CA2668714A1 - Methods of treating, diagnosing or detecting cancer - Google Patents

Abstract

The invention provides, inter alia, methods for treating cancer, compositions for treating cancer, and methods and compositions for diagnosing and/or detecting cancer. In particular, the present invention provides compositions and methods for treating, diagnosing and detecting cancers associated with FZDlO overexpression.

Description

METHODS OF TREATING, DIAGNOSING OR DETECTING CANCER
[0001] FIELD OF THE INVENTION

[0002] The present invention relates generally to the field of oncology. More particularly, the invention relates to methods for treating cancer, compositions for treating cancer, and methods and compositions for diagnosing and/or detecting cancer.

[0003] BACKGROUND OF THE INVENTION

[0004] Cancer is the second leading cause of death in the United States.
Although "cancer" is used to describe many different types of cancer, i.e. breast, prostate, lung, colon, pancreas, each type of cancer differs both at the phenotypic level and the genetic level. The unregulated growth characteristic of cancer occurs when the expression of one or more genes becomes dysregulated due to mutations, and cell growth can no longer be controlled.

[0005] Frizzled antigens are a family of G protein coupled receptor-like receptors that have binding sites for Wnt protein ligands, which are secreted molecules that act as upregulators of gene expression. Members of this family of receptor-ligand pairs play a role in embryonic development, and may play a role in cellular proliferation and the ultimate fate of cells during embryogenesis. Most of frizzled receptors are coupled to the (3-catenin canonical signaling pathway, which leads to the activation of dishevelled proteins, inhibition of GSK-3 kinase, nuclear accumulation of 0-catenin and activation of Wnt target genes. A
second signaling pathway involving PKC and calcium fluxes has been seen for some family members, but it is not yet clear if it represents a distinct pathway or if it can be integrated in the canonical pathway.

[0006] There are 18 Wnt and 10 Frizzled genes, all of which are highly homologous in structure, which have been identified thus far from the human genome database (Science, 291:1304-1351 (2001)). There are also 5 secreted Frizzled forms. Each Fz gene encodes an integral membrane protein with a large extracellular portion, seven putative transmembrane domains, and a cytoplasmic tail (Wang, et al., J. Biol. Chem. 271, 4468-4476(1996); Vinson et al., Nature (London) 338, 263-264(1989)). Near the NH2 terminus of the extracellular portion is a cysteine-rich domain (CRD) that is well conserved among other members of the FZ family (Wang, et al., J. Biol. Chem. 271, 4468-4476(1996)). The CRD, comprised of 110-amino acid residues, including 10 invariant cysteines, is the putative binding site for Wnt ligands (Bhanot et al., Nature (London) 382, 225-230(1996)).

[0007] Frizzled receptors can dimerize and that dimerization is correlated with activation of the Wnt/B-catenin pathway (Carron et al., Journal of Cell Science, 116:2541-2550 (2003)).

[0008] Frizzled-10 (FZD10) has been cloned and characterized (Koike et al., Biochem Biophys Res Commun., 262(1):39-43(1999)). Nucleotide sequence analysis showed that human FZD10 gene encodes a seven-transmembrane-receptor of 581 amino acids, with the N-terminal cysteine-rich domain and the C-terminal Ser/Thr-Xxx-Val motif. Large amounts of FZD10 mRNA, 4.0 kb in size, were detected in the placenta and fetal kidney, followed by fetal lung and brain. In adult brain, FZD10 mRNA was abundant in the cerebellum. The FZD10 gene was mapped to human chromosome 12q24.33. FZD10 shares 65.7% amino-acid identity with Frizzled-9 (FZD9). FZD10 and FZD9 constitute a subfamily among the Frizzled genes.

[0009] There is a need to identify compositions and methods that modulate molecules that play a role in cancer. The present invention is directed to these, as well as other, important needs.

[00010] SUMMARY OF THE INVENTION

[00011] The materials and methods of the present invention fulfill the aforementioned and other related needs in the art.

[00012] In one embodiment of the invention, a composition comprising a FZD10 modulator and one or more pharmaceutically acceptable carriers, is provided wherein said FZD10 modulator has one or more of the activities selected from the group consisting of Dishevelled phosphorylation, c-Jun phosphorylation, and 0-catenin phosphorylation. In another embodiment, the composition is provided wherein the composition is a sterile injectable. In still another embodiment, the composition is provided wherein the FZD10 modulator is an inhibitor that decreases phosphorylation of Dishevelled, decreases phosphorylation of c-Jun, and increases phosphorylation of 0-catenin. In a related embodiment, the composition is provided wherein the FZD10 modulator decreases phosphorylation of Dishevelled or wherein the FZD10 modulator inhibits FZD10 binding to Dishevelled.

[00013] In another embodiment of the invention, the aforementioned composition is provided wherein the FZD10 modulator is an oligonucleotide, a small molecule, a mimetic, a soluble receptor, a decoy, or an antibody. In another embodiment, the composition is provided wherein the FZD10 modulator is a monoclonal antibody which binds to FZD10 with an affinity of at least 1x108Ka. In another embodiment, the composition is provided wherein the FZD10 modulator is a monoclonal antibody which binds to FZD10 with an affinity of at least 1x107 Ka, at least 1x106Ka at least 1x105Ka at least 1x104Ka. In yet another embodiment, the composition is provided wherein the FZD10 modulator is a monoclonal antibody which selectively binds FZD 10 and modulates one or more FZD10-related biological activities. In still another embodiment the monoclonal antibody is a human antibody, a humanized antibody or chimeric antibody. In a related embodiment, the monoclonal antibody binds to an epitope of FZD10, the epitope selected from the group consisting of the first extracellular domain (ECD), the second ECD, the third ECD, the fourth ECD and the ligand-binding domain. In still another embodiment, the monoclonal antibody binds to an epitope of the ECD of FZD10. In yet another embodiment, the monoclonal antibody binds to an epitope of the the first ECD. In another embodiment, the monoclonal antibody does not bind to the first ECD. In still another embodiment, the monoclonal antibody does not cross-react with FZD9. In yet nother embodiment, the monoclonal antibody is conjugated to another diagnostic or therapeutic agent.

[00014] In another embodiment of the invention, a method of treating cancer or a cancer symptom in a patient in need thereof is provided comprising administering to the patient a therapeutically effective amount of an aforementioned FZD10 modulator. In another embodiment, the aforementioned method is provided wherein the FZD10 modulator decreases phosphorylation of Dishevelled. In yet another embodiment, the aforementioned method is provided wherein the FZD10 modulator inhibits FZD10 expression by at least 50%
as compared to a control. In another embodiment, the aforementioned method is provided wherein the FZD10 modulator is an oligonucleotide, a small molecule, a mimetic, a soluble receptor, a decoy receptor, or an antibody. In a related embodiment, the FZD10 modulator is a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a human antibody, a humanized antibody, a single-chain antibody, or a Fab fragment. In another embodiment, the aforementioned method is provided wherein the antibody is labeled. In a related embodiment, the label is an enzyme, radioisotope, toxin or fluorophore.

