three VHHVR sequences selected from: (a) HVR-H1 comprising amino acid sequence SEQ ID NO 17; (b) HVR-H2 comprising amino acid sequence SEQ ID NO. 18; (c) HVR-H3 comprising the amino acid sequence seq id No. 21, the second VH domain comprising at least one, at least two, or all three VHHVR sequences selected from: (d) HVR-H1 comprising amino acid sequence SEQ ID NO: 22; (e) HVR-H2 comprising amino acid sequence SEQ ID NO: 23; and (f) HVR-H3 comprising amino acid sequence SEQ ID NO: 24. In one embodiment, an isolated bispecific antibody that binds two different regions of LRP6 comprises a first VH domain comprising all three VHHVR sequences from: (a) HVR-H1 comprising amino acid sequence SEQ ID NO 17; (b) HVR-H2 comprising amino acid sequence SEQ ID NO. 18; (c) HVR-H3 comprising the amino acid sequence seq id no:21, the second VH domain comprising all three VHHVR sequences from: (d) HVR-H1 comprising amino acid sequence SEQ ID NO: 22; (e) HVR-H2 comprising amino acid sequence SEQ ID NO: 23; and (f) HVR-H3 comprising amino acid sequence SEQ ID NO: 24.

In one embodiment, an isolated bispecific antibody that binds two different regions of LRP6 comprises a first VH domain comprising at least one, at least two, or all three VHHVR sequences selected from: (a) HVR-H1 comprising amino acid sequence SEQ ID NO: 20; (b) HVR-H2 comprising amino acid sequence SEQ ID NO. 18; (c) HVR-H3 comprising the amino acid sequence of seq id No. 19, the second VH domain comprising at least one, at least two, or all three VHHVR sequences selected from: (d) HVR-H1 comprising amino acid sequence SEQ ID NO: 22; (e) HVR-H2 comprising amino acid sequence SEQ ID NO: 23; and (f) HVR-H3 comprising amino acid sequence SEQ ID NO: 24. In one embodiment, an isolated bispecific antibody that binds two different regions of LRP6 comprises a first VH domain comprising all three VHHVR sequences from: (a) HVR-H1 comprising amino acid sequence SEQ ID NO: 20; (b) HVR-H2 comprising amino acid sequence SEQ ID NO. 18; (c) HVR-H3 comprising the amino acid sequence seq id No. 19, the second VH domain comprising all three VHHVR sequences from: (d) HVR-H1 comprising amino acid sequence SEQ ID NO: 22; (e) HVR-H2 comprising amino acid sequence SEQ ID NO: 23; and (f) HVR-H3 comprising amino acid sequence SEQ ID NO: 24.

In one embodiment, the bispecific antibody in the above embodiments further comprises at least one, at least two, or all three VLHVR sequences selected from: (a) HVR-L1 comprising amino acid sequence SEQ ID NO. 25; (b) HVR-L2 comprising amino acid sequence SEQ ID NO. 26; (c) HVR-L3 comprising amino acid sequence SEQ ID NO: 27.

In one embodiment, the bispecific antibody in the above embodiments further comprises at least one, at least two, or all three VLHVR sequences selected from: (a) HVR-L1 comprising amino acid sequence SEQ ID NO. 25; (b) HVR-L2 comprising amino acid sequence SEQ ID NO. 26; (c) HVR-L3 comprising amino acid sequence SEQ ID NO: 28.

One embodiment provides an isolated bispecific antibody that binds to two different regions of LRP6, wherein the antibody comprises a first VH comprising the amino acid sequence of seq id No. 15 and a second VH selected from the group consisting of: VH comprising the amino acid sequences SEQ ID NO 9, SEQ ID NO 11 and SEQ ID NO 13. In one embodiment, such an antibody further comprises a VL comprising the amino acid sequence of seq id No. 10 or seq id No. 12. In one embodiment, the bispecific antibody comprises a first VH comprising the amino acid sequence of seq id No. 15 and a second VH comprising the amino acid sequence of seq id No. 9 and a VL comprising the amino acid sequence of seq id No. 10.

In one embodiment, the bispecific antibody inhibits signaling induced by a Wnt isoform selected from the group consisting of Wnt3 and Wnt3a and inhibits signaling induced by a Wnt isoform selected from the group consisting of Wnt1, 2b, 6, 8a, 9b, and 10 b. In one embodiment, the bispecific antibody further inhibits signaling induced by a Wnt isoform selected from the group consisting of Wnt4, 7a, 7b, and 10 a. In one embodiment, the bispecific antibody inhibits autocrine Wnt signaling.

One aspect of the invention provides a bispecific antibody that inhibits signaling induced by a Wnt isoform selected from the group consisting of Wnt3 and Wnt3a and inhibits signaling induced by a Wnt isoform selected from the group consisting of Wnt1, 2b, 6, 8a, 9b, and 10 b. In one embodiment, the bispecific antibody further inhibits signaling induced by a Wnt isoform selected from the group consisting of Wnt4, 7a, 7b, and 10 a.

One aspect of the invention provides an antibody that competes for binding to LRP6 with any of the anti-LRP 6 antibodies described herein, including bispecific antibodies.

Another aspect of the invention provides an antibody that binds to the same two epitopes as the bispecific antibody described herein. In one embodiment, one of the two epitopes comprises amino acid residues R28, E51, D52, V70, S71, E73, L95, S96, D98, E115, R141 and N185 of LRP6. In one embodiment, one of the two epitopes comprises amino acid residues R28, E51, D52, V70, S71, E73, L95, S96, D98, E115, R141, N185, R29, W188, K202, P225, H226, S243 and F266 of LRP6.

Another aspect of the invention provides an isolated nucleic acid encoding an anti-LRP 6 antibody described herein. Another aspect provides a host cell comprising such a nucleic acid.

One aspect of the invention provides an immunoconjugate comprising an anti-LRP 6 antibody described herein and a cytotoxic agent. Another aspect provides a pharmaceutical formulation comprising an anti-LRP 6 antibody described herein and a pharmaceutically acceptable carrier.

One aspect of the invention provides an anti-LRP 6 antibody described herein for use as a medicament. One aspect provides an anti-LRP 6 antibody described herein for use in treating a cancer or a bone disorder. One aspect provides an anti-LRP 6 antibody described herein for use in inhibiting signaling induced by a first Wnt isoform and potentiating signaling induced by a second Wnt isoform. One aspect provides for the use of an anti-LRP 6 antibody described herein in the manufacture of a medicament useful for treating, for example, cancer or a bone disorder.

One aspect of the invention provides a method of treating an individual having cancer, such as non-small cell lung cancer, breast cancer, pancreatic cancer, ovarian cancer, renal cancer, and prostate cancer, comprising administering to the individual an effective amount of an anti-LRP 6 antibody described herein.

Brief Description of Drawings

FIG. 1A. The figure shows the inhibition of Wnt luciferase reporter activity induced in HEK293 cells with 0.1mg/ml purified Wnt3a by antibodies against lrp6.E3-E4 protein.

FIG. 1B. HEK293 cells not stimulated or induced with Wnt3a and treated with the indicated LRP6 antibody or purified protein were analyzed by Western blot.

FIG. 1C. The figure shows that the YW210.09 antibody potentiates Wnt reporter gene activity in a manner proportional to Wnt3a concentration in HEK293 cells.

Fig. 2A. The figure shows concentration-dependent inhibition and potentiation of autocrine Wnt signaling in PA-1 teratocarcinoma cells transfected with luciferase reporter and treated with LRP6 antibody (alone or in combination) or Fzd8CRD-Fc protein.

Fig. 2B. The figure shows the results of qPCR expression analysis of Wnt-induced genes SAX1 and GAD1 and Wnt-repressed gene LEFTY2 in PA-1 cells treated with or without 0.3mg/ml Wnt3a protein and with 10mg/ml yw211.31antibody, anti-gD monoclonal antibody (negative control) or Fzd8CRD-Fc protein (positive control). Data were normalized to samples from cells without added (NA) Wnt3 a.

Fig. 3. This summary table shows the effect of LRP6 antibody and Fzd8CRD-Fc protein on autocrine signaling in cell lines.

Fig. 4A. The figure depicts the results of qPCR expression analysis of AXIN2mRNA in four cell lines treated with 25 μ g/ml yw211.31.57 antibody or Fzd8CRD-Fc protein, with and without (NA)0.2 μ g/ml wnt3 a.

Fig. 4B. The figure depicts Wnt-induced gene expression in NCI-H23 cells potentiated by yw211.31.57 and antagonized by YW210.09 antibody (30 μ g/ml). The CD4-Fc protein (30. mu.g/ml) served as a negative control.

Fig. 4C. The figure depicts Wnt-induced gene expression in M14 cells potentiated by yw211.31.57 and antagonized by YW210.09 antibody (30 μ g/ml).

Fig. 4D. The figure shows concentration-dependent inhibition of Wnt3 a-stimulated signaling by yw211.31.57 antibody in Hs578T cells stably integrated with a Wnt luciferase reporter.

Fig. 4E. The figure shows concentration-dependent potentiation of autocrine Wnt signaling by yw211.31.57 antibody in Hs578T cells stably integrated with a Wnt luciferase reporter.

Fig. 4F. The figure shows that EKVX cells transfected with a Wnt luciferase reporter demonstrate potentiation of autocrine Wnt signaling (NA) and antagonism of Wnt3 a-induced signaling by yw211.31.57 antibody.

Fig. 4G. This figure shows that antibody-mediated potentiation of autocrine Wnt signaling is inhibited by 5 μ g/ml fzd8CRD-Fc protein.

Fig. 5. Summary table of the effect of 10mg/mlLRP6 antibody or Fzd8CRD-Fc protein on the signaling induced by expression constructs transfected with Wnt isoform in HEK293 or Hs578T cell lines stably integrated with Wnt luciferase reporter. Expression of Wnt luciferase reporter was normalized to cell number and additionally to levels in cells transfected with the same expression construct but not treated with protein.

Fig. 6. Summary of the effect of 10mg/ml lrp6 antibody or Fzd8CRD-Fc protein on signaling in HEK293 cell lines stably integrated with Wnt luciferase reporter. Signaling is induced by expression constructs transfected with chimeric proteins consisting of Wnt isoform fusion FZD isoforms or LRP6. Expression of Wnt luciferase reporter was normalized to cell number and additionally to levels in cells transfected with the same expression construct but not treated with protein.

Fig. 7. Summary table of the effect of 10mg/ml lrp6 antibody or antibody combination on signaling induced by expression constructs transfected with Wnt isoforms in cell lines stably integrated with Wnt luciferase reporter. Expression of Wnt luciferase reporter was normalized to cell number and additionally to levels in cells transfected with the same expression construct but not treated with protein.

Fig. 8A. The figure shows that the combination of yw211.31.57 and YW210.09 antibodies inhibited signaling in HEK293 cells stably integrated with Wnt luciferase reporter transfected to express Wnt3a, Wnt1, or both Wnt3a and Wnt 1. anti-gD antibody and Fzd8CRD-Fc protein are shown as negative and positive controls, respectively, for inhibition of Wnt signaling.

Fig. 8B. The figure shows that the combination of yw211.31.57 and YW210.09 antibodies potentiates autocrine Wnt signaling in Hs578T cells.

Fig. 8C. The figure shows that the combination of yw211.31.57 and YW210.09 antibodies potentiates autocrine Wnt signaling in EKVX cells.

FIGS. 9A and B. Biolayer interferometry with biotinylated LRP6E1-E4 protein immobilized on a streptavidin biosensor indicated that yw211.31.57 antibody inhibited Wnt3a and Wnt9b from binding to LRP6, while YW210.09 antibody only inhibited Wnt9b binding.

Fig. 9C. Biolayer interferometry with smaller, non-overlapping LRP6 fragments showed that Wnt3a bound to the E3-E4 region, and that this interaction was blocked by the intact or one-armed YW211.31antibody (one-arm yw211.31antibody).

Fig. 9D. Biolayer interferometry with smaller, non-overlapping LRP6 fragments showed that the YW210.09 antibody bound to the LRP6E1-E2 protein fragment and competed with Wnt9b binding.

Fig. 9E. This biolayer interferometry shows that yw211.31.57 and YW210.09 antibodies, when added sequentially in either order, bind together to immobilized lrp6.E1-E4 protein, confirming separate epitopes.

Fig. 10A. This figure shows that MMTV-Wnt1 allograft tumor growth regressed when mice are treated with YW210.09 antibody, similar to that observed with Fzd8CRD-Fc protein.

Fig. 10B. The figure shows that the Ntera-2 xenograft tumors in mice treated with the full or one arm YW211.31antibody exhibited reduced SP5mRNA expression, but not the YW210.09 antibody, according to qPCR analysis.

Fig. 10C. The figure shows that mouse calvaria explants in culture treated with YW210.09 antibody significantly increased Bone Mineral Density (BMD) of calcified parietal bone, similar to RANK-Fc protein treatment, but yw211.31.62 antibody did not.

Fig. 11A. The figure shows that bispecific anti-LRP 6 antibodies generated in e.coli or HEK293 cells similarly inhibited the Wnt luciferase reporter activity induced in HEK293 cells with 0.1 μ g/ml purified Wnt3a in a concentration-dependent manner. IC50 values were 0.032 and 0.014. mu.g/ml, respectively.

FIG. 11B. The figure shows the effect of the indicated control buffer (PBS), antibody combination, or Fzd8CRD-Fc protein (10. mu.g/ml each) on autocrine Wint signaling in PA-1 and M14 cells stably integrated with Wnt luciferase reporter and reporter transfected CAL-51 cells treated with the indicated control buffer (PBS), antibody combination, or Fzd8CRD-Fc protein (10. mu.g/ml each), with (C) or without (B) stimulation at 0.1. mu.g/ml Wnt3 a.

FIG. 11C. The figure shows the effect of treatment with control buffer (PBS), antibody combination, or Fzd8CRD-Fc protein (10. mu.g/ml each) on PA-1 and M14 cells stably integrated with Wnt luciferase reporter and CAL-51 cells transfected with the reporter, stimulated with 0.1. mu.g/ml Wnt3 a.

Fig. 12. Summary of the effect of antibodies or Fzd8CRD protein (10 μ g/ml) on signaling induced by expression constructs transfected with Wnt isoform in HEK293 or Hs578T cell lines stably integrated with Wnt luciferase reporter.

Fig. 13A. Western analysis of HEK293 cells transfected with or without Wnt3a and treated with the indicated antibody or Fzd8CRD-Fc protein (5. mu.g/ml) for 18 h. The upper and lower gels show β -actin or GAPDH protein levels, respectively, as sample loading controls.

FIG. 13B. Figure showing that M14 xenograft tumors in SCID-bg mice treated with 30mg/kgLRP6 bispecific antibody or Fzd8CRD protein but not with control anti-gD antibody for 16 hours show reduced AXIN2 and APCDD1mRNA expression according to qPCR analysis.

Fig. 14. Detailed view of CDRH3 interaction with residues of the LRP6 groove, showing an important network of interactions by the NAVK motif.

Fig. 15. Details of the interactions performed by CDRH1,2, L1,2 and 3.

Fig. 16. The heavy chain variable region (VH) of the exemplary anti-LRP 6 antibody, shows kabat cdrs.

Fig. 17. The light chain variable region (VL) of an exemplary anti-LRP 6 antibody, showing kabat cdrs.

Detailed Description

I. Definition of

For purposes herein, an "acceptor human framework" refers to a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework as defined below. An acceptor human framework "derived" from a human immunoglobulin framework or human consensus framework may comprise its identical amino acid sequence, or it may contain amino acid sequence variations. In some embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is identical in sequence to a VL human immunoglobulin framework sequence or a human consensus framework sequence.

"affinity" refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). As used herein, unless otherwise indicated, "binding affinity" refers to an intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be expressed in terms of the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including the methods described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.

An "affinity matured" antibody refers to an antibody that has one or more alterations in one or more hypervariable regions (HVRs) which result in an improved affinity of the antibody for an antigen compared to a parent antibody that does not possess such alterations.

The terms "anti-LRP 6 antibody" and "antibody that binds to LRP 6" refer to an antibody that is capable of binding LRP6 with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent for targeting LRP6. In one embodiment, the extent to which an anti-LRP 6 antibody binds an unrelated, non-LRP 6 protein is less than about 10% of the binding of the antibody to LRP6 as measured, for example, by a Radioimmunoassay (RIA). In certain embodiments, an antibody that binds LRP6 has ≦ 1 μ M ≦ 100nM, ≦ 10nM, ≦ 1nM, ≦ 0.1nM, ≦ 0.01nM, or ≦ 0.001nM (e.g., 10 nM)^-8M or less, e.g. 10^-8M to 10^-13M, e.g. 10^-9M to 10^-13M) dissociation constant (Kd). In certain embodiments, an anti-LRP 6 antibody binds to the LRP6 epitope that is conserved among LRP6 from different species.

The term "antibody" herein is used in the broadest sense and encompasses a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

An "antibody fragment" refers to a molecule distinct from an intact antibody that comprises a portion of the intact antibody that binds to an antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab '-SH, F (ab')₂(ii) a A diabody; a linear antibody; single chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

An "antibody that binds to the same epitope" as a reference antibody refers to an antibody that blocks binding of the reference antibody to its antigen by 50% or more in a competition assay, and conversely, the reference antibody blocks binding of the antibody to its antigen by 50% or more in a competition assay. Exemplary competition assays are provided herein.

The terms "cancer" and "cancerous" refer to or describe a physiological condition in mammals that is typically characterized by unregulated cell growth/proliferation. Examples of cancer include, but are not limited to, carcinoma, lymphoma (e.g., Hodgkin's lymphoma and non-Hodgkin's lymphoma), blastoma, sarcoma, and leukemia. More specific examples of such cancers include squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer (livercacer), bladder cancer, hepatoma (hepatoma), breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, prostate cancer, vulval cancer, thyroid cancer, liver cancer (hepatorcinoma), leukemia, and other lymphoproliferative disorders, and various types of head and neck cancer.

"chemotherapeutic agent" refers to a chemical compound useful for the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents (alkylating agents), such as thiotepa and cyclophosphamide (cyclophosphamide)Alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines (aziridines), such as benzotepa (benzodepa), carboquone (carboquone), metoclopramide (meteredepa), and uretepa (uredepa); ethyleneimines and methylmelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimetalmamine; annona squamosa lactones (acetogenin) (especially bractesin (b)ullatacin) and bullatacin (bullatacinone)); -9-tetrahydrocannabinol (dronabinol),) β -lapachone (lapachone), lapachol (lapachol), colchicines (colchicines), betulinic acid (betulinic acid), camptothecin (camptothecin) (including the synthetic analogue topotecan (topotecan)CPT-11 (irinotecan),) Acetyl camptothecin, scopoletin (scopoletin), and 9-aminocamptothecin); bryostatin; callystatin; CC-1065 (including its adozelesin (adozelesin), carvelesin (carzelesin), and bizelesin (bizelesin) synthetic analogs); podophyllotoxin (podophylotoxin); podophyllinic acid (podophyllinic acid); teniposide (teniposide); cryptophycins (especially cryptophycins 1 and 8); dolastatin (dolastatin); duocarmycins (including synthetic analogs, KW-2189 and CB1-TM 1); eiscosahol (eleutherobin); pancratistatin; sarcodictyin; spongistatin (spongistatin); nitrogen mustards (nitrogenmustards), such as chlorambucil (chlorambucil), chlorambucil (chlorenaphazine), cholorophosphamide (cholorophosphamide), estramustine (estramustine), ifosfamide (ifosfamide), mechlorethamine (mechlorethamine), mechlorethamine hydrochloride (mechlorethamine oxydichloride), melphalan (melphalan), neomustard (novembichin), benzene mustard cholesterol (pherenesterestrine), prednimustine (prednimustine), triamcinolone (trofosfamide), uracil mustard (uracilmustard); nitrosoureas such as carmustine (carmustine), chlorouretocin (chlorozotocin), fotemustine (fotemustine), lomustine (lomustine), nimustine (nimustine) and ramustine (ranimustine); antibiotics, such as enediynes (enediynes) (e.g. calicheamicin), especially calicheamicinWhich are calicheamicin gamma 1I and calicheamicin omega I1 (see, e.g., Nicolaouetal, Angew, ChemIntl, Ed. Engl.,33:183-186 (1994)), CDP323, an oral α -4 integrin inhibitor, an anthracycline (dynemicin) including dynemicin A, an epothilone (esperamicin), and neocarzinostatin (neocarzinostatin) and related chromoprotein enediyne antibiotic chromophores), aclacinomycin (aclacinomycin), actinomycin (actinomycin), anthranomycin (anthrandamycin), azaserine (azaserine), bleomycin (bleubomycin), actinomycin C (cactinomycin), carbamicin, carmycin (carminomycin), carminomycin (carminomycin), doxorubicin (doxorubicin), doxorubicin (5), doxorubicin (doxorubicin-6), doxorubicin (doxorubicin, doxorubicin (doxorubicin), doxorubicin (doxorubicin, doxorubicin (doxorubicin), doxorubicin (doxorubicin ), doxorubicin (doxorubicin), doxorubicin (doxorubicin, doxorubicin (doxorubicin), doxorubicin (doxorubicin), doxorubicin (doxorubicin, doxorubicinMorpholino doxorubicin, cyano morpholino doxorubicin, 2-pyrrol doxorubicin and doxorubicin hydrochloride liposome injectionLiposomal doxorubicin TLCD-99PEGylated liposomal doxorubicinAnd doxorubicin), epirubicin (epirubicin), esorubicin (esorubicin), idarubicin (idarubicin), marijumycin (marcellomycin), mitomycins (mitomycins) such as mitomycin C, mycophenolic acid (mycophenolic acid), norramycin (nogalamycin), olivomycin (olivomycin), pelomycin (peplomycin), pofiomycin (potfiromycin), puromycin (puromycin), triiron doxorubicin (quelamycin), rodobicin (rodorubicin), streptonigrin (streptonigrin), streptozocin (streptozotocin), tubercidin (tubicidin), ubenimex (enimenimimex), purified staudin (zinostatin), zorubicin (zorubicin); resist againstMetabolites, such as methotrexate, gemcitabine (gemcitabine)Tegafur (tegafur)Capecitabine (capecitabine)Epothilone (epothilone) and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteroyltriglutamic acid (pteropterin), trimetrexate (trimetrexate); purine analogs such as fludarabine (fludarabine), 6-mercaptopurine (mercaptoprine), thiamiprine (thiamiprine), thioguanine (thioguanine); pyrimidine analogs such as ancitabine (ancitabine), azacitidine (azacitidine), 6-azauridine, carmofur (carmofur), cytarabine (cytarabine), dideoxyuridine (dideoxyuridine), deoxyfluorouridine (doxifluridine), enocitabine (enocitabine), floxuridine (floxuridine); androgens such as carotinoid (calusterone), dromostanolonenepropionate (dromostanolone), epitioandrostane (mepitiostane), testolactone (testolactone); anti-adrenal agents such as aminoglutethimide (aminoglutethimide), mitotane (mitotane), trilostane (trilostane); folic acid supplements such as folinic acid (folinic acid); acetoglucurolactone (acegultone); an aldophosphamide glycoside (aldophosphamideglycoside); aminolevulinic acid (aminolevulinic acid); eniluracil (eniluracil); amsacrine (amsacrine); bestrabuucil; bisantrene; edatrexate (edatraxate); desphosphamide (defosfamide); dimecorsine (demecolcine); diazaquinone (diaziqutone); elfornitine; ammonium etiolate (ellitiniumacetate); an epothilone; etoglut (etoglucid); gallium nitrate; hydroxyurea (hydroxyurea); lentinan (lentinan); lonidamine (lonidamine); maytansinoids (maytansinoids), such as maytansine (maytansine) and ansamitocins (ansamitocins); mitoguazone (mitoguzone); mitoxantrone (mitoxantr)one); mopidamol (mopidamol); diamine nitracridine (nitrarine); pentostatin (pentostatin); methionine mustard (phenamett); pirarubicin (pirarubicin); losoxantrone (losoxantrone); 2-ethyl hydrazide (ethylhydrazide); procarbazine (procarbazine);polysaccharide complexes (jhsnaral products, Eugene, OR); razoxane (rizoxane); rhizomycin (rhizoxin); sisofilan (sizofiran); helical germanium (spirogermanium); tenuazonic acid (tenuazonic acid); triimine quinone (triaziquone); 2, 2', 2 "-trichlorotriethylamine; trichothecenes (trichothecenes), especially the T-2 toxin, verrucin A, rorodin A and snake-fish (anguidin); urethane (urethan); vindesine (vindesine)Dacarbazine (dacarbazine); mannitol mustard (mannomustine); dibromomannitol (mitobronitol); dibromodulcitol (mitolactol); pipobromane (pipobroman); a polycytidysine; cytarabine (arabine) ("Ara-C"); thiotepa (thiotepa); taxols (taxoids), such as paclitaxel (paclitaxel)Albumin-engineered nanoparticle dosage forms paclitaxel (ABRAXANETM) and docetaxel (doxetaxel)Chlorambucil (chlorambucil); 6-thioguanine (thioguanine); mercaptopurine (mercaptoprine); methotrexate (methotrexate); platinum analogs, such as cisplatin (cissplatin), oxaliplatin (oxaliplatin) (e.g., cisplatin)) And carboplatin (carboplatin); changchun medicineClass (vincas), which prevents tubulin polymerization to form microtubules, includes vinblastine (vinblastine)Vincristine (vincristine)Vindesine (vindesine)And vinorelbine (vinorelbine)Etoposide (VP-16); ifosfamide (ifosfamide); mitoxantrone (mitoxantrone); leucovorin (leucovorin); oncostatin (novantrone); edatrexate (edatrexate); daunomycin (daunomycin); aminopterin (aminopterin); ibandronate (ibandronate); topoisomerase inhibitor RFS 2000; difluoromethyl ornithine (DMFO); retinoids, such as tretinoids, including bexarotene (bexarotene)Diphosphonates (bisphosphates), such as clodronate (e.g. clodronate)Or) Etidronate sodium (etidronate)NE-58095, zoledronic acid/zoledronate (zoledronic acid/zoledronate)Alendronate (alendronate)Pamidronate (pamidronate)Tiludronate (tirudronate)Or risedronate (risedronate)And troxacitabine (1, 3-dioxolane nucleoside cytosine analogues), antisense oligonucleotides, in particular antisense oligonucleotides which inhibit the expression of genes in signaling pathways involving abnormal cell proliferation, such as, for example, PKC- α, Raf, H-Ras and epidermal growth factor receptor (EGF-R), vaccines, such asVaccines and gene therapy vaccines, e.g.A vaccine,A vaccine anda vaccine; topoisomerase 1 inhibitors (e.g. topoisomerase 1 inhibitors)) (ii) a rmRH (e.g. rmRH)）；BAY439006(sorafenib;Bayer)；SU-11248(sunitinib,Pfizer); perifosine (perifosine), COX-2 inhibitors (such as celecoxib (celecoxib) or etoricoxib (etoricoxib)), proteosome inhibitors (such as PS 341); bortezomibCCI-779; tipifarnib (R11577); orafenaib, ABT 510; bcl-2 inhibitors, such as oblimersensodiumpixantrone; EGFR inhibitors (see definition below); tyrosine kinase inhibitors (see definition below); serine-threonine kinase inhibitors, such as rapamycin (rapamycin) (sirolimus,) (ii) a Farnesyl transferase inhibitors such as lonafarnib (SCH6636, SARASARTM); and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing; and combinations of two or more of the above, such as CHOP (abbreviation for cyclophosphamide, doxorubicin, vincristine and prednisolone combination therapy) and FOLFOX (oxaliplatin)^TM) Abbreviation for treatment regimen combining 5-FU and folinic acid).