[00015] In another embodiment, the aforementioned method is provided wherein the antibody has a binding affinity less than about 1x105Ka for a polypeptide other than FZD10.
In still another embodiment, the aforementioned method is provided wherein the modulator is a monoclonal antibody. In a related embodiment, the monoclonal antibody binds to FZD10 with an affinity of at least 1x108Ka. In still another embodiment, the monoclonal antibody binds to an epitope of FZD10, the epitope selected from the group consisting of the first ECD, the second ECD, the third ECD, the fourth ECD and ligand-binding domain. In another embodiment, the monoclonal antibody binds to an epitope of the first ECD of FZD10.
In an alternative embodiment, the monoclonal antibody does not bind to the first ECD of FZD10. In another embodiment, the aforementioned method is provided wherein the monoclonal antibody does not bind to FZD9.

[00016] In still another embodiment of the invention, the aforementioned method is provided wherein the monoclonal antibody decreases phosphorylation of Dishevelled, decreases phosphorylation of c-Jun, and increases phosphorylation of (i-catenin. In another embodiment, the aforementioned method is provided wherein the cancer is ovarian, colon, lung, stomach or uterine cancer.

[00017] In another embodiment, the aforementioned method is provided wherein the FZD10 modulator is an oligonucleotide. In yet another embodiment, the aforementioned method further comprises the administration of a traditional cancer therapeutic to the patient.
In another embodiment, the aforementioned method further comprises the treatment of the patient with one or more of chemotherapy, radiation therapy or surgery. In still another embodiment, the aforementioned method is provided wherein the cancer symptom is selected from the group consisting of pain, death, weight loss, weakness, difficulty eating, blood in stool, nausea, vomiting, liver metastases, lung metastases, bone metastases, abdominal fullness, bloating, fluid in peritoneal cavity, vaginal bleeding, constipation, abdominal distension, perforation of colon, acute peritonitis (infection, fever, pain), vomiting blood, and difficulty swallowing.

[00018] Another embodiment of the invention provides a method of modulating a related biological activity in a patient, the method comprising administering to the patient an amount of an aforementioned FZD10 modulator effective to modulate the FZD10 biological activity. In another embodiment, the FZD10 modulator is a monoclonal antibody which selectively binds FZD10. In still another embodiment, the patient has or is predisposed to one or more of ovarian, colon, lung, or uterine cancer. In yet another embodiment, the FZD10 modulator is an antibody and is administered to the subject via in vivo therapeutic antibody gene transfer.

[00019] In another embodiment of the invention, a method of identifying a patient susceptible to FZD10 therapy is provided comprising detecting the presence or absence of evidence of FZD10 expression in the sample, wherein the presence of evidence of FZD10 expression in the sample is indicative of a patient who is a candidate for FZD10 therapy and the absence of evidence of FZD10 expression in the sample is indicative of a patient who is not a candidate for FZD10 therapy; administering a therapeutically effective amount of an aforementioned composition to the patient if the patient is a candidate for FZD10 therapy; and administering a traditional cancer therapeutic to the patient if the patient is not a candidate for FZD10 therapy. In a related embodiment, the expression of FZD10 is increased at least 30%
compared to a control. In another related embodiment, the expression of FZD10 is increased at least 50% compared to a control. In still another related embodiment, the expression of FZD10 is increased at least 100% compared to a control.
[00020] In another embodiment, the aforementioned method is provided wherein evidence of FZD10 expression is detected by measuring FZD10 RNA. In another embodiment, evidence of FZD10 expression is detected by measuring FZD10 expression products. In another embodiment, the aforementioned method is provided wherein the patient has or is predisposed to one or more of ovarian, colon, lung, or uterine cancer. In another embodiment, the aforementioned method further comprises the administration of a traditional cancer therapeutic to the patient.

[00021] In another embodiment, a method of inhibiting cancer cell growth in a patient in need thereof is provided comprising administering a therapeutically effective amount of an aforementioned FZD 10 modulator to the patient. In another embodiment, the aforementioned method is provided wherein the FZD10 modulator is a monoclonal antibody which specifically binds FZD10 and decreases phosphorylation of Dishevelled.

[00022] In another embodiment of the invention, a method of inhibiting a cancer cell phenotype in a patient in need thereof is provided, the method comprising administering to the patient a therapeutically effective amount of an aforementioned FZD10 modulator. In another embodiment, the aforementioned method is provided wherein the cancer cell phenotype is one or more of colony formation in soft agar and tubular network formation in a three dimensional basement membrane or extracellular membrane preparation. In another embodiment, the aforementioned method further comprises the administration of a traditional cancer therapeutic to the patient. In still another embodiment, the aforementioned method further comprises the treatment of the patient with one or more of chemotherapy, radiation therapy or surgery. In yet another embodiment, the aforementioned method is provided wherein the cancer cells are selected from the group consisting of ovarian, colon, lung, or uterine cancer cells.

[00023] In still another embodiment of the invention, a method for detecting a tumor in a patient is provided comprising administering to the patient a composition comprising an aforementioned FZD10 modulator linked to an imaging agent and detecting the localization of the imaging agent in the patient. In another embodiment, the aforementioned method is provided wherein the FZD10 modulator is selected from the group consisting of a small molecule, an oligonucleotide, a mimetic, a soluble receptor, a decoy receptor, or an antibody.
In still another embodiment, the aforementioned method is provided wherein the composition comprises an anti-FZD10 antibody conjugated to an imaging agent. In a related embodiment, the imaging agent is 18F , 43K , 52Fe, 57Co, 67Cu, 67Ga > 77 Br, $7 MSr, 86Y>
90Y> 99MTc, 111In1123I
, 125I 127C5 129Cs 1311 132I 197Hg 203Pb, or 2o6Bi.

[00024] In another embodiment, the aforementioned method is provided wherein the FZD10 modulator is selected from the group consisting of a small molecule, an oligonucleotide, a mimetic, a soluble receptor, a decoy receptor, or an antibody. In another embodiment, the aforementioned method is provided wherein the FZD10 modulator is a monoclonal antibody which selectively binds FZD10 and decreases phosphorylation of Dishevelled. In yet another embodiment, the aforementioned method is provided wherein the antiobody is conjugated to a toxic effector molecule.

[00025] In still another embodiment of the invention, a method of expressing an anti-FZD10 antibody in a CHO or myeloma cell is provided wherein the anti-FZD10 antibody inhibits one or more FZD10-related biological activities, the method comprising expressing a nucleic acid encoding the anti-FZD10 antibody in said CHO or myeloma cell. In yet another embodiment, a method of identifying a cancer inhibitor, the cancer characterized by overexpression of FZD10 compared to a control is provided, the method comprising contacting a cell expressing FZD10 with a candidate compound and determining whether an FZD10-related biological activity is induced, wherein the modulated FZD10-related biological activity is selected from the group consisting of phosphorylation of Dishevelled, c-Jun, and (3-catenin, wherein modulation of the FZD10-related biological activity is indicative of a cancer inhibitor.

[00026] These and other aspects of the present invention will be elucidated in the following detailed description of the invention.

[00027] BRIEF DESCRIPTION OF THE DRAWINGS

[00028] Figure 1 shows the up-regulation of FZD10 in lung, ovary and uterine cancers.

[00029] Figure 2 shows the up-regulation of FZD10 in LCM dissected colon cancer.

[00030] Figure 3 shows FZD10 quatitative RT-PCR of primary colon, lung and ovarian cancer patients.

[00031] Figure 4 shows the surface expression of FZD10 in different cancer cell lines.

[00032] Figure 5 shows siRNAs reduce surface expression of FZD10 protein and mRNA
in NCI-H460 cells.

[00033] Figure 6 shows FZD10 knockdown inhibits growth and induces apoptosis in NCI-H460 NSCLC cell line.

[00034] Figure 7 shows FZD10 knockdown inhibits growth and induces apoptosis in EKVX NSCLC cell line.

[00035] Figure 8 shows FZD10 knockdown does not inhibit growth and induces apoptosis in APC mutated colon cancer cell line SW620.

[00036] Figure 9 shows FZD10 knockdown does not inhibit growth and induces apoptosis in APC mutated colon cancer cell line Co1o320DM.

[00037] Figure 10 shows anti-FZD1- antibodies recognize FZD10 in cancer cell lines.

[00038] Figure 11 shows over expression of FZD9 or FZD10 resulted in hyper-phosphorylation of both Dv12 and Dv13.

[00039] DETAILED DESCRIPTION

[00040] The present invention provides methods and compositions for the treatment, diagnosis and imaging of cancer, in particular for the treatment, diagnosis and imaging of FZD10-related cancer.

[00041] Definitions [00042] Various definitions are used throughout this document. Most words have the meaning that would be attributed to those words by one skilled in the art.
Words specifically defined either below or elsewhere in this document have the meaning provided in the context of the present invention as a whole and as are typically understood by those skilled in the art.

[00043] The practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, molecular biology, immunology and pharmacology, within the skill of the art. Such techniques are explained fully in the literature.
See, e.g., Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990); Methods In Enzymology (S. Colowick and N. Kaplan, eds., Academic Press, Inc.); and Handbook of Experimental Immunology, Vols. I-IV
(D.M. Weir and C.C. Blackwell, eds., 1986, Blackwell Scientific Publications); and Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989).

[00044] As used herein, the singular forms "a," "an" and "the" include plural references unless the content clearly dictates otherwise. Thus, for example, reference to "an antibody"
includes a mixture of two or more such antibodies.

[00045] As used herein, the term "about" refers to +/- 30%, +/- 20%, +/- 10%, or +/- 5% of a value. As used herein, the terms "FZD10" or "Frizzled-10" refers to a receptor of SEQ ID
NO:2, or naturally occurring splice or allelic variant thereof. Wnt-1, Wnt3a (Munji, R. N et al., Developmental Biology 271 (2): 605-605, 2004-abstract from Meeting), Wnt-2 (Saitoh, T
et al., International Journal of Oncology 20 (1): 117-120, 2002), Wnt-7a (Kawakami, Y. et al., Dev Growth Differ 42 (6): 561-9, 2000), Wnt7b (Wang, Z. et al., Mol Cell Bio125 (12): 5022-30, 2005 ), Wnt-8 (Terasaki, H. et al., Int J Mol Med 9(2):107-12, 2002), Wnt-3 and non-WNT ligand HIG2 (Togashi, A. et al., Cancer Res 65 (11): 4817-26, 2005 ) are believed to be ligands of Frizzled-10 .

[00046] In GeneCard, FZD10 is also known as FZ-10, frizzled (Drosophila) homolog 10, frizzled homolog 10 (Drosophila), Frizzled 10 precursor, Frizzled-10, Fz-10, FzE7, FzE7, hFz10, and hFz10. The sequence of the FZD10 is set forth in accession number NM_007197 The nucleotide sequence and amino acid sequence are set out in SEQ ID NO:1 and SEQ ID
NO:2, respectively. Frizzled-10 is a 7-transmembrane receptor of 581 amino acids that has a N-terminal cysteine-rich extracellular domain at positions 29-150 of SEQ ID
NO:2 that contains the putative extracellular binding domain. Other domains are located at: positions 21-221 of SEQ ID NO:2 as large first extracellular domain; positions 284-312 of SEQ ID
NO:2 as second extracellular domain; positions 373-394 of SEQ ID NO:2 as third extracellular domain; and positions 465-503 of SEQ ID NO:2 as fourth extracellular domain;
positions 226-246 of SEQ ID NO:2 as first transmembrane domain; positions 263-283 of SEQ
ID NO:2 as second transmembrane domain; positions 312-332 of SEQ ID NO:2 as third transmembrane domain; positions 352-372 of SEQ ID NO:2 as fourth transmembrane domain;
positions 394-414 of SEQ ID NO:2 as fifth transmembrane domain; positions 444-464 of SEQ
ID NO:2 as sixth transmembrane domain; positions 503-523 of SEQ ID NO:2 as seventh transmembrane domain.

[00047] The terms "polypeptide" and "protein", are used interchangeably and refer to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. The term includes fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequence, fusions with heterologous and homologous leader sequences, with or without N-terminal methionine residues;
immunologically tagged proteins; and the like.

[00048] The terms "individual", "subject", "host" and "patient" are used interchangeably and refer to any subject for whom diagnosis, treatment, or therapy is desired, particularly humans. Other subjects may include cattle, dogs, cats, guinea pigs, rabbits, rats, mice, horses, and the like. In some preferred embodiments the subject is a human.

[00049] As used herein, "cancer" refers to primary or metastatic cancers. The term "cancer cells" refers to cells that are transformed. These cells can be isolated from a patient who has cancer, or be cells that are transformed in vitro to become cancerous. Cancer cells can be derived from many types of samples including any tissue or cell culture line.
In some embodiments the cancer cells are hyperplasias, tumor cells, or neoplasms. In some embodiments, the cancer cells are isolated from ovarian, uterine, colon, esophageal, breast, prostate, leukemia, melanoma, lung, brain, liver, pancreas, and lymphoma cancers. In some embodiments, the cancer cells are taken from established cell lines that are publicly available.
In some embodiments, cancer cells are isolated from pre-existing patient samples or from libraries comprising cancer cells. In some embodiments, cancer cells are isolated and then implanted in a different host, e.g., in a xenograft. In some embodiments cancer cells are transplanted and used in a SCID mouse model. In some embodiments, the cancer is ovarian, colon, lung or uterine cancer.