As defined herein, a chemotherapeutic agent includes the class of "anti-hormonal agents" or "endocrine therapeutic agents" that act to modulate, reduce, block or inhibit the effects of hormones that promote cancer growth. They may themselves be hormones, including but not limited to: antiestrogens with mixed agonist/antagonist properties including tamoxifen (tamoxifen) (NOLVADEX), 4-hydroxytamoxifen, toremifene (toremifene)Idoxifene (idoxifene), droloxifene (droloxifene), raloxifene (raloxifene)Trioxifene (trioxifene), naloxifene (keoxifene), and Selective Estrogen Receptor Modulators (SERMs), such as SERM 3; pure antiestrogens without agonist properties, such as fulvestrantAnd EM800 (such agents may block Estrogen Receptor (ER) dimerization, inhibit DNA binding, increase ER turnover, and/or suppress ER levels); aromatase inhibitors, including steroidal aromatase inhibitors, such as formestane (formestane) and exemestane (exemestane)And non-steroidal aromatase inhibitors, such as anastrozole (anastrozole)Letrozole (letrozole)And aminoglutethimide (aminoglutethimide), and other aromatase inhibitors, including vorozole (vorozole)Megestrol acetate (megestrolacete)Fadrozole (fadrozole) and 4(5) -imidazole; luteinizing hormone releasing hormone agonists, including leuprolide (leuprolide) ((leuprolide))And) Goserelin (goserelin), buserelin (buserelin) and triptorelin (triptorelin); sex steroids (sexsteroids) including pregnanins (progestines) such as megestrol acetate and medroxyprogesterone acetate (medroxyprogesterone acetate), estrogens such as diethylstilbestrol (diethylstilbestrol) and pramelin (premarin), and androgens/retinoids such as fluoxymesterone (fluoroxymesterone), all trans retinoic acid (transretinic acid) and fenretinide (fenretinide); onapristone (onapri)stone); anti-pregnenones; estrogen receptor down-regulators (ERD); anti-androgens such as flutamide (flutamide), nilutamide (nilutamide), and bicalutamide (bicalutamide); and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing; and combinations of two or more of the foregoing.

The term "chimeric" antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species.

The "class" of an antibody refers to the type of constant domain or constant region that its heavy chain possesses. There are 5 major classes of antibodies: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG₁、IgG₂、IgG₃、IgG₄、IgA₁And IgA₂The constant domains of the heavy chains corresponding to different classes of immunoglobulins are designated α, γ, and μ, respectively.

As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents cellular function and/or causes cell death or destruction. Cytotoxic agents include, but are not limited to: radioisotope (e.g. At)²¹¹、I¹³¹、I¹²⁵、Y⁹⁰、Re¹⁸⁶、Re¹⁸⁸、Sm¹⁵³、Bi²¹²、P³²、Pb²¹²And radioactive isotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate), doxorubicin (adriamycin), vinca alkaloids (vinca alkaloids) (vincristine), vinblastine (vinblastine), etoposide (etoposide)), doxorubicin (doxorubicin), melphalan (melphalan), mitomycin (mitomycin) C, chlorambucil (chlorembucil), daunorubicin (daunorubicin), or other intercalating agents); a growth inhibitor; enzymes and fragments thereof, such as nucleolytic enzymes; (ii) an antibiotic; toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof; and various antitumor or anticancer agents disclosed hereinafter.

"Effector function" refers to those biological activities attributable to the Fc region of an antibody and which vary with the antibody isotype. Examples of antibody effector functions include: c1q binding and Complement Dependent Cytotoxicity (CDC); fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down-regulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

An "effective amount" of a pharmaceutical agent (e.g., a pharmaceutical formulation) refers to an amount effective to achieve the desired therapeutic or prophylactic result over the necessary dosage and period of time.

The term "Fc region" is used herein to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxy-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as described in Kabat et al, sequence of proteins of immunological interest, 5 th edition public health service, national institutes of health, Bethesda, MD, 1991.

"framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. In general, the FRs of a variable domain consist of 4 FR domains: FR1, FR2, FR3 and FR 4. Thus, HVR and FR sequences typically occur in the following order in VH (or VL): FR1-H1(L1) -FR2-H2(L2) -FR3-H3(L3) -FR 4.

The terms "full length antibody", "intact antibody" and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to a native antibody structure or having a heavy chain comprising an Fc region as defined herein.

The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to a cell into which an exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells," which include primary transformed cells and progeny derived therefrom, regardless of the number of passages. Progeny may not be identical in nucleic acid content to the parent cell, but may contain mutations. Included herein are mutant progeny that have the same function or biological activity as screened or selected in the originally transformed cell.

"human antibody" refers to an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human or human cell or derived from a non-human source using a repertoire of human antibodies or other human antibody coding sequences. This definition of human antibodies specifically excludes humanized antibodies comprising non-human antigen binding residues.

"human consensus framework" refers to a framework representing the amino acid residues most commonly found in the selection of human immunoglobulin VL or VH framework sequences. Typically, the selection of human immunoglobulin VL or VH sequences is from a subset of variable domain sequences. Typically, the sequence subgroups are subgroups as in Kabat et al, sequence of proteins of immunologicalcalemtest, fifth edition, NIHPublication91-3242, BethesdamD (1991), volumes 1-3. In one embodiment, for VL, the subgroup is as in Kabat et al, supra for subgroup kappa I. In one embodiment, for the VH, the subgroup is as in Kabat et al, supra, subgroup III.

A "humanized" antibody is a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise at least one, and typically two, substantially the entire variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. Optionally, the humanized antibody may comprise at least a portion of an antibody constant region derived from a human antibody. An antibody, e.g., a "humanized form" of a non-human antibody, refers to an antibody that has undergone humanization.

As used herein, the term "hypervariable region" or "HVR" refers to each region of an antibody variable domain which is hypervariable in sequence and/or which forms structurally defined loops ("hypervariable loops"). Typically, a native 4 chain antibody comprises 6 HVRs; three in VH (H1, H2, H3) and three in VL (L1, L2, L3). HVRs typically comprise amino acid residues from hypervariable loops and/or from "complementarity determining regions" (CDRs), the latter being of highest sequence variability and/or involved in antigen recognition. Exemplary hypervariable loops are present at amino acid residues 26-32(L1), 50-52(L2), 91-96(L3), 26-32(H1), 53-55(H2) and 96-101 (H3). (Chothia and Lesk, J.mol.biol.196:901-917 (1987)). Exemplary CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and CDR-H3) are present at amino acid residues 24-34 of L1, 50-56 of L2, 89-97 of L3, 50-65 of 31-35B, H2 of H1 and 95-102 of H3 (Kabat et al, sequencesof proteins of immunologicalcalest, 5 th edition blic health service, national institutes of health, Bethesda, MD (1991)). In addition to CDR1 in VH, the CDRs generally comprise amino acid residues that form hypervariable loops. CDRs also contain "specificity determining residues", or "SDRs", which are residues that contact the antigen. SDR is contained within a CDR region called a shortened-CDR, or a-CDR. Exemplary a-CDRs (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2, and a-CDR-H3) are present at amino acid residues 31-34 of L1, 50-55 of L2, 89-96 of L3, 50-58 of 31-35B, H2 of H1, and 95-102 of H3 (see Almagro and nsson, Front. biosci.13: 1619-. Unless otherwise indicated, HVR residues and other residues (e.g., FR residues) in the variable domains are numbered herein according to Kabat et al, supra.

An "immunoconjugate" refers to an antibody conjugated to one or more heterologous molecules, including but not limited to cytotoxic agents.

An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

An "isolated" antibody refers to an antibody that has been separated from components of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis), or chromatography (e.g., ion exchange or reverse phase HPLC). For a review of methods for assessing antibody purity, see, e.g., Flatman et al, J.Chromatogr.B848:79-87 (2007).

An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from components of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

An "isolated nucleic acid encoding an anti-LRP 6 antibody" refers to one or more nucleic acid molecules encoding the heavy and light chains of an antibody (or fragments thereof), including such nucleic acid molecules in a single vector or in different vectors, and such nucleic acid molecules present at one or more locations in a host cell.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except, for example, for possible variant antibodies containing naturally occurring mutations or occurring during the production of a monoclonal antibody preparation, such variants are typically present in very small amounts. Unlike polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies to be used in accordance with the present invention can be generated by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods that utilize transgenic animals containing all or part of a human immunoglobulin locus, such methods and other exemplary methods for generating monoclonal antibodies are described herein.

"naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or a radioactive label. The naked antibody may be present in a pharmaceutical formulation.

"Natural antibody" refers to a naturally occurring immunoglobulin molecule having a different structure. For example, a native IgG antibody is an heterotetrameric glycan protein of about 150,000 daltons, consisting of two identical light chains and two identical heavy chains that are disulfide-bonded. From N to C-terminus, each heavy chain has one variable region (VH), also called variable or heavy chain variable domain, followed by three constant domains (CH 1, CH2, and CH 3). Similarly, from N-to C-terminus, each light chain has a variable region (VL), also known as the variable light domain or light chain variable domain, followed by a Constant Light (CL) domain. Antibody light chains can be classified into one of two types, called kappa (κ) and lambda (λ), based on their constant domain amino acid sequences.

The term "package insert" is used to refer to instructions for use typically contained in commercial packaging for a therapeutic product that contains information regarding the indications, usage, dosage, administration, combination therapy, contraindications, and/or warnings relating to the use of such therapeutic products.

"percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with amino acid residues in the reference polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and without considering any conservative substitutions as part of the sequence identity. Comparison for the purpose of determining percent amino acid sequence identity can be performed in a variety of ways within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or megalign (dnastar) software. One skilled in the art can determine suitable parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment over the full length of the sequences being compared. However, for purposes of the present invention,% amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was written by Genentech, inc, and the source code has been submitted to the us copyright office (USCopyrightOffice, washington d.c.,20559) along with the user document, where it is registered with us copyright registration number TXU 510087. The ALIGN-2 program is publicly available from Genentech, Inc., south san Francisco, Calif., or may be compiled from source code. The ALIGN2 program should be compiled for use on UNIX operating systems, including digital UNIXV4.0D. All sequence comparison parameters were set by the ALIGN-2 program and were not changed.

In the case of employing ALIGN-2 to compare amino acid sequences, the% amino acid sequence identity of a given amino acid sequence a relative to (to), with (with), or against (against) a given amino acid sequence B (or may be stated as having or comprising a given amino acid sequence a with respect to, with, or against a given amino acid sequence B) is calculated as follows:

fractional X/Y times 100

Wherein X is the number of amino acid residues scored as identical matches in the A and B alignments of the sequence alignment program by the program ALIGN-2, and wherein Y is the total number of amino acid residues in B. It will be appreciated that if the length of amino acid sequence a is not equal to the length of amino acid sequence B, then the% amino acid sequence identity of a relative to B will not equal the% amino acid sequence identity of B relative to a. Unless otherwise specifically indicated, all% amino acid sequence identity values used herein are obtained using the ALIGN-2 computer program as described in the preceding paragraph.

The term "pharmaceutical formulation" refers to a preparation that is in a form that allows the biological activity of the active ingredient contained therein to be effective, and that is free of other components having unacceptable toxicity to a subject that will receive administration of the formulation.

"pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation that is different from the active ingredient and is not toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

As used herein, the term "LRP 6" refers to any native source from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses "full length," unprocessed LRP6, as well as any form of LRP6 that results from processing in a cell. The term also encompasses naturally occurring variants of LRP6, such as splice variants or allelic variants. An exemplary amino acid sequence of human LRP6 is shown in SEQ ID NO. 29. See also NCBI accession number AAI 43726; strausberg, R.L., et al, Proc.Natl.Acad.Sci.U.S.A.99: 16899-; he, X, et, Development,131: 1663-; chen, M., et., J.biol.chem.,284:35040-35048 (2009).

As used herein, "treatment" refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and may be performed either prophylactically or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment/lessening of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and regression or improved prognosis. In some embodiments, antibodies of the invention are used to delay the development of or slow the progression of disease.

The term "variable region" or "variable domain" refers to a domain in an antibody heavy or light chain that is involved in binding of the antibody to an antigen. The heavy and light chain variable domains of natural antibodies (VH and VL, respectively) generally have similar structures, with each domain comprising 4 conserved Framework Regions (FR) and 3 hypervariable regions (HVRs). (see, e.g., Kindt et al KubyImmunology, 6 th edition, W.H.FreemanndCo., page 91 (2007)). A single VH or VL domain may be sufficient to confer antigen binding specificity. In addition, antibodies that bind a particular antigen can be isolated by screening libraries of complementary VL or VH domains using VH or VL domains, respectively, from antibodies that bind the antigen. See, for example, Portolano et al, J.Immunol.150:880-887(1993); Clarkson et al, Nature352:624-628 (1991).

As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors which are self-replicating nucleic acid structures and vectors which integrate into the genome of a host cell into which they are introduced. Certain vectors are capable of directing the expression of a nucleic acid to which they are operably linked. Such vectors are referred to herein as "expression vectors".

Compositions and methods

The invention provides anti-LRP 6 antibodies that have the unexpected ability to inhibit signaling by some Wnt isoforms and potentiate signaling by other isoforms. As described in the examples, the two characterized anti-LRP 6 antibodies further showed opposite activity to most wnts, with one antibody antagonizing and the other potentiating. These two antibodies bind to different regions of LRP6 (as different Wnt isoforms) and inhibition of signaling results from blocking Wnt binding.

Based on their functional interaction with the anti-LRP 6 antibody of the invention, the 14 Wnt isoforms tested can be grouped into three classes: wnt3 and Wnt3a were inhibited by anti-LRP 6 antibody YW211.31 and potentiated by anti-LRP 6 antibody YW 210.09; wnt1, 2B, 6, 8A, 9B and 10B were potentiated by anti-LRP 6 antibody YW211.31 and antagonized by anti-LRP 6 antibody YW 210.09; while Wnt4, 7A, 7B, and 10A were potentiated by anti-LRP 6 antibody YW211.31 and were not inhibited by anti-LRP 6 antibody YW210.09 (fig. 3C). These classifications clearly do not match the proposed Wnt gene system, although the Wnt3/3a subfamily is the most evolutionarily divergent (choetal, 2010). Combinations of anti-LRP 6 antibodies that inhibit different classes of Wnt isoforms may be used to provide effective therapeutics for treating diseases associated with Wnt signaling.

Antibody-mediated dimerization of LRP6 only potentiates signaling when Wnt isoforms are also able to bind the complex, presumably recruiting FZD. Endogenous autocrine Wnt signaling in different tumor cell lines can be antagonized or potentiated by the LRP6 antibody. This complexity of co-receptor-ligand interactions may allow differential modulation of Wnt isoform signaling, and antibodies may be used to differentially manipulate Wnt signaling in specific tissues or disease states.

In some embodiments, anti-LRP 6 antibodies are capable of inhibiting autocrine or endogenous Wnt signaling in some cell types and potentiating autocrine signaling in other cell types. In some embodiments, an anti-LRP 6 antibody mediates LRP6 dimerization and enhances or potentiates signaling in the presence of Wnt isoforms that simultaneously bind LRP6. In some embodiments, an anti-LRP 6 antibody potentiates Wnt signaling by inhibiting the binding of a Wnt antagonist, such as a DKK isoform and SOST.

anti-LRP 6 antibodies are useful for selectively modulating processes activated or inhibited by Wnt isoform-induced signaling. Such processes include, for example, cell proliferation, cell fate specification (specification) and stem cell self-renewal in different cancer types, and developmental processes. anti-LRP 6 may be used, for example, to treat Wnt-mediated disorders such as cancer and disorders of the bone or skeletal system and vascular disorders. Examples of cancers that can be treated using anti-LRP 6 antibodies include small cell lung cancer, non-small cell lung cancer, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma (hepatoma), breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer (including renal cell carcinoma), liver cancer, prostate cancer. Examples of bones or bone disorders that may be treated using anti-LRP 6 antibodies include osteoporosis, osteoarthritis, bone fractures, and bone injuries. Examples of vascular disorders that can be treated using anti-LRP 6 antibodies include retinal vascular diseases such as Norrie (Norrie) disease, osteoporosis-pseudoglioma syndrome (OPPG), Familial Exudative Vitreoretinopathy (FEVR), retinopathy of prematurity (ROP), diabetic retinopathy, age-related macular degeneration, retinopathy of prematurity, coronary (Coats) disease and coronary-like reactions, and retinal artery or vein occlusion and myocardial related conditions such as myocardial infarction and ischemic heart disease.

Thus, one aspect of the invention provides an antibody that binds to LRP6, wherein the antibody inhibits signaling induced by one Wnt isoform and potentiates signaling induced by another Wnt isoform. In one embodiment, the antibody inhibits Wnt3 and/or Wnt3a signaling. In one embodiment, the antibody potentiates signaling by Wnt1, 2b, 4,6, 7a, 7b, 8a, 9b, 10a, and/or 10 b. In one embodiment, the antibody inhibits Wnt3 and/or Wnt3a signaling and potentiates Wnt1, 2b, 4,6, 7a, 7b, 8a, 9b, 10a, and/or 10b signaling. In one embodiment, the antibody inhibits Wnt3 and Wnt3a signaling and potentiates Wnt1, 2b, 4,6, 7a, 7b, 8a, 9b, 10a, and 10b signaling. In one embodiment, an anti-LRP 6 antibody binds to the E3-E4 region (first and second β -helices) of LRP6.

In another embodiment, the antibody inhibits Wnt1, 2b, 6, 8a, 9b, and/or 10b signaling. In one embodiment, the antibody potentiates Wnt3 and/or Wnt3a signaling. In one embodiment, the antibody inhibits Wnt1, 2b, 6, 8a, 9b, and/or 10b signaling and potentiates Wnt3 and/or Wnt3a signaling. In one embodiment, the antibody inhibits Wnt1, 2b, 6, 8a, 9b, and/or 10b signaling and potentiates Wnt3 and/or Wnt3a signaling. In one embodiment, an anti-LRP 6 antibody binds to the E1-E2 region (third and fourth beta-helices (beta-propellers)) of LRP6.

Another aspect of the invention provides multispecific anti-LRP 6 antibodies. As shown in the examples, in some embodiments, multispecific antibodies have the benefit of inhibiting all three Wnt isoforms. In one embodiment, the anti-LRP 6 antibody is a multispecific antibody capable of binding two or more different regions or epitopes of LRP6. In one embodiment, the multispecific antibody is a bispecific antibody capable of specifically binding two different regions of LRP6. In one embodiment, the bispecific antibody binds to the E1-E2 region of LRP6 and binds to the E3-E4 region of LRP6. In one embodiment, the multispecific antibody inhibits signaling induced by a Wnt isoform selected from the group consisting of Wnt3 and Wnt3a and inhibits signaling induced by a Wnt isoform selected from the group consisting of Wnt1, 2b, 6, 8a, 9b, and 10 b. In one embodiment, the multispecific antibody inhibits signaling induced by a Wnt isoform selected from the group consisting of Wnt3 and Wnt3a and inhibits signaling induced by a Wnt isoform selected from the group consisting of Wnt1, 2b, 6, 8a, 9b, and 10b and further inhibits signaling induced by a Wnt isoform selected from the group consisting of Wnt4, 7a, 7b, and 10 a. In one embodiment, the multispecific antibody inhibits Wnt signaling induced by a combination of Wnt1 and Wnt3 a. In one embodiment, the multispecific antibody inhibits autocrine Wnt signaling.

In certain embodiments, the multispecific antibody is a bispecific antibody that inhibits signaling induced by a Wnt isoform selected from the group consisting of Wnt3 and Wnt3a and inhibits signaling induced by a Wnt isoform selected from the group consisting of Wnt1, 2b, 6, 8a, 9b, and 10 b. In certain embodiments, the multispecific antibody is a bispecific antibody that inhibits signaling induced by a Wnt isoform selected from the group consisting of Wnt3 and Wnt3a and inhibits signaling induced by a Wnt isoform selected from the group consisting of Wnt1, 2b, 6, 8a, 9b, and 10b and further inhibits signaling induced by a Wnt isoform selected from the group consisting of Wnt4, 7a, 7b, and 10 a. In certain embodiments, the multispecific antibody is a bispecific antibody that inhibits signaling induced by the combination of Wnt1 and Wnt3 a. In one embodiment, the multispecific antibody is a bispecific antibody that inhibits signaling induced by the combination of Wnt1 and Wnt3a more effectively than a monospecific antibody combination having the same specificity as a bispecific antibody.

In certain embodiments, the multispecific antibody is a bispecific antibody that inhibits autocrine Wnt signaling more effectively than a monospecific antibody combination having the same specificity as a bispecific antibody.

In certain embodiments, an anti-LRP 6 antibody or a multispecific anti-LRP 6 antibody inhibits Wnt signaling by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more. Inhibition of Wnt signaling can be determined using assays known in the art and described herein. For example, inhibition of Wnt signaling can be determined using a Wnt reporter assay, such as the Wnt luciferase reporter assay described in the examples.

Inhibition of Wnt signaling can also be determined by monitoring expression of Wnt target genes, such as APCDD1, AXIN2, GAD1, leafy 2, and SAX1, as described in the examples.

In certain embodiments, the anti-LRP 6 antibody or multispecific anti-LRP 6 antibody inhibits expression of a Wnt target gene, such as APCDD1, AXIN2, GAD1, LEFTY2, and SAX1, by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%. In one embodiment, expression of the Wnt target gene is determined using a gene expression assay, such as PCR, including qPCR.

Another aspect of the invention provides antibodies that bind to LRP6 and compete for binding with any of the anti-LRP 6 antibodies described herein. Another aspect of the invention provides an antibody that binds to the same epitope on LRP6 as any of the anti-LRP 6 antibodies described herein.

A. Exemplary anti-LRP 6 antibodies

One aspect of the invention provides an anti-LRP 6 antibody that is a monoclonal antibody, including chimeric, humanized, or human antibodies. In one embodiment, the anti-LRP 6 antibody is generated using a phage library. In one embodiment, the anti-LRP 6 antibody is an antibody fragment, such as an Fv, Fab ', scFv, diabody, or F (ab')₂And (3) fragment. In another embodiment, the antibody is a full length antibody, e.g., a complete IgG 1antibody or other antibody class or isotype, as defined herein.

In one embodiment, the anti-LRP 6 antibody comprises a heavy chain sequence comprising the amino acid sequence of table 2. In one embodiment, the anti-LRP 6 antibody comprises a light chain sequence comprising the amino acid sequence of table 2. In one embodiment, an anti-LRP 6 antibody comprises a heavy chain sequence and a light chain sequence comprising the amino acid sequences of table 2.

In one embodiment, the anti-LRP 6 antibody comprises a heavy chain sequence comprising the amino acid sequence of seq id No. 1. In one embodiment, the anti-LRP 6 antibody comprises a light chain sequence comprising the amino acid sequence of seq id No. 2. In one embodiment, the anti-LRP 6 antibody comprises a heavy chain sequence comprising the amino acid sequence of seq id No. 1 and a light chain sequence comprising the amino acid sequence of seq id No. 2.

In one embodiment, the anti-LRP 6 antibody comprises a heavy chain sequence comprising the amino acid sequence of seq id No. 3. In one embodiment, the anti-LRP 6 antibody comprises a light chain sequence comprising the amino acid sequence of seq id No. 4. In one embodiment, the anti-LRP 6 antibody comprises a heavy chain sequence comprising the amino acid sequence of seq id No. 3 and a light chain sequence comprising the amino acid sequence of seq id No. 4.

In one embodiment, the anti-LRP 6 antibody comprises a heavy chain sequence comprising the amino acid sequence of seq id No. 5. In one embodiment, the anti-LRP 6 antibody comprises a light chain sequence comprising the amino acid sequence of seq id No. 6. In one embodiment, the anti-LRP 6 antibody comprises a heavy chain sequence comprising the amino acid sequence of seq id No.5 and a light chain sequence comprising the amino acid sequence of seq id No. 6.

In one embodiment, the anti-LRP 6 antibody comprises a heavy chain sequence comprising the amino acid sequence of seq id No. 7. In one embodiment, the anti-LRP 6 antibody comprises a light chain sequence comprising the amino acid sequence of seq id No. 8. In one embodiment, the anti-LRP 6 antibody comprises a heavy chain sequence comprising the amino acid sequence of seq id No.7 and a light chain sequence comprising the amino acid sequence of seq id No. 8.

In one embodiment, the anti-LRP 6 antibody comprises a heavy chain variable domain (VH) from an amino acid sequence of table 3. In one embodiment, the anti-LRP 6 antibody comprises a light chain variable domain (VL) from an amino acid sequence of table 3. In one embodiment, an anti-LRP 6 antibody comprises a VH and a VL from the amino acid sequences of table 3.

In one embodiment, the anti-LRP 6 antibody comprises a heavy chain variable domain (VH) from the heavy chain of amino acid sequence seq id no: 1. In one embodiment, the anti-LRP 6 antibody comprises a light chain variable domain (VL) from the light chain sequence of amino acid sequence seq id No. 2. In one embodiment, the anti-LRP 6 antibody comprises a VH from the heavy chain of amino acid sequence seq id No. 1 and a VL from the light chain of amino acid sequence seq id No. 2.

In one embodiment, the anti-LRP 6 antibody comprises a heavy chain variable domain (VH) comprising the amino acid sequence of seq id No. 9. In one embodiment, the anti-LRP 6 antibody comprises a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO. 10. In one embodiment, the anti-LRP 6 antibody comprises a VH comprising the amino acid sequence of seq id No. 9 and a VL comprising the amino acid sequence of seq id No. 10.

In one embodiment, the anti-LRP 6 antibody comprises a heavy chain variable domain (VH) from the heavy chain of amino acid sequence seq id No. 3. In one embodiment, the anti-LRP 6 antibody comprises a light chain variable domain (VL) from the light chain sequence of amino acid sequence seq id No. 4. In one embodiment, the anti-LRP 6 antibody comprises a VH from the heavy chain of amino acid sequence seq id No. 3 and a VL from the light chain sequence of amino acid sequence seq id No. 4.

In one embodiment, the anti-LRP 6 antibody comprises a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 11. In one embodiment, the anti-LRP 6 antibody comprises a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 12. In one embodiment, the anti-LRP 6 antibody comprises a VH comprising the amino acid sequence of seq id No. 11 and a VL comprising the amino acid sequence of seq id No. 12.

In one embodiment, the anti-LRP 6 antibody comprises a heavy chain variable domain (VH) from the heavy chain of amino acid sequence seq id No. 5. In one embodiment, the anti-LRP 6 antibody comprises a light chain variable domain (VL) from the light chain sequence of amino acid sequence seq id No. 6. In one embodiment, the anti-LRP 6 antibody comprises a VH from the heavy chain of amino acid sequence seq id No.5 and a VL from the light chain sequence of amino acid sequence seq id No. 6.

In one embodiment, the anti-LRP 6 antibody comprises a heavy chain variable domain (VH) comprising the amino acid sequence of seq id No. 13. In one embodiment, the anti-LRP 6 antibody comprises a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 14. In one embodiment, the anti-LRP 6 antibody comprises a VH comprising the amino acid sequence of seq id No. 13 and a VL comprising the amino acid sequence of seq id No. 14.

In one embodiment, the anti-LRP 6 antibody comprises a heavy chain variable domain (VH) from the heavy chain of amino acid sequence seq id No. 7. In one embodiment, the anti-LRP 6 antibody comprises a light chain variable domain (VL) from the light chain sequence of amino acid sequence SEQ ID NO. 8. In one embodiment, the anti-LRP 6 antibody comprises a VH from the heavy chain of amino acid sequence seq id No.7 and a VL from the light chain sequence of amino acid sequence seq id No. 8.

In one embodiment, the anti-LRP 6 antibody comprises a heavy chain variable domain (VH) comprising the amino acid sequence of seq id No. 15. In one embodiment, the anti-LRP 6 antibody comprises a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO. 16. In one embodiment, the anti-LRP 6 antibody comprises a VH comprising the amino acid sequence of seq id No. 15 and a VL comprising the amino acid sequence of seq id No. 16.

Another aspect of the invention provides a multispecific anti-LRP 6 antibody. In one embodiment, the multispecific antibody comprises a heavy chain comprising an amino acid sequence of at least one of seq id No. 1, seq id No. 3, seq id No.5, or seq id No. 7. In one embodiment, the multispecific antibody comprises a heavy chain comprising the amino acid sequence of at least two of seq id No. 1, seq id No. 3, seq id No.5, or seq id No. 7. In one embodiment, the multispecific antibody is a bispecific antibody comprising a heavy chain comprising an amino acid sequence of at least one of seq id No. 1, seq id No. 3, seq id No.5, or seq id No. 7. In one embodiment, the bispecific antibody comprises a first heavy chain comprising the amino acid sequence of seq id No. 1 and a second heavy chain comprising the amino acid sequence of seq id No. 7. In one embodiment, the bispecific antibody comprises a first heavy chain comprising the amino acid sequence of seq id No. 3 and a second heavy chain comprising the amino acid sequence of seq id No. 7. In one embodiment, the bispecific antibody comprises a first heavy chain comprising the amino acid sequence of seq id No.5 and a second heavy chain comprising the amino acid sequence of seq id No. 7.

In one embodiment, the multispecific anti-LRP 6 antibody comprises a VH from a heavy chain of an amino acid sequence of at least one of seq id No. 1, seq id No. 3, seq id No.5, or seq id No. 7. In one embodiment, the multispecific antibody comprises a VH from a heavy chain of amino acid sequences of at least two of seq id No. 1, seq id No. 3, seq id No.5, or seq id No. 7. In one embodiment, the multispecific antibody is a bispecific antibody comprising a VH from the heavy chain of amino acid sequence seq id No. 1, seq id No. 3, seq id No.5, or seq id No. 7. In one embodiment, the bispecific antibody comprises a VH derived from the heavy chain of amino acid sequence SEQ ID NO. 1 and a VH derived from the heavy chain of amino acid sequence SEQ ID NO. 7. In one embodiment, the bispecific antibody comprises a VH derived from the heavy chain of amino acid sequence seq id No. 3 and comprises a VH derived from the heavy chain of amino acid sequence seq id No. 7. In one embodiment, the bispecific antibody comprises a VH derived from the heavy chain of amino acid sequence seq id No.5 and comprises a VH derived from the heavy chain of amino acid sequence seq id No. 7.

In one embodiment, the multispecific anti-LRP 6 antibody comprises a VH comprising the amino acid sequence of at least one of seq id No. 9, seq id No. 11, seq id No. 13, or seq id No. 15. In one embodiment, the multispecific antibody comprises a VH comprising the amino acid sequence of at least two of seq id No. 9, seq id No. 11, seq id No. 13, or seq id No. 15. In one embodiment, the multispecific antibody is a bispecific antibody comprising a VH comprising the amino acid sequence of seq id No. 9, seq id No. 11, seq id No. 13, or seq id No. 15. In one embodiment, the bispecific antibody comprises a first VH comprising the amino acid sequence of seq id No. 9 and a second VH comprising the amino acid sequence of seq id No. 15. In one embodiment, the bispecific antibody comprises a first VH comprising the amino acid sequence of seq id No. 11 and a second VH comprising the amino acid sequence of seq id No. 15. In one embodiment, the bispecific antibody comprises a first VH comprising the amino acid sequence of seq id No. 13 and a second VH comprising the amino acid sequence of seq id No. 15.

In one embodiment, an anti-LRP 6 antibody comprises at least one, two, three, four, five, or six HVRs selected from: (a) HVR-H1, which is derived from the HVR-H1 amino acid sequence of Table 4; (b) HVR-H2, which is derived from the HVR-H2 amino acid sequence of Table 4; (c) HVR-H3, which is derived from the HVR-H3 amino acid sequence of Table 4; (d) HVR-L1, which is derived from the HVR-L1 amino acid sequence of Table 4; (e) HVR-L2, which is derived from the HVR-L2 amino acid sequence of Table 4; and (f) HVR-L3, which is derived from the HVR-L3 amino acid sequence of Table 4.

In one embodiment, the anti-LRP 6 antibody comprises a VH comprising at least one, two, three, four, five, or six HVRs selected from: (a) HVR-H1 of the heavy chain of SEQ ID NO. 1; (b) HVR-H2 of the heavy chain of SEQ ID NO. 1; (c) HVR-H3 of the heavy chain of SEQ ID NO. 1; (d) HVR-L1 of the light chain of SEQ ID NO. 2; (e) HVR-L2 of the light chain of SEQ ID NO. 2; and (f) HVR-L3 of the light chain of SEQ ID NO: 2.

In one embodiment, the anti-LRP 6 antibody comprises at least one, at least two, or all three VHHVR sequences selected from: (a) HVR-H1 of the heavy chain of SEQ ID NO. 1; (b) HVR-H2 of the heavy chain of SEQ ID NO. 1; (c) HVR-H3 of the heavy chain of SEQ ID NO. 1. In one embodiment, the antibody comprises HVR-H3 of the heavy chain of SEQ ID NO: 1. In another embodiment, the antibody comprises HVR-H3 of the heavy chain of SEQ ID NO. 1 and HVR-L3 of the light chain of SEQ ID NO. 2. In yet another embodiment, the antibody comprises HVR-H3 of the heavy chain of SEQ ID NO. 1, HVR-L3 of the light chain of SEQ ID NO.2, and HVR-H2 of the heavy chain of SEQ ID NO. 1. In yet another embodiment, an antibody comprises (a) HVR-H1 of the heavy chain of SEQ ID NO: 1; (b) HVR-H2 of the heavy chain of SEQ ID NO. 1; and (c) HVR-H3 of the heavy chain of SEQ ID NO: 1.

In one embodiment, the anti-LRP 6 antibody comprises at least one, at least two, or all three VHHVR sequences selected from: (a) HVR-H1, comprising the amino acid sequence of SEQ ID NO: 17; (b) HVR-H2, comprising the amino acid sequence of SEQ ID NO: 18; (c) HVR-H3, comprising the amino acid sequence of SEQ ID NO: 19. In one embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 19. In another embodiment, an antibody comprises HVR-H3 and HVR-L3, wherein HVR-H3 comprises the amino acid sequence of SEQ ID NO:19 and HVR-L3 comprises the amino acid sequence of SEQ ID NO: 27. In yet another embodiment, an antibody comprises HVR-H3, HVR-L3, and HVR-H2, wherein HVR-H3 comprises amino acid sequence SEQ ID NO:19, HVR-L3 comprises amino acid sequence SEQ ID NO:27, and HVR-H2 comprises amino acid sequence SEQ ID NO: 18. In yet another embodiment, an antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 17; (b) HVR-H2, comprising the amino acid sequence of SEQ ID NO: 18; and (c) HVR-H3, comprising the amino acid sequence of SEQ ID NO: 19.

In one embodiment, an anti-LRP 6 antibody comprises at least one, at least two, or all three VLHVR sequences selected from: (a) HVR-L1 of the light chain of SEQ ID NO. 2; (b) HVR-L2 of the light chain of SEQ ID NO. 2; and (c) HVR-L3 of the light chain of SEQ ID NO: 2. In one embodiment, the antibody comprises (a) HVR-L1 of the light chain of SEQ ID NO: 2; (b) HVR-L2 of the light chain of SEQ ID NO. 2; and (c) HVR-L3 of the light chain of SEQ ID NO: 2.

In one embodiment, an anti-LRP 6 antibody comprises (a) a VH domain comprising at least one, at least two, or all three VHHVR sequences selected from: (i) HVR-H1 of the heavy chain of SEQ ID NO:1, (ii) HVR-H2 of the heavy chain of SEQ ID NO:1, and (iii) HVR-H3 of the heavy chain of SEQ ID NO: 1; and (b) a VL domain comprising at least one, at least two, or all three VLHVR sequences selected from: (i) HVR-L1 of the light chain of SEQ ID NO.2, (ii) HVR-L2 of the light chain of SEQ ID NO.2, and (c) HVR-L3 of the light chain of SEQ ID NO. 2.

In one embodiment, an anti-LRP 6 antibody comprises (a) a VH domain comprising at least one, at least two, or all three VHHVR sequences selected from: (i) HVR-H1 comprising the amino acid sequence of seq id no:17, (ii) HVR-H2 comprising the amino acid sequence of seq id no:18, and (iii) HVR-H3 comprising the amino acid sequence of seq id no: 19; and (b) a VL domain comprising at least one, at least two, or all three VLHVR sequences selected from: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:25, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO:26, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27.

In another aspect, the invention provides an antibody comprising (a) HVR-H1 of the heavy chain of SEQ ID NO: 1; (b) HVR-H2 of the heavy chain of SEQ ID NO. 1; (c) HVR-H3 of the heavy chain of SEQ ID NO. 1; (d) HVR-L1 of the light chain of SEQ ID NO. 2; (e) HVR-L2 of the light chain of SEQ ID NO. 2; and (f) HVR-L3 of the light chain of SEQ ID NO: 2.

In another aspect, the invention provides an antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 17; (b) HVR-H2, comprising the amino acid sequence of SEQ ID NO: 18; (c) HVR-H3, comprising the amino acid sequence of SEQ ID NO. 19; (d) HVR-L1, comprising the amino acid sequence of SEQ ID NO. 25; (e) HVR-L2, comprising the amino acid sequence of SEQ ID NO: 26; and (f) HVR-L3, comprising the amino acid sequence of SEQ ID NO: 27.

In one embodiment, an anti-LRP 6 antibody comprises at least one, two, three, four, five, or six HVRs selected from: (a) HVR-H1 of the heavy chain of SEQ ID NO. 3; (b) HVR-H2 of the heavy chain of SEQ ID NO. 3; (c) HVR-H3 of the heavy chain of SEQ ID NO. 3; (d) HVR-L1 of the light chain of SEQ ID NO. 4; (e) HVR-L2 of the light chain of SEQ ID NO. 4; and (f) HVR-L3 of the light chain of SEQ ID NO: 4.

In one embodiment, the anti-LRP 6 antibody comprises at least one, at least two, or all three VHHVR sequences selected from: (a) HVR-H1 of the heavy chain of SEQ ID NO. 3; (b) HVR-H2 of the heavy chain of SEQ ID NO. 3; (c) HVR-H3 of the heavy chain of SEQ ID NO. 3. In one embodiment, the antibody comprises HVR-H3 of the heavy chain of SEQ ID NO. 3. In another embodiment, the antibody comprises HVR-H3 of the heavy chain of SEQ ID NO. 3 and HVR-L3 of the light chain of SEQ ID NO. 4. In yet another embodiment, the antibody comprises HVR-H3 of the heavy chain of SEQ ID NO. 3, HVR-L3 of the light chain of SEQ ID NO.4, and HVR-H2 of the heavy chain of SEQ ID NO. 3. In yet another embodiment, an antibody comprises (a) HVR-H1 of the heavy chain of SEQ ID NO. 3; (b) HVR-H2 of the heavy chain of SEQ ID NO. 3; and (c) HVR-H3 of the heavy chain of SEQ ID NO: 3.

In one embodiment, the anti-LRP 6 antibody comprises at least one, at least two, or all three VHHVR sequences selected from: (a) HVR-H1, comprising the amino acid sequence of SEQ ID NO: 17; (b) HVR-H2, comprising the amino acid sequence of SEQ ID NO: 18; (c) HVR-H3, comprising the amino acid sequence of SEQ ID NO: 21. In one embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 21. In another embodiment, the antibody comprises heavy chain HVR-H3 and HVR-L3, heavy chain HVR-H3 is SEQ ID NO:21, and HVR-L3 comprises amino acid sequence SEQ ID NO: 28. In yet another embodiment, an antibody comprises HVR-H3, HVR-L3, and HVR-H2, wherein HVR-H3 comprises amino acid sequence SEQ ID NO:21, HVR-L3 comprises amino acid sequence SEQ ID NO:28, and HVR-H2 comprises amino acid sequence SEQ ID NO: 18. In yet another embodiment, an antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 17; (b) HVR-H2, comprising the amino acid sequence of SEQ ID NO: 18; and (c) HVR-H3, comprising the amino acid sequence of SEQ ID NO: 21.

In one embodiment, an anti-LRP 6 antibody comprises at least one, at least two, or all three VLHVR sequences selected from: (a) HVR-L1 of the light chain of SEQ ID NO. 4; (b) HVR-L2 of the light chain of SEQ ID NO. 4; and (c) HVR-L3 of the light chain of SEQ ID NO: 4. In one embodiment, the antibody comprises (a) HVR-L1 of the light chain of SEQ ID NO: 4; (b) HVR-L2 of the light chain of SEQ ID NO. 4; and (c) HVR-L3 of the light chain of SEQ ID NO: 4.

In one embodiment, an anti-LRP 6 antibody comprises at least one, at least two, or all three VLHVR sequences selected from: (a) HVR-L1, comprising the amino acid sequence of SEQ ID NO. 25; (b) HVR-L2, comprising the amino acid sequence of SEQ ID NO: 26; and (c) HVR-L3, comprising the amino acid sequence of SEQ ID NO: 28. In one embodiment, an antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 25; (b) HVR-L2, comprising the amino acid sequence of SEQ ID NO: 26; and (c) HVR-L3, comprising the amino acid sequence of SEQ ID NO: 28.

In one embodiment, an anti-LRP 6 antibody comprises (a) a VH domain comprising at least one, at least two, or all three VHHVR sequences selected from: (i) HVR-H1 of the heavy chain of SEQ ID NO. 3, (ii) HVR-H2 of the heavy chain of SEQ ID NO. 3, and (iii) HVR-H3 of the heavy chain of SEQ ID NO. 3; and (b) a VL domain comprising at least one, at least two, or all three VLHVR sequences selected from: (i) HVR-L1 of the light chain of SEQ ID NO:4, (ii) HVR-L2 of the light chain of SEQ ID NO:4, and (c) HVR-L3 of the light chain of SEQ ID NO: 4.

In one embodiment, an anti-LRP 6 antibody comprises (a) a VH domain comprising at least one, at least two, or all three VHHVR sequences selected from: (i) HVR-H1 comprising the amino acid sequence of seq id no:17, (ii) HVR-H2 comprising the amino acid sequence of seq id no:18, and (iii) HVR-H3 comprising the amino acid sequence of seq id no: 21; and (b) a VL domain comprising at least one, at least two, or all three VLHVR sequences selected from: (i) HVR-L1 comprising amino acid sequence SEQ ID NO:25, (ii) HVR-L2 comprising amino acid sequence SEQ ID NO:26, and (c) HVR-L3 comprising amino acid sequence SEQ ID NO: 28.

In another aspect, the invention provides an antibody comprising (a) HVR-H1 of the heavy chain of SEQ ID NO. 3; (b) HVR-H2 of the heavy chain of SEQ ID NO. 3; (c) HVR-H3 of the heavy chain of SEQ ID NO. 3; (d) HVR-L1 of the light chain of SEQ ID NO. 4; (e) HVR-L2 of the light chain of SEQ ID NO. 4; and (f) HVR-L3 of the light chain of SEQ ID NO: 4.

In another aspect, the invention provides an antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 17; (b) HVR-H2, comprising the amino acid sequence of SEQ ID NO: 18; (c) HVR-H3, comprising the amino acid sequence of SEQ ID NO: 21; (d) HVR-L1, comprising the amino acid sequence of SEQ ID NO. 25; (e) HVR-L2, comprising the amino acid sequence of SEQ ID NO: 26; and (f) HVR-L3, comprising the amino acid sequence of SEQ ID NO: 28.

In one embodiment, an anti-LRP 6 antibody comprises at least one, two, three, four, five, or six HVRs selected from: (a) HVR-H1 of the heavy chain of SEQ ID NO. 5; (b) HVR-H2 of the heavy chain of SEQ ID NO. 5; (c) HVR-H3 of the heavy chain of SEQ ID NO. 5; (d) HVR-L1 of the light chain of SEQ ID NO. 6; (e) HVR-L2 of the light chain of SEQ ID NO. 6; and (f) HVR-L3 of the light chain of SEQ ID NO: 6.

In one embodiment, the anti-LRP 6 antibody comprises at least one, at least two, or all three VHHVR sequences selected from: (a) HVR-H1 of the heavy chain of SEQ ID NO. 5; (b) HVR-H2 of the heavy chain of SEQ ID NO. 5; (c) HVR-H3 of the heavy chain of SEQ ID NO. 5. In one embodiment, the antibody comprises HVR-H3 of the heavy chain of SEQ ID NO. 5. In another embodiment, the antibody comprises HVR-H3 of the heavy chain of SEQ ID NO.5 and HVR-L3 of the light chain of SEQ ID NO. 6. In yet another embodiment, the antibody comprises HVR-H3 of the heavy chain of SEQ ID NO.5, HVR-L3 of the light chain of SEQ ID NO.6, and HVR-H2 of the heavy chain of SEQ ID NO. 5. In yet another embodiment, an antibody comprises (a) HVR-H1 of the heavy chain of SEQ ID NO. 5; (b) HVR-H2 of the heavy chain of SEQ ID NO. 5; and (c) HVR-H3 of the heavy chain of SEQ ID NO: 5.

In one embodiment, the anti-LRP 6 antibody comprises at least one, at least two, or all three VHHVR sequences selected from: (a) HVR-H1, comprising the amino acid sequence of SEQ ID NO: 20; (b) HVR-H2, comprising the amino acid sequence of SEQ ID NO: 18; (c) HVR-H3, comprising the amino acid sequence of SEQ ID NO: 19. In one embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 19. In another embodiment, an antibody comprises HVR-H3 and HVR-L3, wherein HVR-H3 comprises the amino acid sequence of SEQ ID NO:19 and HVR-L3 comprises the amino acid sequence of SEQ ID NO: 27. In yet another embodiment, an antibody comprises HVR-H3, HVR-L3, and HVR-H2, wherein HVR-H3 comprises amino acid sequence SEQ ID NO:19, HVR-L3 comprises amino acid sequence SEQ ID NO:27, and HVR-H2 comprises amino acid sequence SEQ ID NO: 18. In yet another embodiment, an antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20; (b) HVR-H2, comprising the amino acid sequence of SEQ ID NO: 18; and (c) HVR-H3, comprising the amino acid sequence of SEQ ID NO: 19.

In one embodiment, an anti-LRP 6 antibody comprises at least one, at least two, or all three VLHVR sequences selected from: (a) HVR-L1 of the light chain of SEQ ID NO. 6; (b) HVR-L2 of the light chain of SEQ ID NO. 6; and (c) HVR-L3 of the light chain of SEQ ID NO: 6. In one embodiment, the antibody comprises (a) HVR-L1 of the light chain of SEQ ID NO: 6; (b) HVR-L2 of the light chain of SEQ ID NO. 6; and (c) HVR-L3 of the light chain of SEQ ID NO: 6.

In one embodiment, an anti-LRP 6 antibody comprises (a) a VH domain comprising at least one, at least two, or all three VHHVR sequences selected from: (i) HVR-H1 of the heavy chain of SEQ ID NO.5, (ii) HVR-H2 of the heavy chain of SEQ ID NO.5, and (iii) HVR-H3 of the heavy chain of SEQ ID NO. 5; and (b) a VL domain comprising at least one, at least two, or all three VLHVR sequences selected from: (i) HVR-L1 of the light chain of SEQ ID NO:6, (ii) HVR-L2 of the light chain of SEQ ID NO:6, and (c) HVR-L3 of the light chain of SEQ ID NO: 6.

In one embodiment, an anti-LRP 6 antibody comprises (a) a VH domain comprising at least one, at least two, or all three VHHVR sequences selected from: (i) HVR-H1 comprising the amino acid sequence of seq id no:20, (ii) HVR-H2 comprising the amino acid sequence of seq id no:18, and (iii) HVR-H3 comprising the amino acid sequence of seq id no: 19; and (b) a VL domain comprising at least one, at least two, or all three VLHVR sequences selected from: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:25, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO:26, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27.

In another aspect, the invention provides an antibody comprising (a) HVR-H1 of the heavy chain of SEQ ID NO. 5; (b) HVR-H2 of the heavy chain of SEQ ID NO. 5; (c) HVR-H3 of the heavy chain of SEQ ID NO. 5; (d) HVR-L1 of the light chain of SEQ ID NO. 6; (e) HVR-L2 of the light chain of SEQ ID NO. 6; and (f) HVR-L3 of the light chain of SEQ ID NO: 6.

In another aspect, the invention provides an antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20; (b) HVR-H2, comprising the amino acid sequence of SEQ ID NO: 18; (c) HVR-H3, comprising the amino acid sequence of SEQ ID NO. 19; (d) HVR-L1, comprising the amino acid sequence of SEQ ID NO. 25; (e) HVR-L2, comprising the amino acid sequence of SEQ ID NO: 26; and (f) HVR-L3, comprising the amino acid sequence of SEQ ID NO: 27.

In one embodiment, an anti-LRP 6 antibody comprises at least one, two, three, four, five, or six HVRs selected from: (a) HVR-H1 of the heavy chain of SEQ ID NO. 7; (b) HVR-H2 of the heavy chain of SEQ ID NO. 7; (c) HVR-H3 of the heavy chain of SEQ ID NO. 7; (d) HVR-L1 of the light chain of SEQ ID NO. 8; (e) HVR-L2 of the light chain of SEQ ID NO. 8; and (f) HVR-L3 of the light chain of SEQ ID NO: 8.

In one embodiment, the anti-LRP 6 antibody comprises at least one, at least two, or all three VHHVR sequences selected from: (a) HVR-H1 of the heavy chain of SEQ ID NO. 7; (b) HVR-H2 of the heavy chain of SEQ ID NO. 7; (c) HVR-H3 of the heavy chain of SEQ ID NO. 7. In one embodiment, the antibody comprises HVR-H3 of the heavy chain of SEQ ID NO. 7. In another embodiment, the antibody comprises HVR-H3 of the heavy chain of SEQ ID NO.7 and HVR-L3 of the light chain of SEQ ID NO. 8. In yet another embodiment, the antibody comprises HVR-H3 of the heavy chain of SEQ ID NO.7, HVR-L3 of the light chain of SEQ ID NO. 8, and HVR-H2 of the heavy chain of SEQ ID NO. 7. In yet another embodiment, an antibody comprises (a) HVR-H1 of the heavy chain of SEQ ID NO. 7; (b) HVR-H2 of the heavy chain of SEQ ID NO. 7; and (c) HVR-H3 of the heavy chain of SEQ ID NO: 7.

In one embodiment, the anti-LRP 6 antibody comprises at least one, at least two, or all three VHHVR sequences selected from: (a) HVR-H1, comprising the amino acid sequence SEQ ID NO: 22; (b) HVR-H2, comprising the amino acid sequence of SEQ ID NO: 23; (c) HVR-H3, comprising the amino acid sequence of SEQ ID NO: 24. In one embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 24. In another embodiment, an antibody comprises HVR-H3 and HVR-L3, wherein HVR-H3 comprises the amino acid sequence of SEQ ID NO:24 and HVR-L3 comprises the amino acid sequence of SEQ ID NO: 27. In yet another embodiment, an antibody comprises HVR-H3, HVR-L3, and HVR-H2, wherein HVR-H3 comprises amino acid sequence SEQ ID NO:24, HVR-L3 comprises amino acid sequence SEQ ID NO:27, and HVR-H2 comprises amino acid sequence SEQ ID NO: 23. In yet another embodiment, an antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 22; (b) HVR-H2, comprising the amino acid sequence of SEQ ID NO: 23; and (c) HVR-H3, comprising the amino acid sequence of SEQ ID NO: 24.