[00050] As used herein, the term "transformed" refers to any alteration in the properties of a cell that is stably inherited by its progeny. In some preferred embodiments, "transformed"
refers to the change of normal cell to a cancerous cell, e.g., one that is capable of causing tumors. In some embodiments, a transformed cell is immortalized.

[00051] As used herein, the term "metastasis" refers to a cancer which has spread to a site distant from the origin of the cancer, e.g. from the primary tumor. Sites of metastasis include without limitation, the bone, lymph nodes, lung, liver, and brain.

[00052] As used herein, the term "clinical endpoint" refers to a measurable event indicative of cancer. Clinical endpoints include without limitation, time to first metastasis, time to subsequent metastasis, size and/or number of metastases, size and/or number of tumors, location of tumors, aggressiveness of tumors, quality of life, pain and the like. Those skilled in the art are credited with the ability to determine and measure clinical endpoints. Methods of measuring clinical endpoints are known to those of skill in the art.

[00053] As used herein, the term "sample" refers to biological material from a patient. The sample assayed by the present invention is not limited to any particular type.
Samples include, as non-limiting examples, single cells, multiple cells, tissues, tumors, biological fluids, biological molecules, or supernatants or extracts of any of the foregoing. Examples include tissue removed for biopsy, tissue removed during resection, blood, urine, lymph tissue, lymph fluid, cerebrospinal fluid, mucous, and stool samples. The sample used will vary based on the assay format, the detection method and the nature of the tumors, tissues, cells or extracts to be assayed. Methods for preparing samples are well known in the art and can be readily adapted in order to obtain a sample that is compatible with the method utilized.

[00054] As used herein, the term "biological molecule" includes, but is not limited to, polypeptides, nucleic acids, and saccharides.

[00055] As used herein, the term "modulating" refers to a change in the quality or quantity of a gene, protein, or any molecule that is inside, outside, or on the surface of a cell. The change can be an increase or decrease in expression or level of the molecule.
The term "modulates" also includes changing the quality or quantity of a biological function/activity including, without limitation, proliferation, secretion, adhesion, apoptosis, cell-to-cell signaling, and the like. For example, in the context of "modulating cell division", the term refers to affecting the rate, amount or degree of cell division. In some embodiments, the methods will completely inhibit cell division. In other embodiments, the methods will decrease the amount of cell division. In other embodiments, the methods will prevent cell division.

[00056] As used herein, the term "N-terminus" refers to the first 10 amino acids of a protein.

[00057] As used herein, the term "C-terminus" refers to the last 10 amino acids of a protein.

[00058] As used herein, the term "cell-cell interaction" refers to an interaction between two or more cells. In some embodiments, the interaction between the cells leads to a cell signal.
Cell-cell interaction can be detected via a number of methods known to those of skill in the art, including, without limitation, the observation of membrane exchange between co-cultured, pre-labeled cells, labeled, for example, with different fluorescent membrane stains including PKH26 and PKH67 (Sigma).

[00059] The term "domain" as used herein refers to a structural part of a biomolecule that contributes to a known or suspected function of the biomolecule. Domains may be co-extensive with regions or portions thereof and may also incorporate a portion of a biomolecule that is distinct from a particular region, in addition to all or part of that region.

[00060] As used herein, the term "ligand binding domain" refers to any portion or region of a receptor retaining at least one qualitative binding activity of a corresponding native sequence FZD 10 receptor.

[00061] The term "region" refers to a physically contiguous portion of the primary structure of a biomolecule. In the case of proteins, a region is defined by a contiguous portion of the amino acid sequence of that protein. In some embodiments a "region" is associated with a function of the biomolecule.

[00062] The term "portion" as used herein refers to a physically contiguous portion of the primary structure of a biomolecule. In the case of proteins, a portion is defined by a contiguous portion of the amino acid sequence of that protein and refers to at least 3-5 amino acids, at least 5-7 amino acids, at least 8-10 amino acids, at least 11-15 amino acids, at least 17-24 amino acids, at least 25-30 amino acids, and at least 30-45 amino acids.
In the case of oligonucleotides, a portion is defined by a contiguous portion of the nucleic acid sequence of that oligonucleotide and refers to at least 9-15 nucleotides, at least 18-30 nucleotides, at least 33-45 nucleotides, at least 48-72 nucleotides, at least 75-90 nucleotides, and at least 90-130 nucleotides. In some embodiments, portions of biomolecules have a biological activity.

[00063] As used herein, the term "agonist" refers to a molecule which is capable of binding to FZD10 and activating one or more biological activities of FZD10. FZD10 agonists include native FZD10 ligands including Wnt-1, Wnt-3 and Wnt-7b, as well as antibodies, other proteins or small molecules that bind to and activate one or more biological activities of [00064] As used herein, the phrase "induce" refers to a stimulation of an activity.

[00065] As used herein, the phrase "FZD10 biological activity" refers to a biological, physiological, or biochemical activity of FZD10 affected by activation of the receptor.
FZD10 may be activated or inhibited by FZD10 modulators including ligands of FZD10 or muteins thereof, oligonucleotide modulators, small molecules, mimetics, decoys or antibodies.
Examples of FZD10 biological activities include without limitation, activation of RHOA, RAC, APC, (3-catenin, JNK, pS63c-Jun, cell adhesion, cell migration, cell survival, cell proliferation, cell apoptosis, dimerization of receptor, internalization of receptor, activation of Disheveled, and induction or stabilization of 0-catenin, or any two or more, or three or more of the preceding activities, and the like.

[00066] As used herein, the phrase "FZD10-related cells/tumors/samples" and the like refers to cells, samples, tumors or other pathologies that are characterized by increased evidence of FZD10 expression relative to non-cancerous and/or non-metastatic cells, samples, tumors, or other pathologies. In some preferred embodiments, FZD10 -related cells, samples, tumors or other pathologies are characterized by increased evidence of FZD10 expression relative to non-metastatic cells, samples, tumors, or other pathologies.

[00067] As used herein, the term "modulator" refers to a composition that modulates one or more physiological or biochemical events associated with cancer. In some preferred embodiments the modulator inhibits one or more biological activities associated with cancer.
In some embodiments the modulator is a small molecule, an antibody as that term is defined herein, a mimetic, a soluble receptor, a decoy receptor or an oligonucleotide modulator as that term is defined herein. In some embodiments the modulator acts by blocking ligand binding or by competing for a ligand-binding site. In some embodiments the modulator acts independently of ligand binding. In some embodiments the modulator does not compete for a ligand binding site. In some embodiments the modulator blocks expression of a gene product involved in cancer. In some embodiments the modulator blocks a physical interaction of two or more biomolecules involved in cancer. In some embodiments modulators of the invention induce or inhibit one or more FZD10 biological activities . In some embodiments the FZD10 modulator inhibits FZD10 expression.