In one embodiment, an anti-LRP 6 antibody comprises at least one, at least two, or all three VLHVR sequences selected from: (a) HVR-L1 of the light chain of SEQ ID NO. 8; (b) HVR-L2 of the light chain of SEQ ID NO. 8; and (c) HVR-L3 of the light chain of SEQ ID NO: 8. In one embodiment, the antibody comprises (a) HVR-L1 of the light chain of SEQ ID NO: 8; (b) HVR-L2 of the light chain of SEQ ID NO. 8; and (c) HVR-L3 of the light chain of SEQ ID NO: 8.

In one embodiment, an anti-LRP 6 antibody comprises (a) a VH domain comprising at least one, at least two, or all three VHHVR sequences selected from: (i) HVR-H1 of the heavy chain of SEQ ID NO.7, (ii) HVR-H2 of the heavy chain of SEQ ID NO.7, and (iii) HVR-H3 of the heavy chain of SEQ ID NO. 7; and (b) a VL domain comprising at least one, at least two, or all three VLHVR sequences selected from: (i) HVR-L1 of the light chain of SEQ ID NO:8, (ii) HVR-L2 of the light chain of SEQ ID NO:8, and (c) HVR-L3 of the light chain of SEQ ID NO: 8.

In one embodiment, an anti-LRP 6 antibody comprises (a) a VH domain comprising at least one, at least two, or all three VHHVR sequences selected from: (i) HVR-H1 comprising the amino acid sequence of seq id no:22, (ii) HVR-H2 comprising the amino acid sequence of seq id no:23, and (iii) HVR-H3 comprising the amino acid sequence of seq id no: 24; and (b) a VL domain comprising at least one, at least two, or all three VLHVR sequences selected from: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:25, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO:26, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27.

In another embodiment, the invention provides an antibody comprising (a) HVR-H1 of the heavy chain of SEQ ID NO. 7; (b) HVR-H2 of the heavy chain of SEQ ID NO. 7; (c) HVR-H3 of the heavy chain of SEQ ID NO. 7; (d) HVR-L1 of the light chain of SEQ ID NO. 8; (e) HVR-L2 of the light chain of SEQ ID NO. 8; and (f) HVR-L3 of the light chain of SEQ ID NO: 8.

In another embodiment, the invention provides an antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 22; (b) HVR-H2, comprising the amino acid sequence of SEQ ID NO: 23; (c) HVR-H3, comprising the amino acid sequence SEQ ID NO: 24; (d) HVR-L1, comprising the amino acid sequence of SEQ ID NO. 25; (e) HVR-L2, comprising the amino acid sequence of SEQ ID NO: 26; and (f) HVR-L3, comprising the amino acid sequence of SEQ ID NO: 27.

In one embodiment, the anti-LRP 6 antibody is a multispecific anti-LRP 6 antibody comprising a first VH domain comprising at least one, at least two, or all three VHHVR sequences selected from: (a) HVR-H1 of the heavy chain of SEQ ID NO. 1; (b) HVR-H2 of the heavy chain of SEQ ID NO. 1; (c) 1 and comprising a second VH domain comprising at least one, at least two, or all three VHHVR sequences selected from: (d) HVR-H1 of the heavy chain of SEQ ID NO. 7; (e) HVR-H2 of the heavy chain of SEQ ID NO. 7; (f) HVR-H3 of the heavy chain of SEQ ID NO. 7.

In one embodiment, the anti-LRP 6 antibody is a multispecific anti-LRP 6 antibody comprising a first VH domain comprising at least one, at least two, or all three VHHVR sequences selected from: (a) HVR-H1, comprising the amino acid sequence of SEQ ID NO: 17; (b) HVR-H2, comprising the amino acid sequence of SEQ ID NO: 18; (c) HVR-H3 comprising the amino acid sequence of seq id No. 19, and comprising a second VH domain comprising at least one, at least two, or all three VHHVR sequences selected from: (d) HVR-H1, comprising the amino acid sequence SEQ ID NO: 22; (e) HVR-H2, comprising the amino acid sequence of SEQ ID NO: 23; (f) HVR-H3, comprising the amino acid sequence of SEQ ID NO: 24.

In one embodiment, the anti-LRP 6 antibody is a multispecific anti-LRP 6 antibody comprising a first VH domain comprising all three VHHVR sequences from: (a) HVR-H1 of the heavy chain of SEQ ID NO. 1; (b) HVR-H2 of the heavy chain of SEQ ID NO. 1; (c) (ii) HVR-H3 of the heavy chain of seq id no:1, and comprising a second VH domain comprising all three VHHVR sequences from: (d) HVR-H1 of the heavy chain of SEQ ID NO. 7; (e) HVR-H2 of the heavy chain of SEQ ID NO. 7; (f) HVR-H3 of the heavy chain of SEQ ID NO. 7.

In one embodiment, the anti-LRP 6 antibody is a multispecific anti-LRP 6 antibody comprising a first VH domain comprising all three VHHVR sequences from: (a) HVR-H1, comprising the amino acid sequence of SEQ ID NO: 17; (b) HVR-H2, comprising the amino acid sequence of SEQ ID NO: 18; (c) HVR-H3 comprising the amino acid sequence seq id No. 19, and comprising a second VH domain comprising all three VHHVR sequences from: (d) HVR-H1, comprising the amino acid sequence SEQ ID NO: 22; (e) HVR-H2, comprising the amino acid sequence of SEQ ID NO: 23; (f) HVR-H3, comprising the amino acid sequence of SEQ ID NO: 24.

In one embodiment, the anti-LRP 6 antibody is a multispecific anti-LRP 6 antibody comprising a first VH domain comprising at least one, at least two, or all three VHHVR sequences selected from: (a) HVR-H1 of the heavy chain of SEQ ID NO. 3; (b) HVR-H2 of the heavy chain of SEQ ID NO. 3; (c) 3 and comprising a second VH domain comprising at least one, at least two, or all three VHHVR sequences selected from: (d) HVR-H1 of the heavy chain of SEQ ID NO. 7; (e) HVR-H2 of the heavy chain of SEQ ID NO. 7; (f) HVR-H3 of the heavy chain of SEQ ID NO. 7.

In one embodiment, the anti-LRP 6 antibody is a multispecific anti-LRP 6 antibody comprising a first VH domain comprising at least one, at least two, or all three VHHVR sequences selected from: (a) HVR-H1, comprising the amino acid sequence of SEQ ID NO: 17; (b) HVR-H2, comprising the amino acid sequence of SEQ ID NO: 18; (c) HVR-H3 comprising the amino acid sequence of seq id No. 21, and comprising a second VH domain comprising at least one, at least two, or all three VHHVR sequences selected from: (d) HVR-H1, comprising the amino acid sequence SEQ ID NO: 22; (e) HVR-H2, comprising the amino acid sequence of SEQ ID NO: 23; (f) HVR-H3, comprising the amino acid sequence of SEQ ID NO: 24.

In one embodiment, the anti-LRP 6 antibody is a multispecific anti-LRP 6 antibody comprising a first VH domain comprising all three VHHVR sequences from: (a) HVR-H1 of the heavy chain of SEQ ID NO. 3; (b) HVR-H2 of the heavy chain of SEQ ID NO. 3; (c) 3 and comprises a second VH domain comprising all three VHHVR sequences from seq id no: (d) HVR-H1 of the heavy chain of SEQ ID NO. 7; (e) HVR-H2 of the heavy chain of SEQ ID NO. 7; (f) HVR-H3 of the heavy chain of SEQ ID NO. 7.

In one embodiment, the anti-LRP 6 antibody is a multispecific anti-LRP 6 antibody comprising a first VH domain comprising all three VHHVR sequences from: (a) HVR-H1, comprising the amino acid sequence of SEQ ID NO: 17; (b) HVR-H2, comprising the amino acid sequence of SEQ ID NO: 18; (c) HVR-H3 comprising the amino acid sequence seq id no:21, and comprising a second VH domain comprising all three VHHVR sequences from: (d) HVR-H1, comprising the amino acid sequence SEQ ID NO: 22; (e) HVR-H2, comprising the amino acid sequence of SEQ ID NO: 23; (f) HVR-H3, comprising the amino acid sequence of SEQ ID NO: 24.

In one embodiment, the anti-LRP 6 antibody is a multispecific anti-LRP 6 antibody comprising a first VH domain comprising at least one, at least two, or all three VHHVR sequences selected from: (a) HVR-H1 of the heavy chain of SEQ ID NO. 5; (b) HVR-H2 of the heavy chain of SEQ ID NO. 5; (c) HVR-H3 of the heavy chain of seq id No.5, and comprising a second VH domain comprising at least one, at least two, or all three VHHVR sequences selected from: (d) HVR-H1 of the heavy chain of SEQ ID NO. 7; (e) HVR-H2 of the heavy chain of SEQ ID NO. 7; (f) HVR-H3 of the heavy chain of SEQ ID NO. 7.

In one embodiment, the anti-LRP 6 antibody is a multispecific anti-LRP 6 antibody comprising a first VH domain comprising at least one, at least two, or all three VHHVR sequences selected from: (a) HVR-H1, comprising the amino acid sequence of SEQ ID NO: 20; (b) HVR-H2, comprising the amino acid sequence of SEQ ID NO: 18; (c) HVR-H3 comprising the amino acid sequence of seq id No. 19, and comprising a second VH domain comprising at least one, at least two, or all three VHHVR sequences selected from: a (d) HVR-H1 comprising the amino acid sequence SEQ ID NO: 22; (e) HVR-H2, comprising the amino acid sequence of SEQ ID NO: 23; (f) HVR-H3, comprising the amino acid sequence of SEQ ID NO: 24.

In one embodiment, the anti-LRP 6 antibody is a multispecific anti-LRP 6 antibody comprising a first VH domain comprising all three VHHVR sequences from: (a) HVR-H1 of the heavy chain of SEQ ID NO. 5; (b) HVR-H2 of the heavy chain of SEQ ID NO. 5; (c) HVR-H3 of the heavy chain of seq id No.5, and comprising a second VH domain comprising all three VHHVR sequences from: (d) HVR-H1 of the heavy chain of SEQ ID NO. 7; (e) HVR-H2 of the heavy chain of SEQ ID NO. 7; (f) HVR-H3 of the heavy chain of SEQ ID NO. 7.

In one embodiment, the anti-LRP 6 antibody is a multispecific anti-LRP 6 antibody comprising a first VH domain comprising all three VHHVR sequences from: (a) HVR-H1, comprising the amino acid sequence of SEQ ID NO: 20; (b) HVR-H2, comprising the amino acid sequence of SEQ ID NO: 18; (c) HVR-H3 comprising the amino acid sequence seq id No. 19, and comprising a second VH domain comprising all three VHHVR sequences from: (d) HVR-H1, comprising the amino acid sequence SEQ ID NO: 22; (e) HVR-H2, comprising the amino acid sequence of SEQ ID NO: 23; (f) HVR-H3, comprising the amino acid sequence of SEQ ID NO: 24.

In any of the above embodiments of the multispecific anti-LRP 6 antibody, the antibody further comprises at least one, at least two, or all three VLHVR sequences selected from: (a) HVR-L1 of the light chain of SEQ ID NO. 2; (b) HVR-L2 of the light chain of SEQ ID NO. 2; and (c) HVR-L3 of the light chain of SEQ ID NO:2 or SEQ ID NO: 4. In one embodiment, the antibody comprises (a) HVR-L1 of the light chain of SEQ ID NO: 2; (b) HVR-L2 of the light chain of SEQ ID NO. 2; and (c) HVR-L3 of the light chain of SEQ ID NO: 2. In one embodiment, the antibody comprises (a) HVR-L1 of the light chain of SEQ ID NO: 2; (b) HVR-L2 of the light chain of SEQ ID NO. 2; and (c) HVR-L3 of the light chain of SEQ ID NO: 4.

In any of the above embodiments of the multispecific anti-LRP 6 antibody, the antibody further comprises at least one, at least two, or all three VLHVR sequences selected from: (a) HVR-L1, comprising the amino acid sequence of SEQ ID NO. 25; (b) HVR-L2, comprising the amino acid sequence of SEQ ID NO: 26; and (c) HVR-L3, comprising the amino acid sequence SEQ ID NO:27 or SEQ ID NO: 28. In one embodiment, an antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 25; (b) HVR-L2, comprising the amino acid sequence of SEQ ID NO: 26; and (c) HVR-L3, comprising the amino acid sequence of SEQ ID NO: 27. In one embodiment, an antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 25; (b) HVR-L2, comprising the amino acid sequence of SEQ ID NO: 26; and (c) HVR-L3, comprising the amino acid sequence of SEQ ID NO: 28.

In one embodiment, the anti-LRP 6 antibody or multispecific anti-LRP 6 antibody comprises HVR-H3 comprising the amino acid sequence NX₁X₂K (SEQ ID NO: 41). In one embodiment, the anti-LRP 6 antibody or multispecific anti-LRP 6 antibody comprises HVR-H3 comprising the amino acid sequence NX₁X₂KN (SEQ ID NO: 42). In one embodiment, the anti-LRP 6 antibody or multispecific anti-LRP 6 antibody comprises HVR-H3 comprising the amino acid sequence NX₁VK (SEQ ID NO: 43). In one embodiment, the anti-LRP 6 antibody or multispecific anti-LRP 6 antibody comprises HVR-H3 comprising the amino acid sequence NX₁IK (SEQ ID NO: 44). In one embodiment, the anti-LRP 6 antibody or multispecific anti-LRP 6 antibody comprises HVR-H3 comprising the amino acid sequence NX₁VKN (SEQ ID NO: 45). In one embodiment, the anti-LRP 6 antibody or multispecific anti-LRP 6 antibody comprises HVR-H3 comprising the amino acid sequence NX₁IKN (SEQ ID NO: 46). In the above embodiment, X₁Is an amino acid and X₂Is I or V; or X₁Is A, S, F, T, Y, or L and X₂Is I or V; or X₁Is A, S, F, T, Y, or L and X₂Is I; or X₁Is A, S, F, T, Y, or L and X₂Is V.

In one embodiment, the anti-LRP 6 antibody or multispecific anti-LRP 6 antibody comprises HVR-H3 comprising the amino acid sequence NAVK (SEQ ID NO: 47). In one embodiment, the anti-LRP 6 antibody or multispecific anti-LRP 6 antibody comprises HVR-H3 comprising the amino acid sequence NAIK (SEQ ID NO: 48). In one embodiment, the anti-LRP 6 antibody or multispecific anti-LRP 6 antibody comprises HVR-H3 comprising the amino acid sequence NAVPN (SEQ ID NO: 49). In one embodiment, the anti-LRP 6 antibody or multispecific anti-LRP 6 antibody comprises HVR-H3 comprising the amino acid sequence NAIKN (SEQ ID NO: 50).

In any of the above embodiments, the anti-LRP 6 antibody is humanized. In one embodiment, an anti-LRP 6 antibody comprises the HVRs of any of the above embodiments, and further comprises an acceptor human framework, e.g., a human immunoglobulin framework or a human consensus framework.

In another aspect, an anti-LRP 6 antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% sequence identity to the VH of the heavy chain of amino acid sequence seq id No. 1, seq id No. 3, seq id No.5, or seq id No. 7. In another aspect, an anti-LRP 6 antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% sequence identity to the amino acid sequence of seq id No. 9, seq id No. 11, seq id No. 13, or seq id No. 15. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-LRP 6 antibody comprising that sequence retains the ability to bind LRP6. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted and/or deleted in the VH of SEQ ID No. 1, SEQ ID No. 3, SEQ ID No.5, or SEQ ID No.7 or in SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13, or SEQ ID No. 15.

In another aspect, an anti-LRP 6 antibody comprises a heavy chain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% sequence identity to the amino acid sequence of seq id No. 1, seq id No. 3, seq id No.5, or seq id No. 7. In certain embodiments, a heavy chain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-LRP 6 antibody comprising that sequence retains the ability to bind LRP6. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted and/or deleted in seq id No. 1, seq id No. 3, seq id No.5, or seq id No. 7.

In certain embodiments, the substitution, insertion, or deletion occurs in a region outside of the HVR (i.e., in the FR). Optionally, the anti-LRP 6 antibody comprises the heavy chain and/or heavy chain VH of seq id no:1, seq id no:3, seq id no:5, or seq id no:7, including post-translational modifications of this sequence.

In another aspect, an anti-LRP 6 antibody is provided, wherein the antibody comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% sequence identity to the VL of the light chain of amino acid sequence seq id No.2, seq id No.4, seq id No.6, or seq id No. 8. In another aspect, an anti-LRP 6 antibody is provided, wherein the antibody comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% sequence identity to the amino acid sequence of SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, or SEQ ID NO. 16. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-LRP 6 antibody comprising that sequence retains the ability to bind LRP6. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted and/or deleted in the VL of SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6, or SEQ ID NO. 8 or in SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, or SEQ ID NO. 16.

In another aspect, an anti-LRP 6 antibody is provided, wherein the antibody comprises a light chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% sequence identity to the amino acid sequence of seq id No.2, seq id No.4, seq id No.6, or seq id No. 8. In certain embodiments, a light chain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-LRP 6 antibody comprising that sequence retains the ability to bind LRP6. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted and/or deleted in seq id No.2, seq id No.4, seq id No.6, or seq id No. 8.

In certain embodiments, the substitution, insertion, or deletion occurs in a region outside of the HVR (i.e., in the FR). Optionally, the anti-LRP 6 antibody comprises the light chain and/or VL sequence in seq id No.2, seq id No.4, seq id No.6, or seq id No. 8, including post-translational modifications of that sequence.

In another aspect, an anti-LRP 6 antibody is provided, wherein the antibody comprises a VH in any of the embodiments provided above and a VL in any of the embodiments provided above.

In yet another aspect, the invention provides antibodies that bind to the same epitope as the anti-LRP 6 antibodies provided herein. For example, in certain embodiments, antibodies are provided that bind the same epitope as an anti-LRP 6 antibody selected from the group consisting of the following anti-LRP 6 antibodies: it comprises a VH sequence SEQ ID NO. 9 and a VL sequence SEQ ID NO. 10, or a VH sequence SEQ ID NO. 11 and a VL sequence SEQ ID NO. 12, or a VH sequence SEQ ID NO. 13 and a VL sequence SEQ ID NO. 14, or a VH sequence SEQ ID NO. 15 and a VL sequence SEQ ID NO. 16. In one embodiment, the anti-LRP 6 antibody binds to an epitope consisting of amino acid residues in the E1-E2 region of LRP6. In one embodiment, the anti-LRP 6 antibody binds to an epitope consisting of amino acid residues in the E3-E4 region of LRP6. In one embodiment, the anti-LRP 6 antibody is a bispecific antibody that binds to an epitope consisting of amino acid residues present in the E1-E2 regions of LRP6 and binds to an epitope consisting of amino acid residues present in the E3-E4 regions of LRP6.

In one embodiment, the anti-LRP 6 antibody binds a conformational epitope comprising residues R28, E51, D52, V70, S71, E73, L95, S96, D98, E115, R141 and N185 of LRP6. In one embodiment, the anti-LRP 6 antibody binds a conformational epitope comprising residues R28, E51, D52, V70, S71, E73, L95, S96, D98, E115, R141, N185, R29, W188, K202, P225, H226, S243, and F266 of LRP6.

In one embodiment, the anti-LRP 6 antibody interacts with at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, or all of amino acid residues R28, E51, D52, V70, S71, E73, L95, S96, D98, E115, R141, and N185 of the E1 β -propeller of LRP6. In yet another embodiment, the anti-LRP 6 antibody further interacts with at least one, at least two, at least three, at least four, at least five, at least six, at least seven of LRP6 residues R29, W188, K202, P225, H226, S243, and F266.

In yet another aspect, an anti-LRP 6 antibody according to any of the above embodiments, alone or in combination, can incorporate any of the features described in sections 1-7 below:

1. affinity of antibody

In certain embodiments, an antibody provided herein has ≦ 1 μ M ≦ 100nM, ≦ 10nM, ≦ 1nM, ≦ 0.1nM, ≦ 0.01nM, or ≦ 0.001nM (e.g., 10 nM)^-8M or less, e.g. 10^-8M to 10^-13M, e.g. 10^-9M to 10^-13M) dissociation constant (Kd).

In one embodiment, Kd is measured by a radiolabeled antigen binding assay (RIA) performed with Fab versions of the antibody of interest and its antigen as described in the assays described below. By using the minimum concentration of (in the presence of unlabeled antigen in the titration series¹²⁵I) The Fab is equilibrated with labeled antigen and then the solution binding affinity of the Fab for the antigen is measured by capturing the bound antigen with an anti-Fab antibody coated plate (see, e.g., Chen et al, J.mol.biol.293:865-881 (1999)). To establish the assay conditions, theWells (Thermoscientific) were coated with 5. mu.g/ml capture anti-Fab antibodies (Cappellabs) in 50mM sodium carbonate (pH9.6) overnight, followed by blocking with 2% (w/v) bovine serum albumin in PBS for 2-5 hours at room temperature (about 23 ℃). On a non-adsorption plate (Nun)c #269620), 100pM or 26pM¹²⁵The I-antigen is mixed with serial dilutions of the Fab of interest (e.g., consistent with the evaluation of anti-VEGF antibodies, Fab-12, by Presta et al, cancer Res.57:4593-4599 (1997)). The Fab of interest was then incubated overnight; however, incubation may continue for longer periods of time (e.g., about 65 hours) to ensure equilibrium is reached. Thereafter, the mixture is transferred to a capture plate and incubated at room temperature (e.g., 1 hour). The solution was then removed and treated with 0.1% polysorbate 20 in PBSThe plate was washed 8 times. After drying the plates, 150. mu.l/well scintillation fluid (MICROSCINT-20) was added^TMPackard) and then in TOPCOUNT^TMPlates were counted on a gamma counter (Packard) for 10 minutes. The concentration at which each Fab gives less than or equal to 20% of the maximum binding is selected for use in competitive binding assays.

According to another embodiment, Kd is determined using a surface plasmon resonance assay-2000 or-3000(BIAcore, inc., Piscataway, NJ) measured at 25 ℃ using an immobilized antigen CM5 chip at about 10 Response Units (RU). Briefly, carboxymethylated dextran biosensor chips (CM5, BIACORE, Inc.) were activated with N-ethyl-N '- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen was diluted to 5. mu.g/ml (about 0.2. mu.M) with 10mM sodium acetate pH4.8 and then injected at a flow rate of 5. mu.l/min to obtain about 10 Response Units (RU) of conjugated protein. After injection of the antigen, 1M ethanolamine was injected to block unreacted groups. For kinetic measurements, injection at 25 ℃ was performed at a flow rate of about 25. mu.l/min in a solution containing 0.05% polysorbate 20 (TWEEN-20)^TM) Two-fold serial dilutions of Fab (0.78 nM to 500 nM) in surfactant PBS (PBST). Using a simple one-to-one Langmuir (Langmuir) binding model (BIACORE)EvaluationSoftware ionization 3.2) calculate the binding rate (k) by simultaneous fitting of the binding and dissociation sensorgrams_on) And dissociation rate (k)_off). Equilibrium dissociation constant (Kd) in the ratio k_off/k_onAnd (4) calculating. See, e.g., Chen et al, J.mol.biol.293:865-881 (1999). If the binding rate is more than 10 according to the above surface plasmon resonance assay⁶M^-1S^-1The binding rate can then be determined using fluorescence quenching techniques, i.e.according to a spectrometer such as an Avivinstruments spectrophotometer equipped with a flow interrupting device or a 8000 series SLM-AMINCO^TMMeasurement in a stirred cuvette in a spectrophotometer (ThermoSpectronic) measured the increase or decrease in fluorescence emission intensity (excitation =295 nM; emission =340nM, 16nM band pass) of 20nM anti-antigen antibody (Fab form) in pbsph7.2 at 25 ℃ in the presence of increasing concentrations of antigen.

2. Antibody fragments

In certain embodiments, the antibodies provided herein are antibody fragments. Antibody fragments include, but are not limited to, Fab '-SH, F (ab')₂For reviews of certain antibody fragments, see Hudson et al Nat. Med.9:129-134(2003) for reviews of scFv fragments, see for example Pluckth ü n, compiled by the Pharmacology of monoclonal antibodies, Vol.113, Rosenburg and Moore, (Springer-Verlag, New York), pp.269-315 (1994); see also WO93/16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458. for Fab and F (ab') containing salvage receptor binding epitope residues and having an extended in vivo half-life₂See U.S. Pat. No.5,869,046 for a discussion of fragments.

Diabodies are antibody fragments with two antigen binding sites, which may be bivalent or bispecific. See, for example, EP404,097, WO1993/01161, Hudson et al, nat. Med.9: 129-. Tri-and tetrabodies are also described in Hudson et al, nat. Med.9: 129-.

Single domain antibodies are antibody fragments that comprise all or part of the heavy chain variable domain or all or part of the light chain variable domain of the antibody. In certain embodiments, the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Pat. No.6,248,516B1).

Antibody fragments can be generated by a variety of techniques, including but not limited to proteolytic digestion of intact antibodies and production of recombinant host cells (e.g., e.coli or phage), as described herein.

3. Chimeric and humanized antibodies

In certain embodiments, the antibodies provided herein are chimeric antibodies. Certain chimeric antibodies are described, for example, in U.S. Pat. No.4,816,567, and Morrison et al, Proc. Natl. Acad. Sci. USA,81:6851-6855 (1984)). In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region. In yet another example, a chimeric antibody is a "class-switched" antibody in which the class or subclass has been altered from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

In certain embodiments, the chimeric antibody is a humanized antibody. Typically, non-human antibodies are humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parent non-human antibody. Generally, a humanized antibody comprises one or more variable domains in which HVRs, e.g., CDRs (or portions thereof), are derived from a non-human antibody and FRs (or portions thereof) are derived from a human antibody sequence. Optionally, the humanized antibody will also comprise at least a portion of a human constant region. In some embodiments, some FR residues in the humanized antibody are replaced with corresponding residues from a non-human antibody (e.g., an antibody from which HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.

Humanized antibodies and Methods for their production are reviewed, for example, in Almagro and Fransson, Front.biosci.13:1619-1633(2008), and further described, for example, in Riechmann et al, Nature332:323-329(1988), Queen et al, Proc. Nat' lAcad. Sci. USA86:10029-10033(1989), U.S. Pat. Nos. 5,821,337,7,527,791,6,982,321 and 7,087,409, Kashmiri et al, Methods36:25-34(2005) (SDR (a-CDR) grafting is described); padlan, mol.Immunol.28:489-498(1991) (describes "resurfacing"); dall' Acqua et al, Methods36:43-60(2005) (describing "FR shuffling"); and Osbourn et al, Methods36:61-68(2005) and Klimka et al, Br.J. cancer, 83:252-260(2000) (describing the "guided selection" method of FR shuffling).

Human framework regions that may be used for humanization include, but are not limited to: framework regions selected using the "best-fit" method (see, e.g., Sims et al J.Immunol.151:2296 (1993)); framework regions derived from consensus sequences of a specific subset of human antibodies from the light or heavy chain variable regions (see, e.g., Carter et al Proc. Natl. Acad. Sci. USA,89:4285(1992); and Presta et al J.Immunol.,151:2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, front.biosci.13:1619-1633 (2008)); and framework regions derived by screening FR libraries (see, e.g., Baca et al, J.biol.chem.272:10678-10684(1997) and Rosok et al, J.biol.chem.271:22611-22618 (1996)).

4. Human antibodies

In certain embodiments, the antibodies provided herein are human antibodies. Human antibodies can be generated using a variety of techniques known in the art. In general, human antibodies are described in vanDijk and vandeWinkel, curr. opin. pharmacol.5:368-74(2001), and Lonberg, curr. opin. immunol.20: 450-.

Human antibodies can be made by administering an immunogen to a transgenic animal that has been modified to produce fully human antibodies or fully antibodies with human variable regions in response to an antigenic challenge. Such animals typically contain all or part of a human immunoglobulin locus, which replaces an endogenous immunoglobulin locus, or which is extrachromosomal or randomly integrated into the animalIn the chromosome(s) of (c). In such transgenic mice, the endogenous immunoglobulin locus has typically been inactivated. For an overview of the method of obtaining human antibodies from transgenic animals, see Lonberg, nat. Biotech.23:1117-1125 (2005). See also, for example, U.S. Pat. Nos. 6,075,181 and 6,150,584, which describe XENOMOUSE^TMA technique; U.S. Pat. No.5,770,429, which describesA technique; U.S. Pat. No.7,041,870, which describes K-MTechnology, and U.S. patent application publication No. US2007/0061900, which describesA technique). The human variable regions from the whole antibodies generated by such animals may be further modified, for example by combination with different human constant regions.

Human antibodies can also be generated by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described (see, e.g., Kozbor J. Immunol.,133:3001(1984); Brodeur et al, monoclonal antibody production techniques and applications, pp 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al, J. Immunol.,147:86 (1991)). Human antibodies generated via human B-cell hybridoma technology are also described in Li et al, Proc.Natl.Acad.Sci.USA,103:3557-3562 (2006). Other methods include those described, for example, in U.S. Pat. No.7,189,826, which describes the production of monoclonal human IgM antibodies from hybridoma cell lines, and Ni, Xiandai Mianyixue,26(4):265-268(2006), which describes human-human hybridomas. The human hybridoma technique (Trioma technique) is also described in Vollmers and Brandlein, Histologyand Histopapathology, 20(3): 927-.

Human antibodies can also be generated by isolating Fv clone variable domain sequences selected from a human-derived phage display library. Such variable domain sequences can then be combined with the desired human constant domains. Techniques for selecting human antibodies from antibody libraries are described below.

5. Library-derived antibodies

Antibodies of the invention can be isolated by screening combinatorial libraries for antibodies having a desired activity or activities. For example, various methods for generating phage display libraries and screening such libraries for antibodies possessing desired binding characteristics are known in the art. Such methods are reviewed, for example, in Hoogenboom et al, Methodesinmolecular biology178:1-37 (O' Brien et al, eds., HumanPress, Totowa, NJ,2001), and further described, for example, in McCafferty et al, Nature348:552-554, Clackson et al, Nature352:624-628(1991), Marks et al, J.Mol.biol.222:581-597(1992), Marks and Bradbury in MethodesinMolecular biology248:161-175(Lo et al, HumanPress, Totowa, NJ,2003), Sidhu et al, J.mol.338 (2): 299-2004 (2004); Lee et al, J.mol.340 (5):1073 (Acetc. 12419, Nature: 119-72, USA) and in Legend et al, Nature # 72 (USA) in Legend).

In some phage display methods, the repertoire of VH and VL genes, respectively, is cloned by Polymerase Chain Reaction (PCR) and randomly recombined in a phage library, which can then be screened for antigen-binding phages, as described in Winter et al, Ann. Rev. Immunol.,12:433-455 (1994). Phage typically display antibody fragments either as single chain fv (scfv) fragments or as Fab fragments. Libraries from immunized sources provide high affinity antibodies to the immunogen without the need to construct hybridomas. Alternatively, the natural repertoire can be cloned (e.g., from humans) to provide a single source of antibodies to a large panel of non-self and also self-antigens in the absence of any immunization, as described by Griffiths et al, EMBOJ,12: 725-. Finally, non-rearranged V gene segments can also be synthesized by cloning non-rearranged V gene segments from stem cells and using PCR primers containing random sequences to encode the highly variable CDR3 regions and effecting rearrangement in vitro, as described by Hoogenboom and Winter, J.mol.biol.,227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example: U.S. Pat. No.5,750,373 and U.S. patent publication Nos. 2005/0079574,2005/0119455,2005/0266000,2007/0117126,2007/0160598,2007/0237764,2007/0292936 and 2009/0002360.

Antibodies or antibody fragments isolated from a human antibody library are considered to be human antibodies or human antibody fragments herein.

6. Multispecific antibodies

In certain embodiments, the antibodies provided herein are multispecific antibodies, e.g., bispecific antibodies. Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites. In certain embodiments, one of the binding specificities is for LRP6 and the other is for any other antigen. In certain embodiments, the bispecific antibody binds two different epitopes of LRP6. Bispecific antibodies can also be used to localize cytotoxic agents to cells expressing LRP6. Bispecific antibodies can be prepared as full length antibodies or antibody fragments.

Techniques for generating multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy-light chain pairs with different specificities (see Milstein and Cuello, Nature305:537(1983)), WO93/08829 and Traunecker et al, EMBO J.10:3655 (1991)) and "bump-in-hole" engineering (see, e.g., U.S. Pat. No.5,731,168). Effects can also be manipulated electrostatically by engineering for the generation of antibody Fc-heterodimer molecules (WO2009/089004a 1); crosslinking two or more antibodies or fragments (see, e.g., U.S. Pat. No.4,676,980, and Brennan et al, Science,229:81 (1985)); the use of leucine zippers to generate bispecific antibodies (see, e.g., Kostelny et al, J.Immunol.,148(5):1547-1553 (1992)); the "diabody" technique used to generate bispecific antibody fragments is used (see, e.g., Hollinger et al, Proc. Natl. Acad. Sci. USA,90: 6444-; and the use of single chain fv (sFv) dimers (see, e.g., Gruber et al, J.Immunol.,152:5368 (1994)); and making a trispecific antibody to generate a multispecific antibody as described, for example, in Tutt et al J.Immunol.147:60 (1991).

Also included herein are engineered antibodies having three or more functional antigen binding sites, including "octopus antibodies" (see, e.g., US2006/0025576a 1).

Antibodies or fragments herein also include "dual action fabs" or "DAFs" comprising an antigen binding site that binds LRP6 and another, different antigen (see, e.g., US 2008/0069820).

7. Antibody variants

In certain embodiments, amino acid sequence variants of the antibodies provided herein are encompassed. For example, it may be desirable to improve the binding affinity and/or other biological properties of an antibody. Amino acid sequence variants of an antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from and/or insertions into and/or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, so long as the final construct possesses the desired characteristics, e.g., antigen binding.

a)Substitution, insertion and deletion variants

In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include HVRs and FRs. Conservative substitutions are shown in table 1 under the heading "conservative substitutions". More substantial variations are provided in table 1 under the heading of "exemplary substitutions" and are described further below with reference to amino acid side chain classes. Amino acid substitutions can be introduced into the antibody of interest and the product screened for a desired activity, such as retained/improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

TABLE 1

Initial residue	Exemplary substitutions	Preferred alternatives
			Ala(A)	Val;Leu;Ile	Val
Arg(R)	Lys;Gln;Asn	Lys
			Asn(N)	Gln;His;Asp,Lys;Arg	Gln
Asp(D)	Glu;Asn	Glu
			Cys(C)	Ser;Ala	Ser
Gln(Q)	Asn;Glu	Asn
			Glu(E)	Asp;Gln	Asp
Gly(G)	Ala	Ala
			His(H)	Asn;Gln;Lys;Arg	Arg
Ile(I)	Leu, Val, Met, Ala, Phe, norleucine	Leu
			Leu(L)	Norleucine, Ile, Val, Met, Ala, Phe	Ile
Lys(K)	Arg;Gln;Asn	Arg
			Met(M)	Leu;Phe;Ile	Leu
Phe(F)	Trp;Leu;Val;Ile;Ala;Tyr	Tyr
			Pro(P)	Ala	Ala
Ser(S)	Thr	Thr
			Thr(T)	Val;Ser	Ser
Trp(W)	Tyr;Phe	Tyr
			Tyr(Y)	Trp;Phe;Thr;Ser	Phe
Val(V)	Ile, Leu, Met, Phe, Ala, norleucine	Leu

According to common side chain properties, amino acids can be grouped as follows:

(1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral, hydrophilic: cys, Ser, Thr, Asn, Gln;

(3) acidic: asp, Glu;

(4) basic: his, Lys, Arg;

(5) residues that influence chain orientation: gly, Pro;

(6) aromatic: trp, Tyr, Phe.

Non-conservative substitutions may entail replacing one of these classes with a member of the other class.

One class of surrogate variants involves replacing one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variants selected for further study will have an alteration (e.g., an improvement) in certain biological properties (e.g., increased affinity, decreased immunogenicity) relative to the parent antibody and/or will substantially retain certain biological properties of the parent antibody. Exemplary surrogate variants are affinity matured antibodies, which can be conveniently generated, for example, using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more HVR residues are mutated and the variant antibodies are displayed on phage and screened for a particular biological activity (e.g., binding affinity).

Changes (e.g., substitutions) can be made to HVRs, for example, to improve antibody affinity. Such changes can be made to HVR "hot spots", i.e., residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e.g., Chowdhury, methods mol. biol.207: 179. 196 (2008)), and/or SDRs (a-CDRs), where the resulting variant VH or VL is tested for binding affinity. Affinity maturation by construction and re-selection of secondary libraries has been described, for example, in Hoogenboom, et al, methods molecular biology178:1-37 (O' Brien et al, eds., HumanPress, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by a variety of methods (e.g., error-prone PCR, strand shuffling, or oligonucleotide-directed mutagenesis). Then, a secondary library is created. The library is then screened to identify any antibody variants with the desired affinity. Another method of introducing diversity involves an HVR-directed method in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are frequently targeted.

In certain embodiments, substitutions, insertions, or deletions may occur within one or more HVRs, so long as such changes do not substantially reduce the ability of the antibody to bind antigen. For example, conservative changes (e.g., conservative substitutions, as provided herein) may be made to HVRs that do not substantially reduce binding affinity. Such changes may be outside of HVR "hotspots" or SDRs. In certain embodiments of the variant VH and VL sequences provided above, each HVR is unaltered, or contains no more than 1,2, or 3 amino acid substitutions.

One method that can be used to identify residues or regions of an antibody that can be targeted for mutagenesis is referred to as "alanine scanning mutagenesis" as described by Cunningham and Wells (1989) Science,244: 1081-1085. In this method, a residue or group of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) are identified and replaced with a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. Further substitutions may be introduced at amino acid positions that indicate functional sensitivity to the initial substitution. Alternatively or additionally, the crystal structure of the antigen-antibody complex is used to identify the contact points between the antibody and the antigen. As alternative candidates, such contact and adjacent residues may be targeted or eliminated. Variants can be screened to determine if they contain the desired property.

Amino acid sequence insertions include amino and/or carboxy-terminal fusions ranging in length from 1 residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include fusions of the N-or C-terminus of the antibody with an enzyme (e.g., for ADEPT) or a polypeptide that extends the serum half-life of the antibody.

b)Glycosylation variants

In certain embodiments, the antibodies provided herein are altered to increase or decrease the degree of glycosylation of the antibody. Addition or deletion of glycosylation sites of an antibody can be conveniently achieved by altering the amino acid sequence such that one or more glycosylation sites are created or eliminated.

In the case of antibodies comprising an Fc region, the carbohydrate to which they are attached may be altered. Natural antibodies produced by mammalian cells typically comprise branched, bi-antennary oligosaccharides, which are typically N-linked to Asn297 of the CH2 domain attached to the Fc region. See, e.g., Wright et al TIBTECH15:26-32 (1997). Oligosaccharides may include various carbohydrates, for example, mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to GlcNAc in the "backbone" of the bi-antennary oligosaccharide structure. In some embodiments, the oligosaccharides in the antibodies of the invention may be modified to create antibody variants with certain improved properties.

In one embodiment, antibody variants are provided that have a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region. For example, the amount of fucose in such antibodies may be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all sugar structures (e.g. complexed, heterozygous and high mannose structures) attached to Asn297, as measured by MALDI-TOF mass spectrometry, e.g. as described in WO 2008/077546. Asn297 refers to the asparagine residue located at about position 297 in the Fc region (Eu numbering of Fc region residues); however, Asn297 may also be located about ± 3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in the antibody. Such fucosylated variants may have improved ADCC function. See, for example, U.S. patent publication No. US2003/0157108(Presta, L.); US2004/0093621(KyowaHakko Kogyo Co., Ltd.). Examples of publications relating to "defucosylated" or "fucose-deficient" antibody variants include: US2003/0157108, WO2000/61739, WO2001/29246, US2003/0115614, US2002/0164328, US2004/0093621, US2004/0132140, US2004/0110704, US2004/0110282, US2004/0109865, WO2003/085119, WO2003/084570, WO2005/035586, WO2005/035778, WO2005/053742, WO2002/031140, Okazaki et al J.mol.biol.336:1239-1249(2004), Yamane-Ohnuki et al Biotech.Bioeng.87:614 (2004). Examples of cell lines capable of producing defucosylated antibodies include protein fucosylation deficient Lec13CHO cells (Ripka et al Arch. biochem. Biophys.249:533-545(1986); U.S. patent application No. US2003/0157108A1, Presta, L; and WO2004/056312A1, Adams et al, inter alia, in example 11), and knock-out cell lines such as alpha-1, 6-fucosyltransferase gene FUT8 knock-out CHO cells (see, e.g., Yamane-Ohnuki et al Biotech. Bioeng.87:614(2004); Kanda, Y. et al, Biotechnol. Bioeng. 94(4):680-688(2006); and WO 2003/085107).

Further provided are antibody variants having bisected oligosaccharides, for example, wherein biantennary oligosaccharides attached to the Fc region of the antibody are bisected by GlcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described, for example, in WO2003/011878 (Jean-Mairet et al); U.S. Pat. No.6,602,684 (Umana et al); and US2005/0123546 (Umana et al). Antibody variants having at least one galactose residue in an oligosaccharide attached to an Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO1997/30087 (Patel et al); WO1998/58964(Raju, S.); and WO1999/22764(Raju, S.).

c)Fc region variants

In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4Fc region) comprising an amino acid modification (e.g., substitution) at one or more amino acid positions.

In certain embodiments, the invention encompasses antibody variants possessing some, but not all, effector functions that make them desirable candidates for applications where the in vivo half-life of the antibody is important, while certain effector functions (such as complement and ADCC) are unnecessary or detrimental. In vitro and/or in vivo cytotoxicity assays may be performed to confirm the reduction/depletion of CDC and/or ADCC activity. For example, Fc receptor (FcR) binding assays may be performed to ensure that the antibody lacks fcyr binding (and therefore potentially lacks ADCC activity), but retains FcRn binding ability. The major cells mediating ADCC, NK cells, express Fc γ RIII only, whereas monocytes express Fc γ RI, Fc γ RII and Fc γ RIII. In ravatch and Kinet, AnFcR expression on hematopoietic cells is summarized in Table 3 on page 464 of nu.Rev.Immunol.9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of molecules of interest are described in U.S. Pat. No.5,500,362 (see, e.g., Hellstrom, I.e., Proc. Nat' l Acad. Sci. USA83: 7059-. Alternatively, non-radioactive assay methods can be employed (see, e.g., ACTI for flow cytometry)^TMNon-radioactive cytotoxicity assays (Celltechnology, Inc. mountain View, CA; and CytoTox)Non-radioactive cytotoxicity assay (Promega, Madison, WI)). Useful effector cells for such assays include Peripheral Blood Mononuclear Cells (PBMC) and Natural Killer (NK) cells. Alternatively/additionally, the ADCC activity of a molecule of interest can be assessed in vivo, for example in an animal model such as that disclosed in Clynes et al Proc. nat' lAcad. Sci. USA95: 652-. A C1q binding assay may also be performed to confirm that the antibody is unable to bind C1q, and therefore lacks CDC activity. See, e.g., the C1q and C3C binding ELISAs in WO2006/029879 and WO 2005/100402. To assess complement activation, CDC assays can be performed (see, e.g., Gazzano-Santoro et al, J.Immunol. methods202:163(1996); Cragg, M.S. et al, Blood101: 1045-. FcRn binding and in vivo clearance/half-life assays can also be performed using methods known in the art (see, e.g., Petkova, s.b. et al, Int' l.immunol.18(12): 1759-.

Antibodies with reduced effector function include those having substitutions in one or more of residues 238,265,269,270,297,327 and 329 of the Fc region (U.S. Pat. No.6,737,056). Such Fc mutants include Fc mutants having substitutions at two or more of amino acid positions 265,269,270,297 and 327, including so-called "DANA" Fc mutants having substitutions of residues 265 and 297 to alanine (U.S. Pat. No.7,332,581).

Certain antibody variants with improved or reduced binding to FcR are described (see, e.g., U.S. Pat. No.6,737,056; WO2004/056312, and Shields et al, J.biol. chem.9(2):6591-6604 (2001)).

In certain embodiments, an antibody variant comprises an Fc region with one or more amino acid substitutions that improve ADCC, e.g., substitutions at positions 298, 333, and/or 334 of the Fc region (EU numbering of residues).

In some embodiments, alterations are made to the Fc region that result in altered (i.e., improved or reduced) C1q binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat. No.6,194,551, WO99/51642, and Idusogene et al J.Immunol.164: 4178-.

Antibodies with extended half-life and improved binding to neonatal Fc receptor (FcRn) responsible for the transfer of maternal IgG to the fetus are described in US2005/0014934A1(Hinton et al), the neonatal Fc receptor (FcRn) and are responsible for the transfer of maternal IgG to the fetus (Guyer et al, J.Immunol.117:587(1976) and Kim et al, J.Immunol.24:249 (1994)). Those antibodies comprise an Fc region having one or more substitutions therein that improve the binding of the Fc region to FcRn. Such Fc variants include those having substitutions at one or more of residues 238,256,265,272,286,303,305,307,311,312,317,340,356,360,362,376,378,380,382,413,424 or 434 of the Fc region, for example, at residue 434 of the Fc region (U.S. patent No.7,371,826).

See also Duncan and Winter, Nature322:738-40(1988), U.S. Pat. No.5,648,260, U.S. Pat. No.5,624,821, and WO94/29351, which focus on other examples of Fc region variants.

d)Cysteine engineered antibody variants

In certain embodiments, it may be desirable to create cysteine engineered antibodies, e.g., "thiomabs," in which one or more residues of the antibody are replaced with cysteine residues. In particular embodiments, the substituted residues are present at accessible sites of the antibody. By replacing those residues with cysteine, the reactive thiol groups are thus localized at accessible sites of the antibody and can be used to conjugate the antibody with other moieties, such as drug moieties or linker-drug moieties, to create immunoconjugates, as further described herein. In certain embodiments, cysteine may be substituted for any one or more of the following residues: v205 of the light chain (Kabat numbering); a118 of the heavy chain (EU numbering); and S400 of the heavy chain Fc region (EU numbering). Cysteine engineered antibodies can be produced as described, for example, in U.S. patent No.7,521,541.

e)Antibody derivatives

In certain embodiments, the antibodies provided herein can be further modified to contain additional non-proteinaceous moieties known in the art and readily available. Suitable moieties for derivatization of the antibody include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3-dioxolane, poly-1, 3, 6-trisAlkanes, ethylene/maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers) and dextran or poly (n-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, propylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in production due to its stability in water. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody can vary, and if more than one polymer is attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the specific properties or functions of the antibody to be improved, whether the antibody derivative will be used for therapy under specified conditions, and the like.

In another embodiment, conjugates of an antibody and a non-proteinaceous moiety that can be selectively heated by exposure to radiation are provided. In one embodiment, the non-proteinaceous moiety is a carbon nanotube (Kam et al, Proc. Natl. Acad. Sci. USA102: 11600-. The radiation can be of any wavelength and includes, but is not limited to, wavelengths that are not damaging to normal cells, but heat the non-proteinaceous moiety to a temperature at which cells in the vicinity of the antibody-non-proteinaceous moiety are killed.

B. Recombinant methods and compositions

Recombinant methods and compositions can be used to generate antibodies, for example, as described in U.S. Pat. No.4,816,567. In one embodiment, isolated nucleic acids encoding an anti-LRP 6 antibody described herein are provided. Such nucleic acids may encode an amino acid sequence comprising a VL of an antibody and/or an amino acid sequence comprising a VH (e.g., a light and/or heavy chain of an antibody). In yet another embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. In yet another embodiment, host cells comprising such nucleic acids are provided. In one such embodiment, the host cell comprises (e.g., has been transformed with): (1) a vector comprising nucleic acids encoding an amino acid sequence comprising a VL of an antibody and an amino acid sequence comprising a VH of an antibody, or (2) a first vector comprising nucleic acids encoding an amino acid sequence comprising a VL of an antibody and a second vector comprising nucleic acids encoding an amino acid sequence comprising a VH of an antibody. In one embodiment, the host cell is eukaryotic, such as a Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., Y0, NS0, Sp20 cell). In one embodiment, a method of producing an anti-LRP 6 antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody under conditions suitable for expression of the antibody, as provided above, and optionally, recovering the antibody from the host cell (or host cell broth).

For recombinant production of an anti-LRP 6 antibody, a nucleic acid encoding the antibody (e.g., as described above) is isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of an antibody).

Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector function are not required. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat. Nos. 5,648,237,5,789,199 and 5,840,523 (see also Charlton, methods molecular biology, Vol.248 (compiled by B.K.C.Lo., HumanaPress, Totowa, NJ,2003), pp.245-254, which describes expression of antibody fragments in E.coli (E.coli)). After expression, the antibody can be isolated from the bacterial cell cytoplasm in a soluble fraction and can be further purified.

In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungi and yeast strains whose glycosylation pathways have been "humanized" resulting in the production of antibodies with partially or fully human glycosylation patterns. See Gerngross, nat. Biotech.22: 1409-.

Host cells suitable for expression of glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. A number of baculovirus strains have been identified which can be used with insect cells, particularly for transfecting spodoptera frugiperda (spodoptera frugiperda) cells.

Plant cell cultures may also be used as hosts. See, for example, U.S. Pat. Nos. 5,959,177,6,040,498,6,420,548,7,125,978 and 6,417,429 (which describe the PLANTIBODIIES technique for producing antibodies in transgenic plants).

Vertebrate cells can also be used as hosts. For example, mammalian cell lines suitable for growth in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed with SV40 (COS-7); human embryonic kidney lines (293 or 293 cells as described, e.g., in Graham et al, J.GenVirol.36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli (sertoli) cells (TM 4 cells, as described, for example, in Mather, biol. reprod.23:243-251 (1980)); monkey kidney cells (CV 1); VERO cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK; bovine mouse (buffaloat) hepatocytes (BRL3A), human lung cells (W138), human hepatocytes (HepG2), mouse mammary tumors (MMT060562), TRI cells, as described, for example, in Mather et al, Annals N.Y.Acad.Sci.383:44-68(1982), MRC5 cells, and FS4 cells other useful mammalian host cell lines include Chinese Hamster Ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al, Proc. Natl.Acad.Sci.USA77:4216(1980)), and myeloma cell lines such as Y0, NS0, and Sp 2/0. for reviews of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methodsida, biology, Vol.K.C.248, Huako.255, Prewawa, 2003, pp.2003 (Towa) Towa 268).

C. Assay method

The anti-LRP 6 antibodies provided herein can be identified, screened, or characterized for their physical/chemical properties and/or biological activities by a variety of assays known in the art.

1. Binding assays and other assays

In one aspect, antibodies of the invention are tested for antigen binding activity, for example, by known methods such as ELISA, Western blot, and the like.

On the other hand, competition assays can be used to identify antibodies that compete with the anti-LRP 6 antibody of the invention for binding to LRP6. In certain embodiments, such competitive antibodies bind to the same epitope (e.g., a linear or conformational epitope) as the epitope bound by the anti-LRP 6 antibody of the invention. A detailed exemplary method for locating epitopes bound by antibodies is described in Morris (1996) "epitome mapping protocols", Methodsinmolecular biology vol.66(HumanaPress, Totowa, N.J.).

In one exemplary competition assay, immobilized LRP6 is incubated in a solution comprising a first labeled antibody that binds to LRP6 and a second unlabeled antibody to be tested for the ability to compete with the first antibody for binding to LRP6. The second antibody may be present in the hybridoma supernatant. As a control, immobilized LRP6 was incubated in a solution containing the first labeled antibody but no second unlabeled antibody. After incubation under conditions that allow the primary antibody to bind to LRP6, excess unbound antibody is removed and the amount of label associated with immobilized LRP6 is measured. If the amount of marker associated with immobilized LRP6 in the test sample is substantially reduced compared to the control sample, this indicates that the second antibody competes with the first antibody for binding to LRP6. See Harlowland (1988) Antibodies, ALaboratoryManuatch.14 (ColdSpringHarbor laboratory, ColdSpringHarbor, NY).

2. Activity assay

In one aspect, assays for identifying anti-LRP 6 antibodies having biological activity are provided. Biological activities may include, for example, inhibiting or potentiating Wnt isoform-mediated signaling, modulating bone mass/content, inhibiting cell proliferation, increasing cell proliferation. Antibodies having such biological activity in vivo and/or in vitro are also provided.

In certain embodiments, antibodies of the invention are tested for such biological activity. Specific assays for testing biological activity are provided in the examples.

D. Immunoconjugates

The invention also provides immunoconjugates comprising the anti-LRP 6 antibodies herein conjugated to one or more cytotoxic agents, such as a chemotherapeutic agent or drug, a growth inhibitory agent, a toxin (e.g., a protein toxin, an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or a fragment thereof), or a radioisotope.