[00068] As used herein, the term "antibody" refers to monoclonal and polyclonal antibodies, single chain antibodies, chimeric antibodies, bifunctional/bispecific antibodies, humanized antibodies, human antibodies, and complementary determining region (CDR)-grafted antibodies, that are specific for the target protein or fragments thereof. As used herein, the term "antibody" refers to intact molecules as well as to fragments thereof, such as Fab, F(ab')2 and Fv, which are capable of binding to the antigenic determinant in question. Further examples of antibodies include a fully assembled antibody, monoclonal antibody, polyclonal antibody, multispecific antibody (e.g., bispecific antibody), single chain antibody, chimeric antibody, humanized antibody, human engineered antibody, human antibody, diabody, triabody, tetrabody, minibody, linear antibody, chelating recombinant antibody, an intrabody, a nanobody, a small modular immunopharmaceutical (SMIP), an antigen-binding-domain immunoglobulin fusion protein, a camelized antibody, a VHH containing antibody, or a mutein of any one of these antibodies that comprise one or more CDR sequences of the antibody, and recombinant peptides comprising the forgoing as long as they exhibit the desired biological activity. (e.g., binding to the extracellular domain of FZD10).

[00069] As used herein, the term "epitope" refers to an antigenic determinant of a polypeptide. In some embodiments an epitope may comprise 3 or more amino acids in a spatial conformation which is unique to the epitope. In some embodiments epitopes are linear or conformational epitopes. Generally an epitope consists of at least 4 such amino acids, at least 5-7 amino acids, and more usually, consists of at least 8-10 such amino acids. Methods of determining the spatial conformation of amino acids are known in the art, and include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance.

[00070] As used herein, the term "oligonucleotide" refers to a series of linked nucleotide residues or bases, and may include naturally occurring or artificial or modified bases. The term "oligonucleotide modulator" includes antisense RNA or DNA, siRNA or RNAi, ribozymes and DNAzymes, and refers to an oligonucleotide that comprises at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more consecutive bases (which may include non-natural, modified bases) that are complementary to a fragment of SEQ ID NO: 1 and modulate expression of its protein product.

[00071] As used herein, the term "decoy receptor" refers to a receptor comprising at least a portion of a polypeptide, mimetic, or other macromolecule capable of binding a ligand.

[00072] As used herein, the term "therapeutically effective amount" is meant to refer to an amount of a medicament which produces a medicinal effect observed as reduction or reverse in one or more clinical endpoints, growth and/or survival of cancer cell, or metastasis of cancer cells in an individual when a therapeutically effective amount of the medicament is administered to the individual. Therapeutically effective amounts are typically determined by the effect they have compared to the effect observed when a composition which includes no active ingredient is administered to a similarly situated individual. The precise effective amount for a subject will depend upon the subject's size and health, the nature and extent of the condition, and the therapeutics or combination of therapeutics selected for administration.
However, the effective amount for a given situation is determined by routine experimentation and is within the judgment of the clinician.

[00073] As used herein, the phrase "modulating cell-cell adhesion" refers to a change in the adhesion or cell to cell contact of one cell with another. In some embodiments, cell adhesion is inhibited by the modulators of the present invention.

[00074] As used herein, the terms "in combination with" or "in conjunction with" refer to administration of the FZD10 modulators of the invention with other therapeutic regimens.
Compositions comprising one or more FZD10 modulator may be administered to persons or mammals suffering from, or predisposed to suffer from, cancer. A FZD10 modulator may also be administered with another therapeutic agent, such as a cytotoxic agent, or cancer chemotherapeutic. Concurrent administration of two therapeutic agents does not require that the agents be administered at the same time or by the same route, as long as there is an overlap in the time period during which the agents are exerting their therapeutic effect. Simultaneous or sequential administration is contemplated, as is administration on different days or weeks.

[00075] As used herein, the term "susceptible" refers to patients for whom FZD10 therapy is an acceptable method of treatment, i.e., patients who are likely to respond positively.
Cancer patients susceptible to FZD10 therapy express high levels of FZD10 relative to those patients not susceptible to FZD10 therapy. Cancer patients who might not be good candidates for FZD10 therapy include cancer patients with tumor samples that lack or have lower levels of FZD10 in or on their cancer cells.

[00076] As used herein, the term "detecting" means to establish, discover, or ascertain evidence of an activity (for example, gene expression) or biomolecule (for example, a polypeptide).

[00077] As used herein, the phrase "homologous nucleotide sequence," or "homologous amino acid sequence," or variations thereof, refers to sequences characterized by a homology, at the nucleotide level or amino acid level, of at least a specified percentage and is used interchangeably with "sequence identity". Homologous nucleotide sequences include those sequences coding for isoforms of proteins. Such isoforms can be expressed in different tissues of the same organism as a result of, for example, alternative splicing of RNA.
Alternatively, isoforms can be encoded by different genes. Homologous nucleotide sequences include nucleotide sequences encoding for a protein of a species other than humans, including, but not limited to, mammals. Homologous nucleotide sequences also include, but are not limited to, naturally occurring allelic variations and mutations of the nucleotide sequences set forth herein. Homologous amino acid sequences include those amino acid sequences which contain conservative amino acid substitutions and which polypeptides have the same binding and/or activity.

[00078] Percent homology or identity can be determined by, for example, the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison WI), using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489). In some preferred embodiments, homology between the probe and target is between about 50% to about 60%. In some embodiments, nucleic acids have nucleotides that are about 60%, preferably about 70%, more preferably about 80%, more preferably about 85%, more preferably about 90%, more preferably about 92%, more preferably about 94%, more preferably about 95%, more preferably about 97%, more preferably about 98%, more preferably about 99% and most preferably about 100% homologous to SEQ ID NO: 1, or a portion thereof.

[00079] Homology may also be at the polypeptide level. In some embodiments, polypeptides are about 60%, about 70%, about 80%, about 85%, about 90%, about 92%, about 94%, about 95%, about 97%, about 98%, about 99% and about 100% homologous to SEQ ID
NO:2 or a portion thereof.