In one embodiment, the immunoconjugate is an antibody-drug conjugate (ADC) in which the antibody is conjugated to one or more drugs, including but not limited to maytansinoids (see U.S. Pat. nos. 5,208,020, 5,416,064, and european patent EP0425235B 1); auristatins such as monomethyl auristatin drug modules DE and DF (MMAE and MMAF) (see U.S. Pat. nos. 5,635,483 and 5,780,588 and 7,498,298); dolastatin (dolastatin); calicheamicin (calicheamicin) or a derivative thereof (see U.S. Pat. Nos. 5,712,374,5,714,586,5,739,116,5,767,285,5,770,701,5,770,710,5,773,001 and 5,877,296; Hinman et al, cancer Res.53: 3336-; anthracyclines such as daunomycin (daunomycin) or doxorubicin (doxorubicin) (see Kratz et al, Current Med. chem.13: 477-; methotrexate; vindesine (vindesine); taxanes (taxanes) such as docetaxel (docetaxel), paclitaxel, larotaxel, tesetaxel and ortataxel; trichothecenes (trichothecenes); and CC 1065.

In another embodiment, the immunoconjugate comprises an antibody as described herein conjugated to an enzymatically active toxin or fragment thereof, the enzymatically active toxins include, but are not limited to diphtheria a chain, non-binding active fragments of diphtheria toxin, exotoxin a chain (from pseudomonas aeruginosa), ricin a chain, abrin a chain, modeccin a chain, α -sarcin (sarcin), aleurites fordii (aleutis fordii) toxic protein, dianthus chinensis (dianthin) toxic protein, phytolacca americana (phytolacacaricanaria) protein (PAPI, PAPII and PAP), momordica charantia (momocarratia) inhibitor, curculin (curcin), crotin (crotin), saponaria officinalis (sapaonaria officinalis) inhibitor, gelonin (gelonin), mitomycin (mitrellin), tricin (curcin), curculin (trichothecin), enomycin (trichothecin), and trichothecin (trichothecin).

In another embodiment, the immunoconjugate comprises an antibody as described herein conjugated to a radioactive atom to form a radioconjugate. A variety of radioisotopes are available for use in generating radioconjugates. Examples include At²¹¹、I¹³¹、I¹²⁵、Y⁹⁰、Re¹⁸⁶、Re¹⁸⁸、Sm¹⁵³、Bi²¹²、P³²、Pb²¹²And radioactive isotopes of Lu. Where a radioconjugate is used for detection, it may contain a radioactive atom for scintigraphic studies, for example tc99m or I123, or a spin label for Nuclear Magnetic Resonance (NMR) imaging (also known as magnetic resonance imaging, mri), such as again iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

A variety of bifunctional protein coupling agents may be used to generate conjugates of the antibody and cytotoxic agent, such as N-succinimidyl 3- (2-pyridyldithio) propionate (SPDP), succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), Iminothiolane (IT), 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 bis (p-diazoniumbenzoyl) -ethylenediamine), diisothiocyanates (such as toluene 2, 6-diisocyanate), and bis-active fluorine compounds (such as 1, 5-difluoro-2, 4-dinitrobenzene) is used. For example, a ricin immunotoxin may be prepared as described in Vitetta et al, Science238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelator for conjugating radionucleotides to antibodies. See WO 94/11026. The linker may be a "cleavable linker" that facilitates release of the cytotoxic drug in the cell. For example, acid-labile linkers, peptidase-sensitive linkers, photolabile linkers, dimethyl linkers, or disulfide-containing linkers can be used (Chari et al, cancer Res52: 127-.

Immunoconjugates or ADCs herein expressly encompass, but are not limited to, such conjugates prepared with crosslinking agents including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl- (4-vinylsulfone) benzoate), which are commercially available (e.g., from pierce biotechnology, inc., Rockford, il., u.s.a.).

E. Methods and compositions for diagnosis and detection

In certain embodiments, any of the anti-LRP 6 antibodies provided herein can be used to detect the presence of LRP6 in a biological sample. As used herein, the term "detecting" encompasses quantitative or qualitative detection. In certain embodiments, the biological sample comprises a cell or tissue.

In one embodiment, anti-LRP 6 antibodies for use in diagnostic or detection methods are provided. In yet another aspect, methods of detecting the presence of LRP6 in a biological sample are provided. In certain embodiments, the methods comprise contacting the biological sample with an anti-LRP 6 antibody under conditions that allow the anti-LRP 6 antibody to bind to LRP6, as described herein, and detecting whether a complex is formed between the anti-LRP 6 antibody and LRP6. Such methods may be in vitro or in vivo. In one embodiment, an anti-LRP 6 antibody is used to select a subject suitable for treatment with an anti-LRP 6 antibody, e.g., wherein LRP6 is a biomarker for selecting patients.

Exemplary disorders that can be diagnosed using the antibodies of the invention include cancer and disorders of the skeletal system.

At a certain pointIn some embodiments, labeled anti-LRP 6 antibodies are provided. Labels include, but are not limited to, labels or moieties that are directly detectable (such as fluorescent, chromogenic, electron-dense, chemiluminescent, and radioactive labels), and moieties that are indirectly detectable, such as enzymes or ligands, for example, via enzymatic reactions or molecular interactions. Exemplary labels include, but are not limited to, radioisotopes³²P、¹⁴C、¹²⁵I、³H and¹³¹I. fluorophores such as rare earth chelates or luciferin and derivatives thereof, rhodamine (rhodamine) and derivatives thereof, dansyl, umbelliferone, luciferases, e.g., firefly and bacterial luciferases (U.S. Pat. No.4,737,456), luciferin, 2, 3-dihydrophthalazinedione, horseradish peroxidase (HRP), alkaline phosphatase, β -galactosidase, glucoamylase, lysozyme, carbohydrate oxidase, e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase (which are coupled to an enzyme employing a hydrogen peroxide oxidation dye precursor such as HRP), lactoperoxidase, or microperoxidase, biotin/avidin, spin labels, phage labels, stable free radicals, and the like.

F. Pharmaceutical formulations

Pharmaceutical formulations of the anti-LRP 6 antibody as described herein are prepared by mixing such antibodies of the desired purity with one or more optional pharmaceutically acceptable carriers (Remington's pharmaceutical sciences 16 th edition, Osol, a. eds. (1980)) in a lyophilized formulation or as an aqueous solution. Generally, pharmaceutically acceptable carriers are nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexane diamine chloride; benzalkonium chloride, benzethonium chloride; phenol, butanol or benzyl alcohol; hydrocarbyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low scoreA quantum (less than about 10 residues) polypeptide; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and/or a non-ionic surfactant, such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further comprise an interstitial drug dispersant such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), e.g., human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 (r: (r) ())Baxter International, Inc.). Certain exemplary shasegps and methods of use, including rHuPH20, are described in U.S. patent publication nos. 2005/0260186 and 2006/0104968. In one aspect, the sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinase.

An exemplary lyophilized antibody formulation is described in U.S. Pat. No.6,267,958. Aqueous antibody formulations include those described in U.S. Pat. No.6,171,586 and WO2006/044908, the latter formulation comprising a histidine-acetate buffer.

The formulations herein may also contain more than one active ingredient necessary for the particular indication being treated, preferably those compounds whose activities are complementary and do not adversely affect each other. Such active components are suitably present in combination in amounts effective for the desired purpose.

The active ingredient may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (e.g., hydroxymethylcellulose or gelatin-microcapsules and poly (methylmethacylate) microcapsules, respectively), in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or in macroemulsions. Such techniques are disclosed, for example, in Remington's pharmaceutical sciences, 16 th edition, Osol, A. eds (1980).

Sustained release formulations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules.

Formulations for in vivo administration are generally sterile. Sterility can be readily achieved, for example, by filtration through sterile filtration membranes.

G. Therapeutic methods and compositions

Any of the anti-LRP 6 antibodies provided herein can be used in therapeutic methods.

In one aspect, an anti-LRP 6 antibody for use as a medicament is provided. In other aspects, anti-LRP 6 antibodies for use in treating Wnt-mediated disorders, such as cancer or skeletal or bone disorders, are provided. In certain embodiments, anti-LRP 6 antibodies for use in methods of treatment are provided. In certain embodiments, the invention provides an anti-LRP 6 antibody for use in a method of treating an individual having cancer or a bone or bone disorder, the method comprising administering to the individual an effective amount of an anti-LRP 6 antibody. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g., as described below. In other embodiments, the invention provides anti-LRP 6 antibodies for use in inhibiting signaling induced by a first Wnt isoform and potentiating signaling induced by a second Wnt isoform. In certain embodiments, the invention provides an anti-LRP 6 antibody for use in a method of inhibiting signaling induced by a first Wnt isoform and potentiating signaling induced by a second Wnt isoform in an individual, the method comprising administering to the individual an effective amount of an anti-LRP 6 antibody to inhibit signaling induced by the first Wnt isoform and potentiate signaling induced by the second Wnt isoform. An "individual" according to any of the above embodiments is preferably a human.

In a further aspect, the invention provides the use of an anti-LRP 6 antibody in the manufacture or manufacture of a medicament. In one embodiment, the medicament is for treating a Wnt-mediated disorder, such as cancer or a skeletal or bone disorder. In yet another embodiment, the medicament is for use in a method of treating a Wnt-mediated disorder, such as cancer or a skeletal or bone disorder, the method comprising administering to an individual having a Wnt-mediated disorder an effective amount of the medicament. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g., as described below. An "individual" according to any of the above embodiments may be a human.

In yet another aspect, the invention provides methods of treating Wnt-mediated disorders, such as cancer or skeletal or bone disorders. In one embodiment, the method comprises administering to an individual having such a Wnt-mediated disorder an effective amount of an anti-LRP 6 antibody. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, as described below. An "individual" according to any of the above embodiments may be a human.

In one embodiment, the Wnt-mediated disorder is a cancer, such as, for example, non-small cell lung cancer, breast cancer, pancreatic cancer, ovarian cancer, renal cancer, or prostate cancer. In another embodiment, the Wnt-mediated disorder is a skeletal or bone disorder, such as, for example, osteoporosis, osteoarthritis, a bone fracture, or a bone injury.

One embodiment provides a method of treating an individual having cancer comprising administering to the individual an effective amount of an antibody that binds to LRP6 and inhibits signaling induced by a Wnt isoform selected from the group consisting of Wnt3 and Wnt3a and an antibody that binds to LRP6 and inhibits signaling induced by a Wnt isoform selected from the group consisting of Wnt1, 2b, 6, 8a, 9b, and 10 b. Another embodiment provides a method of treating an individual having cancer comprising administering to the individual an effective amount of an antibody that binds to LRP6 and inhibits signaling induced by Wnt3 and Wnt3a and an antibody that binds to LRP6 and inhibits signaling induced by Wnt1, 2b, 6, 8a, 9b, and 10 b. Another embodiment provides a method of treating an individual having cancer comprising administering to the individual an effective amount of an antibody that binds to LRP6 and inhibits signaling induced by Wnt3 and Wnt3a and an antibody that binds to LRP6 and inhibits signaling induced by Wnt1, 2b, 4,6, 7a, 7b, 8a, 9b, 10a, and 10 b.

In yet another aspect, the invention provides a method for potentiating Wnt signaling induced by a Wnt isoform in an individual comprising administering to the individual an effective amount of an anti-LRP 6 antibody potentiating signaling induced by the Wnt isoform and the Wnt isoform to potentiate Wnt signaling induced by the Wnt isoform.

In yet another aspect, the invention provides a pharmaceutical formulation comprising any of the anti-LRP 6 antibodies provided herein, e.g., for use in any of the above-described therapeutic methods. In one embodiment, the pharmaceutical formulation comprises any of the anti-LRP 6 antibodies provided herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical formulation comprises any of the anti-LRP 6 antibodies provided herein and at least one additional therapeutic agent, e.g., as described below.

The antibodies of the invention may be used alone or in combination with other agents in therapy. For example, an antibody of the invention can be co-administered with at least one additional therapeutic agent. In certain embodiments, the additional therapeutic agent is a chemotherapeutic agent. In another embodiment, the agent is an antibody effective for treating cancer or treating bone or a bone disorder. Such combination therapies noted above encompass combined administration (where two or more therapeutic agents are contained in the same or different formulations), and separate administration, in which case the antibody of the invention may be administered prior to, concurrently with, and/or after administration of the other therapeutic agent and/or adjuvant. The antibodies of the invention may also be used in combination with radiotherapy.

The antibodies (and any other therapeutic agent) of the invention may be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing may be by any suitable route, for example by injection, such as intravenous or subcutaneous injection, depending in part on whether administration is transient or chronic. Various dosing schedules are contemplated herein, including but not limited to a single administration or multiple administrations over multiple time points, bolus administration, and pulse infusion.

The antibodies of the invention should be formulated, dosed and administered in a manner consistent with good medical practice. Factors to be considered in this regard include the particular condition being treated, the particular mammal being treated, the clinical status of the individual patient, the cause, the site of drug delivery, the method of administration, the schedule of administration, and other factors known to practitioners. The antibody need not be, but may optionally be, formulated with one or more drugs currently used to prevent or treat the condition. The effective amount of such other drugs will depend on the amount of antibody present in the formulation, the type of condition being treated, and other factors discussed above. These agents are generally used at the same dosage and with the administration routes described herein, or at about 1-99% of the dosages described herein, or at any dosage and by any route, as empirically determined/clinically determined appropriate.

For the prevention or treatment of disease, the appropriate dosage of the antibody of the invention (when used alone or in combination with one or more other therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, the prophylactic or therapeutic purpose for which the antibody is administered, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician. The antibody is suitable for administration to a patient in one or a series of treatments. Depending on the type and severity of the disease, about 1. mu.g/kg to 15mg/kg (e.g., 0.1mg/kg to 10mg/kg) of the antibody may be administered to the patient as a first candidate amount, whether, for example, by one or more separate administrations, or by continuous infusion. A typical daily dose may range from about 1. mu.g/kg to 100mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment should generally be continued until the desired suppression of the condition occurs. An exemplary dose of antibody will be about 0.05mg/kg to about 10 mg/kg. Thus, one or more doses of about 0.5mg/kg, 2.0mg/kg, 4.0mg/kg, or10 mg/kg (or any combination thereof) may be administered to a patient. The above-described dose can be administered intermittently, such as once per week or once every three weeks (e.g., such that the patient receives from about 2 to about 20, or, for example, about 6 doses of antibody). An initial higher loading dose may be administered followed by one or more lower doses. The progress of the treatment is readily monitored by conventional techniques and assays.

It is understood that any of the above-described formulations or therapeutic methods may be practiced using the immunoconjugates of the invention in place of or in addition to an anti-LRP 6 antibody.

H. Article of manufacture

In another aspect of the invention, an article of manufacture is provided that contains materials useful for the treatment, prevention and/or diagnosis of the conditions described above. The article comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. The container may be formed from a variety of materials such as glass or plastic. The container contains a composition effective, alone or in combination with another composition, in the treatment, prevention, and/or diagnosis of a condition, and may have a sterile access port (e.g., the container may be a vial or intravenous solution bag having a stopper penetrable by a hypodermic needle). At least one active agent in the composition is an antibody of the invention. The label or package insert indicates the use of the composition to treat the selected condition. In addition, the article of manufacture can comprise (a) a first container having a composition contained therein, wherein the composition comprises an antibody of the invention; and (b) a second container having a composition contained therein, wherein the composition comprises an additional cytotoxic or otherwise therapeutic agent. The article of manufacture in this embodiment of the invention may further comprise a package insert indicating that the composition may be used to treat a particular condition. Alternatively, or in addition, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution, and dextrose solution. It may further comprise other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles and syringes.

It will be appreciated that any of the above-described preparations may include an immunoconjugate of the invention in place of or in addition to the anti-LRP 6 antibody.

Example III

The following are examples of the methods and compositions of the present invention. It is to be understood that various other embodiments may be implemented in view of the general description provided above.

Example 1

Experimental protocols

Cell culture and cell assay

Cell lines EKVX and M14 were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and 2mM glutamine; JHH-1 cells were cultured in Williams's Medium E with the same supplements. All other cell lines were obtained from the American Type Culture Collection (ATCC) and maintained as recommended.

Cells were transfected with FuGENE6 transfection reagent (Roche) in 24-well plates according to the manufacturer's recommendations. For luciferase reporter assays, a mixture of expression plasmid DNA was transfected: 7.5ngTOPglow (upstate) or TOPbrite (Zhang et al, 2009) firefly luciferase Wnt reporter, 0.5ngpRL-SV40Renilla luciferase (Promega), and 1ngLEF 1. Cells were treated with antibody for 16-20h, starting 24h after transfection. Wnt3a protein (purified according to X, or purchased from R & DSystems) was added to the cells, starting 1h after antibody treatment was initiated. Cells were harvested in 150ul lysis buffer (DeAlmeida et al, 2007) and luminescence was measured on 30-50ul lysates using the dual Glo luciferase system (Promega) and envisionMultilabeReader (PerkinElmer). For transfection efficiency, firefly luciferase levels were normalized to Renilla luciferase levels, and Relative Luciferase Units (RLU) were additionally normalized to levels in cells not stimulated with Wnt3 a.

HEK293 and Hs578T cell lines stably integrated with the TOPbrite reporter were selected for hygromycin resistance. Expression of Wnt luciferase reporter was normalized to cell number based on stably integrated SV 40-driven Renilla luciferase (for HEK293 cells) or based on MultiTox-Fluor cell viability assay (Promega) (for Hs578T cells).

Wnt chimeric constructs were generated by cloning full-length Wnt1 or Wnt3a upstream of full-length FZD4, FZD5, or LRP6 in pRK5 expression vectors. A linker of 24 amino acids (GGGSGGGT)₃Inserted between Wnt and FZD or LRP6 sequences (Cong et al, 2004).

One-armed YW211.31antibody variants were generated in E.coli by co-expression of the YW211.31.62 heavy and light chains and a truncated Fc domain using the "node-in-hole" engineering technique (Ridgway, J.B.B.et al, protein engineering9:617-621 (1996).)₂Fragments (Sigma-Aldrich) were incubated with one arm of YW211.31antibody for 1h, after which the mixture was added to the cells.

For Western analysis, 1.2X10⁶One HEK293 cell was seeded onto a 10-cm dish and 3 days later with 10. mu.g/ml antibody, or X. mu.g/ml DKK1 (R)&DSystems) or Fzd8CRD-Fc (DeAlmeida et al, 2007) protein treatment for 1h, followed by addition of 0.2ug/ml Wnt3a protein, further treatment for 1h cells were washed twice with cold PBS and lysed on ice in 0.5ml lysis buffer 20. mu.g of protein was electrophoretically resolved on a denaturing SDS-polyacrylamide gel (4-12%), transferred to nitrocellulose membrane, and treated with a standard for phospho-and total-LRP 6 (CellSignaling technology), β -catenin (BDTransduction)Antibodies to onLaboratories), β -actin and GAPDH proteins were visualized using an infrared labeled secondary antibody (RocklandImmunochogicals) and an Odyssey imager (LI-COR).

For quantitative real-time PCR (qpcr) expression analysis, RNA was isolated from cells using RNeasy kit (QIAGEN) and the reaction was performed on a 7900HT fast real-time PCR system (applied biosystems) using TaqMan one-step RT-PCR master-grade mixed reagent kit (applied biosystems). Relative RNA levels were calculated using the Δ Δ Ct method and normalized to human GAPDH or mouse Rpl19RNA levels within the same sample and additionally normalized to samples from cells without added (NA) Wnt3a, antibodies, or other proteins. The primer and probe sets (listed 5 'to 3' for forward primer, reverse primer and probe sequences) were SP 5: AATGCTGCTGAACTGAATAGAAA (SEQ ID NO: 32), AACCGGTCCTAGCGAAAA (SEQ ID NO: 33), CCGAGCACTGTTTCAAATCTCCCA (SEQ ID NO: 34); ZNRF 3: TGAGAGTGTGACATTGTTGGAA (SEQ ID NO: 35), GTAAAATCTGTGTGCAATTATCATGT (SEQ ID NO: 36), AATCATTGAAAATGACTAACACAAGACCCTGTAAAT (SEQ ID NO: 37); mouse Mmp 7: TGAGGACGCAGGAGTGAA (SEQ ID NO: 38), CCCAGAGAGTGGCCAAAT (SEQ ID NO: 39), CCTGTTTGCTGCCACCCATGA (SEQ ID NO: 40).

Primers and probes for human APCDD1, axin2, GAD1, LEFTY2 and SAX1, and for mouse Rpl19 and axin2, respectively, were previously described (DeAlmeida et al, 2007; Liu et al, 2010).

GAPDH primers and probes were purchased from applied biosystems. For reporter genes and qPCR assays, all values represent the mean and standard deviation of triplicate or quadruplicate experimental replicates.

LRP6 antibody screening and affinity maturation

The coding regions E1-E2 (amino acids A19-R644 of SEQ ID NO: 29) and E3-E4 (amino acids V629-G1244 of SEQ ID NO: 29) of the human LRP6cDNA fragment were cloned separately into a mammalian expression vector containing the HSV signal sequence and the human IgGFc region as protein tags (SEQ ID NO: 30 (E1-E2-fc); SEQ ID NO: 31 (E3-E4-fc)). The LRP6.E1-E2-Fc and LRP6.E3-E4-Fc proteins were expressed in CHO cells by transient transfection and purified by protein A/G affinity chromatography. Human synthetic Fab phage display libraries were also screened using LRP6.E1-E2-Fc and LRP6.E3-E4-Fc proteins, respectively. After selection on immobilized LRP6 protein, phage clones were isolated and binding to LRP6-Fc fusion protein fragment, but not Fc protein, was confirmed by phage ELISA. Phage Fab clones were then reformatted for expression as human IgG1 monoclonal antibodies. 24 unique antibody heavy chain clones directed to LRP6.E1-E2-Fc and 22 clones directed to LRP6.E3-E4-Fc were transfected and transiently expressed in HEK293 cells with common Herceptin derived human kappa light chain and IgG proteins were purified by affinity chromatography. Subsequent large-scale antibody preparations were generated by transient transfection in CHO cells.

Affinity maturation of the YW211.31antibody was performed using His-tagged LRP6.E3-E4 protein. Randomization was performed by soft randomization of selected residues to target three different combinations of CDR loops (H1/L3, H2/L3, and H3/L3) in separate libraries. In addition, targeting the L1/L2/L3CDR combination for hard randomization. In a first round of selection, phages were selected from the randomized library with immobilized lrp6.e3-4-His protein, followed by five rounds of solution phase sorting, in which the concentration of lrp6.e3-4-His was gradually decreased from 300nM to 0.5nM, and a 100-fold excess of lrp6.e3-4-Fc protein was added to deplete antibodies with faster off-rates. 11 phage clones were purified and all showed improved affinity for lrp3.E3-E4 as determined by phage competition ELISA. The sequences of these clones displayed 1 to 6 amino acid changes in CDR-H1, CDR-H3, and CDR-L3. The dissociation rate constants of the purified antibodies were evaluated by surface plasmon resonance analysis using a BIAcore instrument.

Biolayer interference LRP6 protein binding assay

Biolayer interferometry is performed as previously described (Bourhis et al, 2010). Briefly, biotinylated, His-tagged LRP6 protein was purified from baculovirus-infected insect cells using the AviTag system (GeneCopoeia). Binding kinetics were measured on the OctetRED system (ForteBio) using a streptavidin high binding FA sensor loaded with 20 μ g/ml lrp6 protein. Purified human Wnt3a and mouse Wnt9b were obtained from R & DSystems without vector and yielded purified DKK1 protein as previously described (Bourhis et al, 2010).

Tumor and bone Studies

Tumors from MMTV-Wnt1 transgenic mice were passaged in the mammary fat pad of C57BL/6 mice, dissociated mechanically and enzymatically, resuspended in Matrigel and Hank's Balanced Salt Solution (HBSS), and injected into the mammary fat pad of athymic NCr nude mice (Taconnic). Once the tumor volume reaches 250-800mm³The process is initiated. For each treatment group, ten mice were administered 30mg/kg of antibody or protein Intraperitoneally (IP) every two days. Tumor volume was analyzed using caliper measurements.

Ntera-2 xenograft tumor growth and in vivo studies were performed as previously described (DeAlmeida et al, 2007). Briefly, NU/NU athymic nude mice (Charles river) were injected subcutaneously with 1 million Ntera-2 cells per mouse (in 50% Matrigel in HBSS), divided into groups of four or five animals (once the mean tumor volume reached 535-595 mm)³) And a dose of IP100mg/kg antibody or 30mg/kg Fzd8CRD-Fc protein was injected. Tumor and serum samples were collected 16h after treatment. Tumors were homogenized using the TissueLyser system (QIAGEN) and RNA was extracted using RNeasy kit (QIAGEN).

Calvaria were harvested and cultured as described by Mohammad et al, 2008. Briefly, calvaria were dissected from young 2-day-old mice, cut in half, and separated from dura mater, blood vessels, and scalp. Calvaria were cultured in tissue culture plates for 1 day in BGJb medium supplemented with 0.1% bovine serum albumin and 100U/ml penicillin and streptomycin, followed by treatment with 10 μ g/ml antibody or protein for 7 days. At 5% CO₂In a humidified atmosphere at 37 ℃. Mice calvaria were imaged with a μ CT40 (scanmedicine, Basserdorf, Switzerland) x-ray micro-CT system. micro-CT data were acquired with the following parameters: x-ray tube energy level =45kV, electricityFlow =177 μ a, integration time =300msec, 2000 transmissions. Axial images were acquired with isotropic resolution of 6 μm. Calibration was performed using Hydroxyapatite (HA) phantom. micro-CT scans were analyzed using Analyze (Analyzer DirectInc., Lenexa, KS, USA). A three-dimensional surface depiction of the cross-section and maximum intensity emission is created for each sample. The parietal boundaries were manually drawn using the Trace tool to segment the parietal regions. Within this zone, the sample volume and mean Bone Mineral Density (BMD) were calculated. The threshold value is 0.3gm-HA/cm³Is applied to the zone to calculate the mean BMD of only calcified tissue within the zone. The threshold is also used to calculate the percentage of calcified volume in the apical area by dividing the number of calcified voxels by the apical area voxels. The following parameters were analyzed for each sample: apical area volume, apical area calcified voxel BMD, and apical area percent calcification. Differences between groups were considered significant if the p-value of the Dunnett's test was less than 0.05.

All experiments with mice were performed according to the Genentech scientific research animal care and use committee guidelines.

Example 2

Isolation of Wnt antagonistic and potentiating LRP6 monoclonal antibodies

To develop candidate therapeutic molecules to manipulate Wnt signaling, antibodies were generated that inhibit or enhance signaling induced by Wnt3a protein. Recombinant LRP6.E1-E2-Fc (SEQ ID NO: 30) and LRP6.E3-E4-Fc (SEQ ID NO: 31) proteins were used to screen the human synthetic Fab phage display library and to confirm binding of LRP6 of the isolated phage clones by ELISA. 24 unique antibody heavy chain clones directed to lrp6.E1-E2 and 22 clones directed to lrp6.E3-E4 were isolated, reformatted and expressed the human IgG1 antibody. Six lrp6.E3-E4 antibodies inhibited Wnt luciferase reporter activity in HEK293 cells induced with 0.1mg/ml purified Wnt3a in a concentration-dependent manner (fig. 1 a. error bars for this and all other figures represent standard deviations of at least 3 replicates unless otherwise indicated). These antibodies were designated YW211.03, YW211.08 and YW211.11, YW211.12, YW211.31 and YW 211.33. None of the lrp6.E1-E2 antibodies exhibited this inhibition. The YW211.31antibody recognizing the lrp6.E3-E4 domain was the most potent in inhibiting signaling in Wnt3 a-stimulated HEK293 cells, IC₅₀At approximately 1ug/ml (or 6 nM). YW211.31antibody inhibits Wnt3 a-induced LRP6 phosphorylation and β -catenin protein stabilization, without affecting the level of LRP6 protein, similar to purified Fzd8CRD and DKK1 proteins (figure 1B, showing Western analysis of HEK293 cells either unstimulated or induced with Wnt3a and treated with the indicated LRP6 antibody or purified protein (showing B-actin and GAPDH protein levels as sample loading controls)). RNAi experiments demonstrate that the lower molecular weight band recognized only by B-catenin polyclonal antibodies represents B-catenin protein.

The YW211.31antibody has a binding affinity of about 2nM (according to Surface Plasmon Resonance (SPR)) and 0.6nM (according to Scatchard analysis). To improve the affinity and potential potency of the YW211.31antibody, this clone was affinity matured using His-tagged LRP6E3-E4 protein and a CDR combinatorial library in which selected CDR residues were targeted for randomization. Four phage clones that showed the most affinity improvement according to phage competition ELISA, namely YW211.31.11, YW211.31.11, 35, yw211.31.57 and yw211.31.62, were reformatted and expressed as full length human IgG. The dissociation rate constants for the four affinity matured iggs were reduced, resulting in improved affinities of the best two antibodies, yw211.31.57 and yw211.31.62, of KD0.27 and 0.17nM, respectively. Yw211.31.57 and yw211.31.62 also showed improved potency in inhibiting signaling in Wnt3 a-stimulated HEK293 cells with an IC50 value of about 0.1 μ g/ml (0.6 nM).

None of the antibodies isolated in the screen activated signaling in HEK293 cells in the absence of stimulation by exogenous Wnt3a protein, whereas five LRP6E1-E2 and two E3-E4 antibodies potentiated Wnt3 a-induced signaling by at least 2-fold. In mouse NIH/3T3 cells, YW210.09 (an E1-E2 antibody) also potentiated Wnt3 a-induced signaling by at least 1.5-fold, indicating that it also recognizes mouse LRP6. In HEK293 cells, the extent to which YW210.09 antibody enhanced Wnt3 a-induced signaling was proportional to Wnt3a concentration (fig. 1C). The YW210.09 antibody interacts with the human LRP6E1-E2 protein with a KD of 5nm as measured by SPR analysis. The ELISA test showed that all antagonistic and potentiating antibodies specifically bind only the LRP6 protein fragment used to isolate them, and none recognized both E1-E2 and E3-E4. FACS analysis indicated that soluble LRP6E1-E4 protein effectively and completely blocked binding of yw211.31.57 and YW210.09 to HEK293 cells, indicating that these antibodies do not recognize other cell surface proteins.

Example 3

Effects of LRP6 monoclonal antibody on autocrine Wnt signaling

The ability of LRP6 antibody to antagonize or potentiate endogenous or autocrine Wnt signaling was determined using a variety of tumor cell lines (Bafico et al, 2004; DeAlmeida et al, 2007; Akiri et al, 2009). In teratocarcinoma cell lines PA-1 and NTERA-2, the YW211.31antibody inhibited reporter activity induced by autocrine Wnt signaling with similar potency to that observed for exogenous Wnt3a (fig. 2A, showing concentration-dependent inhibition and potentiation of autocrine Wnt signaling in PA-1 teratocarcinoma cells transfected with luciferase reporter and treated with LRP6 antibody, alone or in combination, or Fzd8CRD-Fc protein (positive control)).

Inhibition of Wnt signaling by YW211.31antibody was also observed on expression of endogenous Wnt target genes in PA1 cells (fig. 2B). Figure 2B shows the results of qPCR expression analysis of Wnt-induced genes SAX1 and GAD1 and Wnt-repressed gene LEFTY2 in PA-1 cells treated with or without 0.3mg/ml Wnt3a protein, and with 10mg/ml yw211.31antibody, anti-gD monoclonal antibody (as negative control), or Fzd8CRD-Fc protein (as positive control sample) (and additionally normalized relative to samples from cells without added (NA) Wnt3 a).

The antibodies partially inhibit SAX1, GAD1, and APCDD1 expression induced by exogenous Wnt3a protein or maintained by endogenous, autocrine Wnt signaling. In contrast, the YW211.31antibody abrogates the repression of LEFTY2 expression by Wnt3a protein or autocrine Wnt signaling. Unlike YW211.31, YW210.09 antibody potentiates both Wnt3 a-induced and autocrine Wnt signaling in PA-1 and NTERA-2 cell lines according to the reporter gene assay (fig. 2A). Although inhibition of Wnt signaling by the yw211.31.57 antibody gradually increased with increasing antibody concentration, potentiation by the potent wyw 210.09 and other antibodies may decrease at high antibody concentrations in some cell types (such as PA-1 cells). This might suggest that receptor LRP6 dimerization is required for potentiation, since high antibody concentrations would favor monovalent interactions and thus limit LRP6 molecular cross-linking. Treatment of PA-1 or NTERA-2 cells with a combination of both yw211.31.57 and YW210.09 antibodies antagonized both Wnt3 a-induced and autocrine Wnt signaling, similar to the effect of yw211.31.57 alone.

To identify additional cell lines displaying autocrine Wnt signaling, cell lines with relatively high Axin 2(Axin2) mRNA expression or phospho-LRP 5/6 were tested for inhibition of Wnt signaling by Fzd8CRD-Fc protein in a Wnt luciferase reporter gene assay. 9 cell lines exhibited autocrine Wnt signaling inhibited by Fzd8CRD-Fc protein, including NSCLC cells NCI-H23 and NCI-H2030 and soft tissue sarcoma cells SW872 and HT-1080, which were previously reported to have endogenous Wnt signaling based on assays using other Wnt antagonists (Guo et al, 2008; Akiri et al, 2009; Nguyen et al, 2009). Figure 3 shows a summary of data analyzed by one-way analysis of variance (ANOVA) (p value < 0.01). Assays were performed using 10mg/ml antibody, except NCI-H358 and HT-1080 cells, which were treated with 1mg/ml of yw211.31.57 or YW210.09 antibody, respectively, to enhance the boosting effect.

Wnt signaling was further induced by exogenous Wnt3a protein in all nine cell lines, and yw211.31.57 antibody inhibited this response to Wnt3a (fig. 3, 4A, 4D, and 4F). Surprisingly, yw211.31.57 antibody potentiated autocrine Wnt signaling in all nine of these cell lines, while YW210.09 potentiated autocrine Wnt signaling in five cell lines and inhibited in three cell lines (fig. 3, 4A-4C, 4E, and 4F). This opposite activity of yw211.31.57 antibody on autocrine and Wnt3 a-induced signaling was observed not only with luciferase reporter but also on expression of endogenous Wnt target genes such as axin2 in six test cell lines (fig. 4A, 4B and 4C). In EKVX and breast cancer Hs578T cell lines, it was confirmed that the increase in Wnt signaling by the YW211.31antibody was dependent on autocrine Wnt, by demonstrating that this increase was blocked by Fzd8CRD-Fc protein (fig. 4G). Potentiation of autocrine Wnt signaling in EKVX and Hs578T cells was also observed with the other five antibody antagonists of Wnt3 a-induced signaling identified in the screen.

In fig. 4A, qPCR expression analysis of axin2mRNA in HT-1080, EKVX, NCI-H358, and Hs578T indicated that yw211.31.57 (25 mg/ml) antibody potentiates autocrine (NA) Wnt signaling and inhibits signaling induced by Wnt3a (0.2 mg/ml), while Fzd8CRD-Fc (25 mg/ml) antagonizes both autocrine (NA) and Wn3 a-induced signaling. In FIGS. 4B and 4C, Wnt-induced gene expression in NCI-H23 (B) and M14 (C) cells was potentiated by YW211.31.57 and antagonized by YW210.09 antibody (30 mg/ml). Wnt3a (0.2 mg/ml) and Fzd8CRD-Fc (30 mg/ml) treatments were shown to serve as positive controls for autocrine Wnt signaling to be potentiated and inhibited, respectively, while CD4-Fc protein (B) or anti-gD antibody (C) served as negative controls (30 mg/ml). For M14 cells (C), axin2 and SP5 expression was more potent potentiated by Wnt3a protein or yw211.31.57 antibody than APCDD1 and ZNRF3 expression. Figures 4D and 4E show that in Hs578T cells stably integrated with Wnt luciferase reporter, yw211.31.57 antibody showed concentration-dependent inhibition of Wnt3 a-stimulated signaling (D) and potentiation of autocrine Wnt signaling (E), while Fzd8CRD-Fc protein inhibited and YW210.09 antibody potentiated signaling with or without (NA) 0.1mg/ml Wnt3a stimulation. RNAi experiments indicated that at least 41% of Wnt3 a-induced signaling in Hs578T cells was dependent on LRP5 expression, and this signaling was predicted to be inhibited by Fzd8CRD-Fc protein rather than yw211.31.57 antibody. In this experiment, SV 40-driven luciferase was not transfected for normalization, and instead, antibody and protein treatment were independently confirmed to have no significant effect on the viability of this cell line. Figures 4F and 4G show that EKVX cells transfected with Wnt luciferase reporter also displayed potentiation of autocrine Wnt signaling and antagonism of Wnt3 a-induced signaling by yw211.31.57 antibody. Antibody-mediated potentiation of autocrine Wnt signaling is inhibited by 5mg/ml Fzd8CRD-Fc protein.

Example 4

Inverse activity of LRP6 antibodies against different Wnt isoforms

The YW211.31antibody inhibits signaling induced by exogenous Wnt3a protein in all cell lines, but can inhibit or potentiate autocrine Wnt signaling in a cell line-dependent manner, suggesting that the particular Wnt isoform driving autocrine signaling dictates the activity of the antibody. Thus, the activity of this antibody on signaling induced by Wnt3a and other Wnt isoforms were determined. Wnt signaling induced by Wnt3a transfection in HEK293 or Hs578T cells was inhibited by yw211.31.57 antibody with similar potency to inhibition of signaling induced by Wnt3a protein treatment. Surprisingly, the signaling induced by Wnt1 expression in both cell lines was potentiated by yw211.31.57 antibody. Both Wnt1 and Wnt3a signaling were inhibited by Fzd8CRD-Fc protein, as expected. Potentiation of Wnt1 signaling was also observed with other Wnt3a antagonist antibodies identified in the screen. The YW210.09 antibody also displayed opposite activity against Wnt3 a-and Wnt 1-induced signaling, which was opposite to yw211.31.57 activity; i.e., potentiation of Wnt3a and inhibition of Wnt1 signaling. The YW210.09 antibody also inhibited Wnt1 signaling in tumor cells cultured in cultures from MMTV-Wnt1 mouse tumors, as observed by a reduction in expression of Wnt target axin2 and Mmp7 to a similar extent as Fzd8CRD-Fc protein treatment. In MMTV-Wnt1 cells, yw211.31.57 antibody failed to potentiate Wnt1 signaling, probably because Wnt1 signaling was already maximal in these cells.

After demonstrating the opposite activity of yw211.31.57 and YW210.09 antibodies on Wnt3a and Wnt 1-initiated Wnt signaling, this assay was also performed on another 11 of the 19 Wnt genes that induced luciferase reporter more than 2-fold in HEK293 cells. FIG. 5 provides a summary of the data, using 10 u g/ml antibody, 10 u g/ml Fzd8CRD implementation of the determination. Only Wnt3a and Wnt3 activities were inhibited by yw211.31.57 antibody, and both were potentiated by YW 210.09. The 7 Wnt isoforms other than Wnt1 were potentiated by yw211.31.57 and inhibited by YW 210.09. The third class of Wnt isoforms (Wnt 7a, 7b, and 10 a) exhibit signaling activity that is not inhibited by any antibody and is potentiated by at least yw211.31.57. In Hs578T cells transfected with different Wnt isoforms, the antibodies displayed most of these same activities. In particular, yw211.31.57 inhibited Wnt3 and Wnt3a and potentiated all 11 other Wnt isoforms that induced the luciferase reporter at least 2-fold. YW210.09 also potentiates Wnt3 and Wnt3a in Hs578T cells, as well as inhibits 5 of 7 Wnt isoforms that are antagonized in HEK293 cells and can be tested in Hs578T cells. The other two Wnt isoforms in this class (i.e., Wnt8a and Wnt9 b) were not affected by the YW210.09 antibody in Hs578T cells. Since RNAi experiments indicate that Wnt3a signaling in Hs578T, but not HEK293 cells, was transduced by both LRP6 and LRP5, Wnt8a and Wnt9b might signal primarily via LRP5 in Hs578T cells. As in HEK293 cells, the third class of Wnt isoforms is not inhibited by either antibody in Hs587T cells, and we can add Wnt4 to this class, Wnt4 does not induce signaling in HEK293 cells. Only the activity of Wnt7b in this class showed differences, i.e. YW210.09 antibody potentiated its signaling in Hs578T but not HEK293 cells. Unlike the Wnt isoform-specific activity of yw211.31.57 and YW210.09 antibodies, Fzd8CRD-Fc protein potently inhibited the activity of all but Wnt6 and Wnt9b in HEK293 cells. In Hs578T cells, autocrine Wnt signaling was potentiated by both yw211.31.57 and YW210.09 antibodies, as was signaling induced by Wnt4, Wnt7a, and Wnt7b expression. Thus, these three Wnt isoforms are candidate Wnt isoforms that drive autocrine signaling in Hs578T cells.

A variety of sirnas directed to Wnt7b, but also other Wnt isoforms, inhibited autocrine signaling in Hs578T cells, identifying specific Wnt proteins that mediate signaling. Since autocrine Wnt signaling in PA-1 cells is inhibited by yw211.31.57 antibody and potentiated by YW210.09, Wnt3 or Wnt3a are likely to activate endogenous signaling in these cells. Indeed, sirnas directed to Wnt3 but not Wnt3a inhibited autocrine Wnt signaling in PA-1 cells. Potentiation of autocrine Wnt signaling and antagonism of YW210.09 by the YW211.31antibody in NCI-H23NSCLC cells and in M14 melanoma cells is consistent with Wnt2RNAi inhibiting endogenous signaling in NCI-H23 cells and with endogenous Wnt1 expression in M14 cells. Using multiple sirnas, we confirmed that Wnt2 expression in NCI-H23 cells and Wnt1 in M14 cells are required for autocrine Wnt signaling.

Since all antibodies isolated from the screen in example 2 that antagonize signaling in Wnt3 a-stimulated HEK293 cells also inhibited Wnt3a stimulation in all other cell lines tested, and also inhibited autocrine Wnt signaling in teratocarcinoma cell lines, it was unexpected that these antibodies potentiated autocrine Wnt signaling in the other 9 cell lines tested. In addition, the YW210.09 antibody potentiates Wnt3a signaling in all cell lines tested and enhances autocrine Wnt signaling in 7 cell lines, but it inhibits endogenous signaling in another 3 lines. These studies showed that different Wnt isoforms (expressed in the same cell line) determine the activity of LRP6 antibodies, and that Wnt3a antagonist and potentiating antibodies also had opposite effects on most other Wnt proteins. Studies have also shown that the introduction of different Wnt isoforms into the same cell line determines the activity of the LRP6 antibody, and that Wnt3a antagonist and potentiating antibodies also have opposite effects on most other Wnt proteins. Based on their functional interactions with two LRP6 antibodies, the 14 Wnt isoforms tested can be grouped into three categories: wnt3 and Wnt3a were inhibited by YW211.31 and potentiated by YW 210.09; wnt1, 2b, 6, 8a, 9b, and 10b were potentiated by YW211.31 and antagonized by YW 210.09; while Wnt4, 7a, 7b, and 10a were potentiated by YW211.31 and not inhibited by YW210.09 (figure 5). These classifications clearly do not fit into the proposed phylogeny of Wnt genes, although the Wnt3/3a subfamily is the most divergent evolutionarily (Cho et al, 2010).

Example 5

Wnt isoforms specify different activities of LRP6 antibodies

Different Wnt isoforms may preferentially bind to different FZD isoforms endogenously expressed in various cell lines and could conceivably account for the differential activity of our LRP6 antibody. To examine this possibility, chimeric proteins covalently linked to different Wnt-FZD pairs were constructed to test whether a particular Wnt or FZD isoform determines the activity of the LRP6 antibody. Wnt3a or Wnt1 fused to either FZD4 or FZD5 potently activated Wnt signaling in HEK293 cells, whereas FZD4 or FZD5 overexpression did not induce Wnt signaling, assuming deletion of endogenous Wnt expression. The yw211.31.57 antibody inhibits signaling activity of Wnt3a fused to either FZD4 or FZD5, and potentiates activity of Wnt1 fused to either FZD4 or FZD5 (figure 6 provides a summary of the data, assays performed using 10 μ g/ml antibody, 10 μ g/ml FZD8 CRD). The YW210.09 antibody showed opposite activity against Wnt1 chimera, inhibiting both. Thus, the activity of the antibody is related to Wnt isoforms, but not FZD isoforms. The Fzd8CRD-Fc protein had no effect on the signaling induced by any four Wnt-Fzd chimeras, consistent with the Wnt-independent Fzd binding site for the chimeras to function.

Expression of a chimeric fusing Wnt1 or Wnt3a to LRP6 induced Wnt signaling more strongly than LRP6 overexpression. Yw211.31.57 and YW210.09 antibodies were unable to inhibit this induction, consistent with the hypothesis that the inhibitory function of the antibodies was dependent on blocking Wnt binding to LRP6 (figure 6).

This study confirms that the isoform of Wnt, but not FZD, determines the activity of the antibody. Chimeric protein fusions of Wnt isoforms with LRP6, but not FZD, were insensitive to inhibition by LRP6 antibodies, suggesting that antagonism may be mediated by blocking ligand-co-receptor interactions. This was confirmed by in vitro binding studies of Wnt3a and YW210.09 antibodies (both competitively binding in the E3-E4 regions of LRP 6) and Wnt9b and YW211.31 antibodies (which competitively bind in the E1-E2 regions). The epitopes of the two LRP6 antibodies each defined binding sites for different classes of Wnt isoforms, one within the E1-E2 and one within the E3-E4 domain. At least a third Wnt binding site was predicted for isoforms not inhibited by either antibody or combination thereof, and it appears likely that each of the four repeat domains binds to a different subset of Wnt isoforms. This modular organization may allow structural divergence of different Wnt and their binding sites to accommodate differential modulation of Wnt-binding and co-receptor binding antagonists such as SFRP and DKK protein isoforms, respectively.

Example 6

Antibody-mediated potentiation of Wnt signaling involves LRP6 dimerization

Potentiation of autocrine Wnt signaling by the YW211.31antibody requires an affinity effect, presumably through LRP6 dimerization. The monovalent Fab fragment of YW211.31 and recombinant one-armed YW211.31antibody at concentrations that inhibit Wnt3 a-induced signaling in EKVX and Hs578T cells exhibited no potentiation of autocrine Wnt signaling in these cell lines. In contrast, the yw211.31fab fragment and one-armed mAb inhibited both autocrine Wnt and Wnt3 a-induced signaling in the PA-1 teratocarcinoma cell line with similar potency as the intact IgG antibody. To test whether cross-linking of one-arm YW211.31antibody would restore Wnt potentiation of the intact IgG molecule, HT-1080 soft tissue sarcoma cell line, which exhibited potentiation of autocrine Wnt and Wnt 2-induced signaling by yw211.31.57 antibody, and Apomab-induced apoptosis, enhanced by Fc cross-linking, were used (Adams et al, 2008). One arm YW211.31antibody had no effect on autocrine Wnt signaling or signaling induced by Wnt2 transfection. Under the crosslinking conditions of anti-Fc antibodies that promote Apomab-mediated apoptosis, it was determined that crosslinking of one-armed antibodies partially reconstitutes the potentiation of both autocrine Wnt and Wnt2 signaling observed with yw211.31.57 intact antibody.

Antibody-mediated Wnt potentiation requires co-receptor dimerization, as one-armed and Fab antibody formats fail to enhance Wnt signaling unless cross-linked. In addition, the cell-based and biochemical data presented herein indicate that Wnt binding to cross-linked LRP6 is also necessary for potentiation of signaling, presumably reflecting the need for Wnt-mediated recruitment of FZD into the complex. A small fraction of over-expressed LRP6 could be identified as a homodimer on the cell surface and the ectodomain is required for dimerization, however it is unclear whether this contributes to Wnt-independent b-catenin signaling induced by LRP6 overexpression (Liu et al, 2003). Deletion of the extracellular domain of LRP6 also activates signaling in a Wnt-independent manner, and forcing this recombinant protein to dimerize extracellularly by different methods can enhance or inhibit this activity (Liu et al, 2003; conn et al, 2004). Wnt induces LRP6 aggregation and phosphorylation at the plasma membrane, both of which require homooligomerization functions of intracellular DVL proteins (bici et al, 2007). These large aggregates also contain axin and GSK3 and are likely to inhibit b-catenin degradation.

Example 7

Antibody-mediated potentiation of Wnt signaling by inhibition of binding of Wnt antagonists

Inhibition of the activity of extracellular LRP6 antagonists, such as DKK1 isoforms and SOST, also potentiate Wnt signaling (Niida et al, 2004). Exogenous DKK1 protein inhibited Wnt 1-induced signaling in HEK293 cells, and yw211.31.57 antibody was able to block this antagonism and potentiate signaling at sufficiently high concentrations even in the presence of DKK1 protein (fig. 4G). In contrast, the YW211.31 one-armed antibody at these same concentrations only very weakly inhibited the antagonism of DKK1 against Wnt1 signaling. The yw211.31.57 intact antibody effectively antagonizes DKK1 activity at all DKK1 concentrations tested, while the one-arm antibody had minimal or no effect even at low DKK1 concentrations. The potent antagonism of exogenous DKK1 activity observed with the intact but one-armed YW211.31antibody may be attributed to Wnt potentiating activity specific for the intact antibody. Alternatively, since DKK1 protein could not completely inhibit Wnt 1-induced signaling in this assay, it was also possible that the intact YW211.31antibody only potentiates the residual signal via LRP6 dimerization.

Since inhibition of DKK1 interaction with LRP6 by antibodies does not necessarily confer Wnt potentiating activity, and DKK1 antagonism appears to require LRP6 dimerization, it is likely that inhibition of DKK1 activity is mediated primarily by potentiation of residual signaling by Wnt binding to co-receptors.

Example 8

Wnt signaling antagonism predominates in LRP6 antibody combinations

The above assays indicate that Wnt3a and Wnt3 bound within the E1-E2 region of LRP6 and were inhibited from binding by yw211.31.57 antibody. Wnt isoforms of the Wnt1 class are predicted to bind to the E3-E4 region, and this binding is blocked by the YW210.09 antibody. Without being bound by any theory, potentiation of Wnt signaling can occur when both Wnt isoforms and antibodies are able to bind to the same LRP6 molecule, presumably requiring Wnt recruitment of FZD and dimerization of the antibody to LRP6. This model predicts that the combination of two antibodies will inhibit signaling induced by either Wnt isoform, since Wnt binding will be blocked by one or the other antibody, although LRP6 dimerization will still be possible. As predicted, simultaneous treatment of HEK293 cells with yw211.31.57 and YW210.09 antibodies inhibited signaling initiated by expression of either Wnt3a or Wnt1 (fig. 7 and 8A). The assay shown in figure 8A was performed in HEK293 cells transfected with either Wnt3a, Wnt1, or expression constructs for both Wnt3a and Wnt1 stably integrated with Wnt luciferase reporter. All antibodies and proteins were used at 10. mu.g/ml each. This analysis was extended to three other Wnt isoforms of the Wnt1 class, and the combination of yw211.31.57 and YW210.09 antibodies was found to inhibit Wnt signaling to a similar extent as YW210.09 alone (figure 7). Both antibodies did not antagonize Wnt signaling when both Wnt3a and Wnt1 were expressed simultaneously, but the combination of both antibodies did inhibit signaling (fig. 8A). One possible explanation for this result is that each antibody inhibits the binding of only one Wnt isoform, and both antibodies are capable of simultaneously binding to LRP6 molecule to block both Wnt binding sites.

A third class of four Wnt isoforms not antagonized by yw211.31.57 or YW210.09 antibodies may bind to sites on LRP6 that are not blocked by either antibody, or they may have the ability to bind to any of the Wnt binding sites defined by the antibodies. For each of these Wnt isoforms, the combination of both antibodies also did not inhibit their signaling, but rather potentiated or did not affect their activity, suggesting that these Wnt isoforms are able to bind to sites that differ from the yw211.31.57 and YW210.09 epitopes (figure 7).

The observed activity of yw211.31.57 and YW210.09 antibody combinations on Wnt signaling induced by exogenous Wnt isoforms also extends to endogenous, autocrine Wnt signaling. In the teratocarcinoma cell lines PA-1 and Ntera-2, in which yw211.31.57 antibody inhibited while YW210.09 potentiated autocrine Wnt signaling, the antibody combination inhibited signaling (fig. 2A). The antibody combination also served to potentiate autocrine Wnt signaling in Hs578T and EKVX cells that were potentiated by both antibodies (fig. 8B and 8C).