[00080] As used herein, the term "probe" refers to nucleic acid sequences of variable length. In some embodiments probes comprise at least about 10 and as many as about 6,000 nucleotides. In some embodiments probes comprise at least 12, at least 14, at least 16, at least 18, at least 20, at least 25, at least 50 or at least 75 consecutive nucleotides. Probes are used in the detection of identical, similar, or complementary nucleic acid sequences.
Longer length probes are usually obtained from natural or recombinant sources, are highly specific to the target sequence, and are much slower to hybridize to the target than are oligomers. Probes may be single- or double-stranded and are designed to have specificity in PCR, hybridization membrane-based, in situ hybridization (ISH), fluorescent in situ hybridization (FISH), or ELISA-like technologies.

[00081] As used herein, the term "mixing" refers to the process of combining one or more compounds, cells, molecules, and the like together in the same area. This may be performed, for example, in a test tube, petri dish, or any container that allows the one or more compounds, cells, or molecules, to be mixed.

[00082] As used herein the term "isolated" refers to a polynucleotide, a polypeptide, an antibody, or a host cell that is in an environment different from that in which the polynucleotide, the polypeptide, or the antibody naturally occurs. Methods of isolating cells are well known to those skilled in the art. A polynucleotide, a polypeptide, or an antibody which is isolated is generally substantially purified.

[00083] As used herein, the term "substantially purified" refers to a compound (e.g., either a polynucleotide or a polypeptide or an antibody) that is removed from its natural environment and is at least 60% free, at least 75% free, at least 85% free, at least 90%, at least 95%, 98%
and 99% free from other components with which it is naturally associated.

[00084] As used herein, the term "binding" means the physical or chemical interaction between two or more biomolecules or compounds. Binding includes ionic, non-ionic, hydrogen bonds, Van der Waals, hydrophobic interactions, etc. Binding can be either direct or indirect, indirect being through or due to the effects of another biomolecule or compound.
Direct binding refers to interactions that do not take place through or due to the effect of another molecule or compound but instead are without other substantial chemical intermediates.

[00085] As used herein, the term "contacting" means bringing together, either directly or indirectly, one molecule into physical proximity to a second molecule. The molecule can be in any number of buffers, salts, solutions, etc. "Contacting" includes, for example, placing a polynucleotide into a beaker, microtiter plate, cell culture flask, or a microarray, or the like, which contains a nucleic acid molecule. Contacting also includes, for example, placing an antibody into a beaker, microtiter plate, cell culture flask, or microarray, or the like, which contains a polypeptide. Contacting may take place in vivo, ex vivo, or in vitro.

[00086] As used herein, the phrase "stringent hybridization conditions" or "stringent conditions" refers to conditions under which a probe, primer, or oligonucleotide will hybridize to its target sequence, but to a minimal number of other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences will hybridize with specificity to their proper complements at higher temperatures.
Generally, stringent conditions are selected to be about 5 C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50%
of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. Since the target sequences are generally present in excess, at Tm, 50% of the probes are hybridized to their complements at equilibrium. Typically, stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30 C for short probes, primers or oligonucleotides (e.g., 10 to 50 nucleotides) and at least about 60 C for longer probes, primers or oligonucleotides. Stringent conditions may also be achieved with the addition of destabilizing agents, such as formamide.

[00087] As used herein, the term "moderate stringency conditions" refers to conditions under which a probe, primer, or oligonucleotide will hybridize to its target sequence, but to a limited number of other sequences. Moderate conditions are sequence-dependent and will be different in different circumstances. Moderate conditions are well-known to the art skilled and are described in, inter alia, Maniatis et al. (Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory; 2nd Edition (December 1989)).

[00088] The nucleic acid compositions described herein can be used, for example, to produce polypeptides, as probes for the detection of mRNA in biological samples (e.g., extracts of human cells) or cDNA produced from such samples, to generate additional copies of the polynucleotides, to generate ribozymes or oligonucleotides (single and double stranded), and as single stranded DNA probes or as triple-strand forming oligonucleotides.
The probes described herein can be used to, for example, determine the presence or absence of the polynucleotides provided herein in a sample. The polypeptides can be used to generate antibodies specific for a polypeptide associated with cancer, which antibodies are in turn useful in diagnostic methods, prognostic methods, and the like as discussed in more detail herein. Polypeptides are also useful as targets for therapeutic intervention, as discussed in more detail herein. Antibodies of the present invention may also be used, for example, to purify, detect, and target the polypeptides of the present invention, including both in vitro and in vivo diagnostic and therapeutic methods. For example, the antibodies are useful in immunoassays for qualitatively and quantitatively measuring levels of the polypeptides of the present invention in biological samples. See, e.g., Harlow et al., Antibodies:
A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988). These and other uses are described in more detail below.

[00089] As used herein the term "imaging agent" refers to a composition linked to an antibody, small molecule, or probe of the invention that can be detected using techniques known to the art-skilled. As used herein, the term "evidence of gene expression" refers to any measurable indicia that a gene is expressed.

[00090] The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, such as antibodies or a polypeptide, genes, and other therapeutic agents. The term refers to any pharmaceutical carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition, and which can be administered without undue toxicity. Suitable carriers can be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates and inactive virus particles. Such carriers are well known to those of ordinary skill in the art.
Pharmaceutically acceptable carriers in therapeutic compositions can include liquids such as water, saline, glycerol and ethanol. Auxiliary substances, such as wetting or emulsifying agents, pH
buffering substances, and the like, can also be present in such vehicles.

[00091] Specific examples of cancers that can be treated by the methods and compositions of the present invention include, but are not limited to, cancers that overexpress FZD 10. The present invention is also applicable to any tumor cell-type where FZD10 plays a role in cell adhesion, migration or repulsion. In some embodiments, the cancer is ovarian, colon, lung or uterine cancer. In some embodiments, the cancers amenable to treatment and/or diagnosis according to the present invention are characterized by overexpression of FZD10. In some embodiments, such cancers exhibit overexpression of FZD10 by at least about 25%, at least about 50%, at least about 75%, at least about 100%, at least about 150%, at least about 200%, or at least about about 300% as compared to a control non-cancerous cell or a non-cancerous tissue.

[00092] The present invention provides methods and compositions that provide for the treatment, inhibition, and management of diseases and disorders associated with FZD10 overexpression as well as the treatment, inhibition, and management of symptoms of such diseases and disorders. Some embodiments of the invention relate to methods and compositions comprising compositions that inhibit cancer cell proliferation and invasion.

[00093] The present invention further provides methods and compositions for the treatment, inhibition, or management of cancer or cancer metastases. Further compositions and methods of the invention include other active ingredients in combination with the FZD 10 modulators of the present invention. In some embodiments, the methods further comprise administering one or more traditional cancer therapeutics to the patient. In some embodiments the methods of the present invention further comprise treating the patient with one or more of chemotherapy, radiation therapy or surgery.

[00094] The present invention also provides methods and compositions for the treatment, inhibition, and management of cancer or other hyperproliferative cell disorder or disease that has become partially or completely refractory to current or standard cancer treatment, such as surgery, chemotherapy, radiation therapy, hormonal therapy, and biological therapy.