Example 9

LRP6 antibody differential inhibition of Wnt binding to multiple sites

The opposite activity of the LRP6 antibodies suggests that yw211.31.57 and YW210.09 interact with different Wnt isoform binding sites on LRP6, and that Wnt binding is competed by an antagonistic antibody but allowed by a potentiating antibody. Biolayer interferometry (see example 1) measuring binding of purified Wnt proteins to purified, immobilized LRP6 ectodomain protein fragments demonstrated that Wnt3a binds to the E3-E4 region of LRP6 (where the epitope of yw211.31.57 antibody resides), and that Wnt9B (which is in the same class of antibody interactions as Wnt 1) binds only to the E1-E2 region (to which YW210.09 antibody also binds) (bouris et al (2010) yw211.31.57 antibody but not YW210.09 inhibits Wnt3a from binding to LRP6.e1-E4 protein fragments (fig. 9A). conversely YW210.09 but not yw211.31.57 inhibits 9B from binding to LRP6.e1-E4 (fig. 9B.) in these assays, antibodies binding to LRP6 proteins were allowed to reach equilibrium, and subsequent dissociation into Wnt binding and shifting phases of the binding pattern of the proteins shows wavelength shifts.

Antibody-mediated inhibition of Wnt binding could also be detected using smaller Wnt-binding fragments, i.e., E3-E4 for Wnt3a and E1-E2 for Wnt9b (fig. 9C and 9D). One-armed YW211.31antibody also inhibits binding of Wnt3a to E3-E4 fragments. Also, yw211.31.57 and YW210.09 were able to sequentially bind lrp6.E3-E4 protein without competition and when added in either order (fig. 9E). The binding competition at different sites on LRP6 protein between Wnt3a and yw211.31.57 antibodies only and between Wnt9b and YW210.09 only was related to the inhibitory activity of each antibody against signaling by specific Wnt isoforms.

Biolayer interferometry previously showed that purified DKK1 protein binds to both E3-E4 and E1-E2 fragments of LRP6, and that this binding inhibits Wnt3a and Wnt9b from binding to these corresponding protein regions (Bourhis et al, 2010). This assay was used to show that yw211.31.57 and YW210.09 antibodies each inhibit DKK1 binding to lrp6.E1-E4 protein. The yw211.31.57 antibody also inhibits DKK1 from binding to lrp6.E3-E4 protein, while the YW210.09 antibody blocks DKK1 from binding to lrp6.E1-E2 fragment. One arm of YW211.31antibody completely retained this inhibitory activity and failed to significantly antagonize exogenous DKK1 activity on Wnt signaling in cells even though it failed to potentiate Wnt signaling. This result suggests that DKK1 antagonism likely did not significantly contribute to antibody-mediated potentiation of Wnt signaling.

Example 10

LRP6 antibody was active against Wnt-driven tumors and bone formation

To begin exploring the anti-tumor therapeutic efficacy of LRP6 antibody, two models of Wnt ligand driven tumors were treated. MMTV-Wnt1 transgenic breast tumor allografts were dependent on Wnt1 expression while Ntera-2 human teratocarcinoma xenografts were driven by autocrine Wnt signaling of unknown Wnt isoforms (DeAlmeida et al, 2007). Tumors were established in athymic mice using isolated cells from MMTV-Wnt1 transgenic mouse mammary tumors, treated every two days with antibody. Rapid and sustained tumor regression was observed with the YW210.09 antibody, similar to the Fzd8CRD-Fc protein (fig. 10A). The yw211.31.57 antibody did not alter tumor growth under these conditions compared to control buffer (PBS) or anti-gD antibody treatment. Mice were administered 30mg/kg antibody or protein every two days (arrows) (FIG. 10A). These results are consistent with the effect of the antibodies described above for MMTV-Wnt1 tumor cells treated in tissue culture on Wnt target gene expression.

Ntera-2 teratocarcinoma cells were also used to establish xenograft tumors in athymic nude mice treated with either antibody or Fzd8CRD-Fc protein. RNA extracted from tumors treated with antibody yw211.31.57, one-armed yw211.31, or a combination of yw211.31.57 and YW210.09 revealed that expression of Wnt target gene SP5 was reduced to 41-57% of the level of tumors from buffer-injected control mice, while Fzd8CRD-Fc protein treatment reduced SP5 expression to 8.0%. SP5mRNA levels were normalized to gapdh mRNA levels within the same tumor and additionally normalized to PBS treated tumors. According to ANOVA, all treatments except YW210.09 exhibited p-values <0.005 compared to PBS control (figure 10B). Fzd8CRD-Fc reduced axin2 expression to only 56.2%, while no significant change in axin2 expression was detected for any antibody treatment. YW210.09 antibody treatment did not significantly affect the expression of either SP5 or axin 2. Assaying serum samples for inhibition or potentiation of Wnt3 a-induced signaling in HEK293 cells confirmed that the injected antibodies and proteins retained at least some in vivo activity throughout the 16-h exposure.

Since activation or potentiation of Wnt signaling increases bone mass by enhancing osteoblast differentiation and function and indirectly by inhibiting osteoclast differentiation (Glass et al, 2005), the activity of LRP6 antibody on mouse calvarial bone in organotypic culture (organotypic culture) was tested. Microdissected calvarial explants were cultured in the presence of antibody or RANK-Fc and then analyzed for parietal volume and density by microcomputerized tomography. Using histogram analysis on control samples, X-ray attenuation ranges are defined for calcified (bone) and non-calcified (cartilage) tissue. YW210.09 antibody treatment significantly increased the mean Bone Mineral Density (BMD) of calcified parietal bone by 7.4%, similar to the 6.8% increase observed for RANK-Fc treatment to inhibit osteoclast differentiation (FIG. 10C; Hsu et al, 1999). Yw211.31.62 antibody treatment did not significantly alter calcified parietal BMD. All treatments were 10. mu.g/ml antibody or protein for 7 days. In fig. 10C, data points represent eight half calvaria from four mice of each treatment group; mean and standard error of mean are shown as horizontal and vertical lines, respectively. Only YW210.09 and RANK-Fc treatment differed significantly from the untreated samples, with p values less than 0.01 and 0.05 for the Dunnett's test, respectively (both t-tests < 0.05).

Antibody or RANK-Fc treatment did not significantly alter the volume of the total parietal region (calcified and non-calcified) and the proportion of calcified bone in this region, suggesting that YW210.09 antibody may enhance mineralization without overall alteration of cell proliferation.

Example 11

LRP6 bispecific antibodies function as pan Wnt inhibitors

Bispecific IgG hybrids with the heavy chain heterodimers YW211.31.62 and YW210.09 were constructed using node-in-hole engineering (Atwell et al, 1997). This LRP6 bispecific antibody produced in e.coli or HEK293 cells antagonized Wnt3 a-induced (0.1 μ g/ml) signaling in HEK293 cells (fig. 11A) and tumor cell lines PA-1, M14 and CAL-51 (fig. 11C). Notably, the bispecific antibody inhibited at least as potently as YW211.31 and did not retain Wnt3a potentiating activity of YW 210.09. The bispecific antibody also inhibited autocrine Wnt signaling in all three tumor cell lines tested (fig. 11B), retaining the inhibitory activity of YW211.31antibody in PA-1 cells and YW210.09 in M14 cells. Interestingly, this bispecific antibody inhibits signaling even though YW211.31 potentiates while YW210.09 does not affect autocrine Wnt signaling in CAL-51 breast cancer cells. This novel antagonistic activity was not observed for the combination of YW211.31 and YW210.09 antibodies. In the above assay, PA-1 and M14 cells stably integrated with Wnt luciferase reporter, and CAL-51 cells transfected with the reporter, were treated with the indicated control buffer (PBS), antibody combination, or Fzd8CRD-Fc protein (10 μ g/ml each) with (11C) or without (11B) stimulation of 0.1 μ g/ml Wnt3 a.

This bispecific antibody potently inhibited all Wnt blockaded by either YW211.31 or YW210.09 when tested for signaling induced by transfection of 13 Wnt isoforms in HEK293 cells (figure 12). The assays summarized in table 12 determined the effect of antibodies or proteins (10 μ g/ml) on signaling induced by expression constructs transfected with Wnt isoforms in HEK293 or Hs578T cell lines stably integrated with Wnt luciferase reporters. Reporter activity is normalized to cell number and additionally to levels in cells transfected with the same expression construct but not treated with antibody or protein. anti-gD was used as a control. Fold change values were considered relevant when they were outside the range observed with control anti-gD antibody treatment: inhibition of less than 0.80 and potentiation of greater than 1.30 in HEK293 cells, and inhibition of less than 0.65 and potentiation of greater than 1.30 in Hs578T cells.

Similarly to the combination of YW211.31 and YW210.09 antibodies, but also unlike either antibody alone, the bispecific antibody blocked signaling induced by the combination of Wnt1 and Wnt3a (figure 12). Unexpectedly, bispecific antibodies also reduced signaling by three wnts not inhibited by homodimeric antibodies alone or in combination. These antagonistic activities of these bispecific antibodies were also observed in Hs578T cells, except perhaps for the effect of the deletion on Wnt7 a-induced signaling.

The ability of the bispecific antibody to inhibit Wnt3 a-induced β -catenin protein stabilization was examined. HEK293 cells transfected with or without Wnt3a were treated with YW211.31, YW210.09, or bispecific antibodies, or with control Fzd8CRD-Fc protein or anti-gD at a concentration of 5 μ g/ml for 18h and the β -catenin protein level and phosphorylated LRP5/6 level were determined by Western blot analysis. Fig. 13A. In HEK293 cells, the bispecific antibody inhibited Wnt 3A-induced β -catenin protein stabilization, similar to YW211.31, unlike YW210.09 (which increases β -catenin levels) (fig. 13A). Both bispecific and YW211.31, but not YW210.09 antibody, blocked the induction of phosphorylated LRP5/6 by Wnt3a, a high molecular weight species. Surprisingly, while YW211.31 and YW210.09 did not affect the steady state levels of total LRP6 protein, bispecific antibodies increased LRP6 protein with or without Wnt3a induction. This stabilized LRP6 may have slightly elevated Ser1490 phosphorylation in the absence of Wnt stimulation, although the bispecific antibody did not affect Wnt reporter activity in HEK293 cells in the absence of Wnt.

The ability of the bispecific antibody to inhibit Wnt signaling in vivo was determined. SCID-bg mice bearing M14 melanoma xenograft tumors were injected with 30mg/kgLRP6 bispecific antibody, Fzd8CRD protein (positive control), or anti-gD antibody (negative control). After 16 hours of treatment, RNA was extracted and checked for Wnt target gene expression by qPCR. mRNA levels were normalized to gapdh mRNA levels within the same tumor and additionally to anti-gD treated tumors. According to ANOVA, all bispecific antibodies and Fzd8CRD treatments showed p-values <0.001 compared to anti-gD controls. As shown in figure 13B, LRP6 bispecific antibodies inhibited Wnt signaling in M14 melanoma cells cultured as xenograft tumors. RNA extracted from the antibody-treated tumors showed a reduction in expression of Wnt target gene axin2 and APCDD1 to levels 46-57% and 35-38% respectively in tumors treated with control anti-gD antibody. These reduced expression levels were similar to those observed with injection of Fzd8CRD protein and indicated that the bispecific antibody was stable and active in vivo.

Example 12

Structure of LRP6E1-yw210.09fab complex.

The crystal structure of the first β -propeller and EGF domain (also called E1) of LRP6 complexed with yw210.09fab was determined by molecular replacement and refined toResolution, R and R free (Rfree) were 0.175 and 0.220, respectively. The crystallographic asymmetric unit consists of one LRP6E1 domain and one yw210.09fab. The electron densities can be read to allow the depiction of residues Ala20 to Lys324 of the E1 domain and residues Asp1 to Glu213 and Glu1 to Lys214 of the Fab light and heavy chain respectively, with the exception of the Fab heavy chain residues Ser127 to Thr131 (all using Kabat numbering).

LRP6E1 domains were assembled in a modular structure comprising β -propeller modules and Epidermal Growth Factor (EGF) -like modules β -propellers were composed of six blades arranged radially, the blades were formed of four-chain antiparallel β -sheets, the N-end edge faced the central channel and the YWTD motif was located in the second chain of each blade LRP6E1 β -propeller structure closely resembles the LDLr structure (Jeon, H., et al, 2001), rmsd being when stacked on 245C- α atomsAlthough the sequence identity is only 36%. most of the conserved residues are concentrated near the YWTD core motif that forms a β -sheet, and are critical to β -propeller structural integrity, while the surface residues are highly diverse, contributing to the functional diversity of these receptors LRP6 uses its EGF-like domain to lock (lockdown) the first and sixth blades of the propeller and maintain its mechanical strengthAnd a shape complementarity of 0.74. EGF moduleThe three residues in the first β -chain of (i.e., Leu296, Leu298, and Met 299) constitute a hydrophobic core that is pushed into the complementary lumen of the β -propeller surrounded by some direct or water-mediated polar interactions these features are also observed in LDLR structures (Jeon, h., et al, 2001; Rudenko, g., et al, 2002).

Yw210.09fab recognizes β a region in the center of the top of the propeller, a region frequently found to be involved in protein-protein interactions (Springer, t.a., 1998). paratope consists of residues from five CDRs, including three heavy chain CDRs (H1, H2, H3) and two light chain CDRs (L1 and L3.) binding of antibodies to β -propellers is buriedThe total area of (a) and the shape complementarity score 0.76. β -propeller top surface one acidic sheet occupies roughly one third of the total area but hardly overlaps with the YW210 epitope. conversely, the heavy and light chains identify discrete regions. direct contact formed by the heavy chain CDRs occupies 80% of the buried area with CDRH3 being singly exclusive over 50%. this segment consists of 17 residues, with the residues His98 to Lys100c forming direct contact with the c-propeller, importantly, Asn100 of the antibody generates a pair of hydrogen bonds with Asn185 of LRP c forming a "handshake" interaction (fig. 16). additionally, the main chain of Val100 c and Lys100c places one carbonyl group that interacts with Arg c of LRP c unusually in the back and two NH groups that interact with the acidic sheet via two water molecules (Wat c and Wat c) in the front c side chain also by binding with the Val and Ser c of LRP c and shows a particularly complementary hydrogen bond with the c H c, al100, LRP 72, a c H-c-H-b-H-c-H interaction with the other propeller side chain binding, a c-H-c-H interaction exhibiting a central H interaction with the side chain binding with the central LRP c-LRP 72-LRP c-H interaction71. E73, L95, S96, D98 and E115. H1 and H2 touch the fifth and sixth paddles, whereas L1 and L3 touch the sixth, first and second paddles (fig. 15) another region involving the LRP6 residue of YW210.09 binding to LRP6, including R29, W188, K202, P225, H226, S243 and f 266. crystal compression interaction does not directly involve the YW210.09 contact LRP6 epitope, indicating that the crystal structure should reflect how the two molecules interact in solution the unique CDRH3nav (seq id no:49) motif and LRP6E1 β -propeller interaction is highly similar to that reported between laminin and nidogen (Takagi, j, cdrl, 2003) in both cases, both via the above-cited handshake β -central channel, and into the conserved protein motif (see the conserved domain of proteins), which is also shown in this publication in the conserved protein motif, i.e. the conserved domain of the amino acid motif, which is found in the conserved protein motif of the dockerin family, i.9. bekk 11, i.7. this publication, i.e. the conserved protein, which is shown in the conserved domain of the dockerin 3, the dockerin, bekk 11, and the dockerin, i.7. this publication No. protein, which is shown in the conserved protein, which is a motif, and/9. this document, and/9. this two variants, which is shown in the conserved protein, and/9. this document, and/11 family, and/11. this protein, and/11, which is also in the conserved protein, which is shown in the conserved protein, which is a conserved protein, which is shown in the conserved protein, which is a conserved domain, is shown in the conserved domain, and which is a conserved protein, which is found in the conserved domain, is also in the conserved domain, which is a protein, which is a conserved domain, is shown in the conserved protein, is a conserved domain, which is a conserved in the previous paragraph, is a conserved domain, is a protein, which is a protein, and which is a conserved under the conserved domain, which is a conserved domain, which is shown in the conserved under the conserved, which is also found in the motif, which is a protein, is a conserved under the conserved protein, which is a protein, which is shown in the motif, which is a conserved under.

Example 13

Exemplary anti-LRP 6 antibodies

The amino acid sequences of certain anti-LRP 6 antibodies are provided in the sequence listing. Tables 2-4 provide descriptions of sequences. An amino acid sequence alignment of the VH and VL domains of a particular anti-LRP 6 antibody is provided in fig. 16 and 17.

TABLE 2

Heavy and light chains

SEQ ID	Description of the invention
		SEQ ID NO：1	YW211.31 heavy chain
SEQ ID NO：2	YW211.31 light chain
		SEQ ID NO：3	YW211.31.57 heavy chain
SEQ ID NO：4	YW211.31.57 light chain
		SEQ ID NO：5	YW211.31.62 heavy chain
SEQ ID NO：6	YW211.31.62 light chain
		SEQ ID NO：7	YW210.09 heavy chain
SEQ ID NO：8	YW210.09 light chain

TABLE 3

Heavy and light chain variable regions

TABLE 4

Heavy and light chain HVRs

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the description and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated by reference in their entirety.

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Claims (42)

1. An isolated antibody that binds LRP6, wherein the antibody is selected from the group consisting of:

(i) an antibody comprising a VH comprising (a) HVR-H1 having the amino acid sequence of seq id no:17, (b) HVR-H2 having the amino acid sequence of seq id no:18, and (c) HVR-H3 having the amino acid sequence of seq id no:19, and a VL comprising (g) HVR-L1 having the amino acid sequence of seq id no:25, (H) HVR-L2 having the amino acid sequence of seq id no:26, and (i) HVR-L3 having the amino acid sequence of seq id no: 27;

(ii) an antibody comprising a VH comprising (a) HVR-H1 having the amino acid sequence of seq id no:17, (b) HVR-H2 having the amino acid sequence of seq id no:18, and (c) HVR-H3 having the amino acid sequence of seq id no:21, and a VL comprising (g) HVR-L1 having the amino acid sequence of seq id no:25, (H) HVR-L2 having the amino acid sequence of seq id no:26, and (i) HVR-L3 having the amino acid sequence of seq id no: 28; and

(iii) an antibody comprising a VH comprising (a) HVR-H1 having amino acid sequence SEQ ID NO:20, (b) HVR-H2 having amino acid sequence SEQ ID NO:18, and (c) HVR-H3 having amino acid sequence SEQ ID NO:19, and a VL comprising (g) HVR-L1 having amino acid sequence SEQ ID NO:25, (H) HVR-L2 having amino acid sequence SEQ ID NO:26, and (i) HVR-L3 having amino acid sequence SEQ ID NO: 27.

2. The antibody of claim 1, wherein the antibody is selected from the group consisting of:

(i) an antibody comprising a VH having an amino acid sequence represented by SEQ ID NO. 9 and a VL having an amino acid sequence represented by SEQ ID NO. 10;

(ii) an antibody comprising a VH having an amino acid sequence represented by SEQ ID NO. 11 and a VL having an amino acid sequence represented by SEQ ID NO. 12; and

(iii) an antibody comprising a VH having an amino acid sequence represented by SEQ ID NO. 13 and a VL having an amino acid sequence represented by SEQ ID NO. 14.

3. The antibody of claim 1, wherein the antibody inhibits signaling induced by a first Wnt isoform and potentiates signaling induced by a second Wnt isoform.

4. The antibody of claim 2, wherein the antibody inhibits signaling induced by a first Wnt isoform and potentiates signaling induced by a second Wnt isoform.

5. The antibody of claim 3, wherein the first Wnt isoform is selected from the group consisting of: wnt3 and Wnt3a, and the second Wnt isoform is selected from the group consisting of: wnt1, 2b, 4,6, 7a, 7b, 8a, 9b, 10a and 10 b.

6. The antibody of claim 4, wherein the first Wnt isoform is selected from the group consisting of: wnt3 and Wnt3a, and the second Wnt isoform is selected from the group consisting of: wnt1, 2b, 4,6, 7a, 7b, 8a, 9b, 10a and 10 b.

7. The antibody of any one of claims 1-6, wherein the antibody binds the E3-E4 region of LRP6.

8. An isolated antibody that binds to LRP6, wherein the antibody comprises a VH comprising (a) HVR-H1 having the amino acid sequence of SEQ ID NO:22, (b) HVR-H2 having the amino acid sequence of SEQ ID NO:23, and (c) HVR-H3 having the amino acid sequence of SEQ ID NO:24, and a VL comprising (g) HVR-L1 having the amino acid sequence of SEQ ID NO:25, (H) HVR-L2 having the amino acid sequence of SEQ ID NO:26, and (i) HVR-L3 having the amino acid sequence of SEQ ID NO: 27.

9. The antibody of claim 8, wherein the antibody comprises the VH having the amino acid sequence of seq id No. 15 and the VL having the amino acid sequence of seq id No. 16.

10. The antibody of claim 8, wherein the antibody inhibits signaling induced by a first Wnt isoform and potentiates signaling induced by a second Wnt isoform.

11. The antibody of claim 9, wherein the antibody inhibits signaling induced by a first Wnt isoform and potentiates signaling induced by a second Wnt isoform.

12. The antibody of claim 10, wherein the first Wnt isoform is selected from the group consisting of: wnt1, 2b, 6, 8a, 9b, and 10b, and the second Wnt isoform is selected from the group consisting of: wnt3 and Wnt3 a.

13. The antibody of claim 11, wherein the first Wnt isoform is selected from the group consisting of: wnt1, 2b, 6, 8a, 9b, and 10b, and the second Wnt isoform is selected from the group consisting of: wnt3 and Wnt3 a.

14. The antibody of any one of claims 8-13, wherein the antibody binds the E1-E2 region of LRP6.

15. An isolated bispecific antibody that binds to two different regions of LRP6, wherein the antibody comprises a first VH domain comprising (a) HVR-H1 of amino acid sequence seq id no:20, (b) HVR-H2 of amino acid sequence seq id no:18, and (c) HVR-H3 of amino acid sequence seq id no:19, a second VH domain comprising (g) HVR-L1 of amino acid sequence seq id no:25, (H) HVR-L2 of amino acid sequence seq id no:26, and (i) HVR-L3 of amino acid sequence seq id no:27, and a second VL domain comprising (b) HVR-H1 of amino acid sequence seq id no:22, HVR-H2 of amino acid sequence 23, and (c) amino acid sequence HVR-L49324 of amino acid sequence seq id no:24, the second VL domain comprising (g) HVR-H49325 of amino acid sequence HVR-L1 of amino acid sequence seq id no:23, the first VL domain comprising (b) HVR-H3645 of amino acid sequence HVR-L2 of amino acid sequence seq id no:23, and (c) HVR-L82923 of amino acid sequence, (h) (ii) HVR-L2 shown by amino acid sequence SEQ ID NO. 26 and (i) HVR-L3 shown by amino acid sequence SEQ ID NO. 27.

16. The bispecific antibody of claim 15, wherein the antibody comprises (a) a first VH having the amino acid sequence of seq id No. 13, (b) a first VL having the amino acid sequence of seq id No. 14, (c) a second VH having the amino acid sequence of seq id No. 15, and (d) a second VL having the amino acid sequence of seq id No. 14.

17. The bispecific antibody of claim 15, wherein the antibody inhibits signaling induced by a Wnt isoform selected from the group consisting of Wnt3 and Wnt3a and inhibits signaling induced by a Wnt isoform selected from the group consisting of Wnt1, 2b, 6, 8a, 9b, and 10 b.

18. The bispecific antibody of claim 16, wherein the antibody inhibits signaling induced by a Wnt isoform selected from the group consisting of Wnt3 and Wnt3a and inhibits signaling induced by a Wnt isoform selected from the group consisting of Wnt1, 2b, 6, 8a, 9b, and 10 b.

19. The bispecific antibody of any one of claims 15-18, wherein the antibody inhibits autocrine Wnt signaling.

20. The bispecific antibody of any one of claims 15-18, wherein the antibody binds the E3-E4 region of LRP6 and the E1-E2 region of LRP6.

21. The bispecific antibody of any one of claims 15-18, wherein the antibody inhibits autocrine Wnt signaling and the antibody binds the E3-E4 region of LRP6 and the E1-E2 region of LRP6.

22. The antibody or bispecific antibody of any one of claims 1-6, 8-13, and 15-18, wherein the antibody is a monoclonal antibody.

23. The antibody or bispecific antibody of claim 7, wherein the antibody is a monoclonal antibody.

24. The antibody or bispecific antibody of claim 14, wherein the antibody is a monoclonal antibody.

25. The antibody or bispecific antibody of claim 19, wherein the antibody is a monoclonal antibody.

26. The antibody or bispecific antibody of claim 20, wherein the antibody is a monoclonal antibody.

27. The antibody or bispecific antibody of claim 21, wherein the antibody is a monoclonal antibody.

28. The antibody or bispecific antibody of any one of claims 1-6, 8-13, and 15-18, wherein the antibody is a chimeric, human, or humanized antibody.

29. The antibody or bispecific antibody of claim 7, wherein the antibody is a chimeric, human or humanized antibody.

30. The antibody or bispecific antibody of claim 14, wherein the antibody is a chimeric, human or humanized antibody.

31. The antibody or bispecific antibody of claim 19, wherein the antibody is a chimeric, human or humanized antibody.

32. The antibody or bispecific antibody of claim 20, wherein the antibody is a chimeric, human or humanized antibody.

33. The antibody or bispecific antibody of claim 21, wherein the antibody is a chimeric, human or humanized antibody.

34. An isolated nucleic acid encoding the antibody or bispecific antibody of any one of claims 1-33.

35. A host cell comprising the nucleic acid of claim 34.

36. A pharmaceutical formulation comprising the antibody or bispecific antibody of any one of claims 1-33 and a pharmaceutically acceptable carrier.

37. Use of the antibody or bispecific antibody of any one of claims 1-33 in the manufacture of a medicament.

38. Use of the antibody or bispecific antibody of any one of claims 1-7 and 15-21 in the manufacture of a medicament for the treatment of cancer.

39. The use of claim 38, wherein the cancer is non-small cell lung cancer, breast cancer, pancreatic cancer, ovarian cancer, renal cancer, or prostate cancer.

40. Use of an antibody according to any one of claims 8 to 14 in the manufacture of a medicament for the treatment of a bone disorder.

41. The use of claim 40, wherein the bone disorder is osteoporosis, osteoarthritis, a bone fracture, or a bone injury.

42. An immunoconjugate comprising the antibody or bispecific antibody of any one of claims 1-33 and a cytotoxic agent.