[00095] The invention also provides diagnostic and/or imaging methods using the FZD10 modulators of the invention, particularly FZD10 antibodies, to diagnose cancer and/or predict cancer progression. In some preferred embodiments, the methods of the invention provide methods of imaging and localizing tumors and/or metastases and methods of diagnosis and prognosis. In some embodiments, the methods of the invention provide methods to evaluate the appropriateness of FZD10 -related therapy.

[00096] FZD10 Modulators [00097] The present invention provides FZD10 modulators for, inter alia, the treatment, diagnosis, detection or imaging of cancer. In some embodiments, the FZD10 modulator is an oligonucleotide, a small molecule, a mimetic, a soluble receptor, a decoy receptor, or an antibody. In some embodiments, the FZD10 modulator induces activation of RHOA, RAC, APC, 0-catenin, JNK, ps63c-Jun, cell adhesion, cell migration, cell survival, cell proliferation, cell apoptosis, dimerization of receptor, internalization of receptor, phosphorylation of Disheveled, and induction or stabilization of 0-catenin, or any two or more, or three or more of the preceding activities, and the like. In some embodiments the FZD10 modulator inhibits FZD10 binding to intracellular adaptor proteins. In some embodiments the FZD10 modulator stimulates binding to intracellular adaptor proteins such as Dishevelled. In some embodiments, the FZD 10 modulator inhibits and/or inactivates the c-Jun and/or the 0-catenin pathways.

[00098] As used herein, the term "intracellular adaptor proteins" refers to a protein that connects different segments of a signaling complex. The adaptor protein may or may not have enzymatic activity. In some embodiments the adaptor protein is Dishevelled.

[00099] In some embodiments, the FZD10 modulator increases 0-catenin phopshorylation, decreases c-Jun phosphorylation, and/or decreases Disevelled phosphorylation by 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, 97%, 98%, 99% or 100%, as compared to a control. Methods are known in the art to determine the level of receptor phosphorylation and can be used to assay candidate FZD10 modulators in order to determine their properties.
Examples of such methods are described herein.

[000100] In some embodiments, the FZD10 modulator inhibits FZD10 expression.
In some embodiments, FZD10 expression is inhibited by 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, 97%, 98%, 99% or 100%, as compared to a control. Methods of determining levels of FZD10 expression are known to those of skill in the art.

[000101] Antibodies [000102] The invention uses antibodies that bind to FZD10 polypeptides expressed by the FZD10 gene and preferably bind specifically to such polypeptides. The term "binds specifically" means that the antibodies have substantially greater affinity for their target FZD10 polypeptide than their affinity for other related polypeptides. As used herein, the term "antibody" refers to intact molecules as well as to fragments thereof, such as Fab, F(ab')2 and Fv, which are capable of binding to the antigenic determinant in question.
Further examples of antibodies include a fully assembled antibody, monoclonal antibody, polyclonal antibody, multispecific antibody (e.g., bispecific antibody), single chain antibody, chimeric antibody, humanized antibody, human engineered antibody, human antibody, diabody, triabody, tetrabody, minibody, linear antibody, chelating recombinant antibody, an intrabody, a nanobody, a small modular immunopharmaceutical (SMIP), an antigen-binding-domain immunoglobulin fusion protein, a camelized antibody, a VHH containing antibody, or a mutein of any one of these antibodies that comprise one or more CDR sequences of the antibody, and recombinant peptides comprising the forgoing as long as they exhibit the desired biological activity. (e.g., binding to the extracellular domain of FZD10). By "substantially greater affinity" we mean that there is a measurable increase in the affinity for the target FZD10 polypeptide of the invention as compared with the affinity for other related polypeptide. Preferably, the affinity is at least 1.5-fold, 2-fold, 5-fold 10-fold, 100-fold, 103-fold, 104-fold, 105-fold, 106-fold or greater for the target FZD10 polypeptide.

[000103] Preferably, the antibodies bind to FZD10 with high affinity, preferably with a dissociation constant of 10-4M, 10-5M, 10-6M or less, preferably 10-7M, 10-$M
or less, most preferably 10-9M or 10-10M or less; subnanomolar affinity (0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3 ,0.2, 0.1 nM or even less) is preferred. The antibodies may bind specifically to the extracellular domain FZD10.

[000104] When the FZD10 polypeptide are to be used to generate antibodies, for example for immunotherapy, the FZD 10 should share at least one epitope or determinant with the full-length protein. By "epitope" or "determinant" herein is meant a portion of a protein that will generate and/or bind an antibody or T-cell receptor in the context of MHC.
Thus, in most instances, antibodies made to a smaller FZD10 polypeptide will be able to bind to the full-length protein. In another embodiment, the epitope is unique; that is, antibodies generated to a unique epitope show little or no cross-reactivity.

[000105] Any polypeptide sequence encoded by the FZD10 gene may be analyzed to determine certain preferred regions of the polypeptide. Regions of high antigenicity are determined from data by DNASTAR analysis by choosing values that represent regions of the polypeptide that are likely to be exposed on the surface of the polypeptide in an environment in which antigen recognition may occur in the process of initiation of an immune response.
For example, the amino acid sequence of a polypeptide encoded by the FZD10 gene sequence may be analyzed using the default parameters of the DNASTAR computer algorithm (DNASTAR, Inc., Madison, Wis.; http://www.dnastar.com/).

[000106] Preferably, antibodies of the invention are specific for a non-conserved region of FZD10. `Non-conserved region' refers to a region of lower homology with other members of the Frizzled family. Preferably, similarity to other Frizzled family members in these non-conserved regions is lower than 50%.

[000107] In one embodiment, the anti-FZD10 antibody is specific for FZD10 and does not cross react with other members of the Frizzled family (e.g., FZD9).
Alternatively, the anti-FZD 10 antibody also blocks other FZD family members that may have redundant function. In another embodiment, the anti-FZD10 antibody also binds to murine FZD10.
Alternatively, the anti-FZD10 antibody does not cross react with murine FZD10.

[000108] Polypeptide features that may be routinely obtained using the DNASTAR
computer algorithm include, but are not limited to, Garnier-Robson alpha-regions, beta-regions, turn-regions, and coil-regions (Garnier et al. J. Mol. Biol., 120: 97 (1978)); Chou-Fasman alpha-regions, beta-regions, and turn-regions (Adv. in Enzymol., 47:45-148 (1978));
Kyte-Doolittle hydrophilic regions and hydrophobic regions (J. Mol. Biol., 157:105-132 (1982)); Eisenberg alpha- and beta-amphipathic regions; Karplus-Schulz flexible regions;
Emini surface-forming regions (J. Virol., 55(3):836-839 (1985)); and Jameson-Wolf regions of high antigenic index (CABIOS, 4(1):181-186 (1988)). Kyte-Doolittle hydrophilic regions and hydrophobic regions, Emini surface-forming regions, and Jameson-Wolf regions of high antigenic index (i.e., containing four or more contiguous amino acids having an antigenic index of greater than or equal to 1.5, as identified using the default parameters of the Jameson-Wolf program) can routinely be used to determine polypeptide regions that exhibit a high degree of potential for antigenicity. One approach for preparing antibodies to a protein is the selection and preparation of an amino acid sequence of all or part of the protein, chemically synthesizing the sequence and injecting it into an appropriate animal, typically a rabbit, hamster or a mouse. Oligopeptides can be selected as candidates for the production of an antibody to the FZD10 protein based upon the oligopeptides lying in hydrophilic regions, which are thus likely to be exposed in the mature protein. Additional oligopeptides can be determined using, for example, the Antigenicity Index, Welling, G.W. et al., FEBS Lett.
188:215-218 (1985), incorporated herein by reference.

[000109] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations that are typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they are synthesized by the homogeneous culture, uncontaminated by other immunoglobulins with different specificities and characteristics.

[000110] The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al., Nature, 256:495 [19751, or may be made by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567). The "monoclonal antibodies" may also be isolated from phage antibody libraries using the techniques described inClackson et al.,Nature,352:624628[1991] end Marks et al., J. Mol. Biol., 222:581-597 (1991), for example.

[000111] Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes. There are five major classes, IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses or isotypes, e.g. IgG1, IgG2, IgG3, IgG4, IgAl and IgA2. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma and mu respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. Different isotypes have different effector functions; for example, IgG1 and IgG3 isotypes have ADCC activity.

[000112] "Antibody fragments" comprise a portion of an intact full length antibody, preferably the antigen binding or variable region of the intact antibody.
Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (Zapata et al., Protein Eng.,8(10):1057-1062 (1995)); single-chain antibody molecules;
and multispecific antibodies formed from antibody fragments. Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, whose name reflects its ability to crystallize readily. Pepsin treatment yields an F(ab')2 fragment that has two "Single-chain Fv" or "sFv"
antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. Preferably, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the Fv to form the desired structure for antigen binding. For a review of sFv see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 1 13, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[000113] The term "hypervariable" region refers to the amino acid residues of an antibody which are responsible for antigen-binding. The hypervariable region comprises amino acid residues from a complementarity determining region or CDR [i.e., residues 24-34 (Ll), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain as described by Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)] and/or those residues from a hypervariable loop (i.e., residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain as described by [Chothia et al., J.
Mol.Biol. 196: 901-917 (1987)].

[000114] "Framework" or FR residues are those variable domain residues other than the hypervariable region residues.

[000115] Polyclonal Antibodies [000116] Methods of preparing polyclonal antibodies are known to the skilled artisan.
Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant. Typically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections. The immunizing agent may include a protein encoded by a nucleic acid of the figures or fragment thereof or a fusion protein thereof. It may be useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized.
Examples of such immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. Examples of adjuvants that may be employed include Freund's complete adjuvant and MPL-TDM
adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate). The immunization protocol may be selected by one skilled in the art without undue experimentation.

[000117] Monoclonal Antibodies [000118] Preferably the antibodies are monoclonal antibodies. Monoclonal antibodies may be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In a hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent.
Alternatively, the lymphocytes may be immunized in vitro. The immunizing agent will typically include a FZD10 polypeptide, or fragment thereof or a fusion protein thereof.
Generally, either peripheral blood lymphocytes ("PBLs") are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103).
Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed. The hybridoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine ("HAT medium"), which substances prevent the growth of HGPRT-deficient cells.

[000119] Monoclonal antibody technology is used in implementing research, diagnosis and therapy. Monoclonal antibodies are used in radioimmunoas says, enzyme-linked immunosorbent assays, immunocytopathology, and flow cytometry for in vitro diagnosis, and in vivo for diagnosis and immunotherapy of human disease. Waldmann, T. A.
(1991) Science 252:1657-1662. In particular, monoclonal antibodies have been widely applied to the diagnosis and therapy of cancer, wherein it is desirable to target malignant lesions while avoiding normal tissue. See, e.g., U.S. Pat. Nos. 4,753,894 to Frankel, et al.; 4,938,948 to Ring et al.; and 4,956,453 to Bjorn et al.

[000120] The monoclonal antibodies to FZD10 may be used in a method of diagnosis of ovarian, colon, lung or uterine cancer either alone, or in conjunction with a method for the detection of the L1 adhesion molecule in serum.

[000121] Antibody fragments [000122] The term "antibody" as used herein includes antibody fragments, as are known in the art, including Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; multispecific antibodies such as bispecific, trispecific, etc.
antibodies; minibody; chelating recombinant antibody; tribodies; bibodies;
intrabodies;
nanobodies; small modular immunopharmaceuticals (SMIP), binding-domain immunoglobulin fusion proteins; camelized antibodies; VHH containing antibodies, chimeric antibodies, etc., and other polypeptides formed from antibody fragments either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA
technologies.

[000123] The term "diabodies" refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites.
Diabodies are described more fully in, for example, EP 404,097; WO 93/11161;
and 30 Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

[000124] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, monovalent fragments consisting of the VL, VH, CL and CH domains each with a single antigen-binding site, and a residual "Fc" fragment, whose name reflects its ability to crystallize readily. Pepsin treatment yields a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region, that has two "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain.
Preferably, the Fv polypeptide further comprises a polypeptide linker between the VH and VL
domains that enables the Fv to form the desired structure for antigen binding, resulting in a single-chain antibody (scFv), in which a VL and VH region are paired to form a monovalent molecule via a synthetic linker that enables them to be made as a single protein chain (Bird et al., Science 242:423-426, 1988, and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988). For a review of sFv see Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 1 13, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994). An Fd fragment consists of the VH and CHl domains.

[000125] Additional antibody fragment include a domain antibody (dAb) fragment (Ward et al., Nature 341:544-546, 1989) which consists of a VH domain.

[000126] "Linear antibodies" comprise a pair of tandem Fd segments (VH -CH1-VH
-CHl) which form a pair of antigen binding regions. Linear antibodies can be bispecific or monospecific (Zapata et al. Protein Eng. 8:1057-62 (1995)).

[000127] A "minibody" consisting of scFv fused to CH3 via a peptide linker (hingeless) or via an IgG hinge has been described in Olafsen, et al., Protein Eng Des Sel.

Apr;17(4):315-23.

[000128] Functional heavy-chain antibodies devoid of light chains are naturally occurring in nurse sharks (Greenberg et al., Nature 374:168-73, 1995), wobbegong sharks (Nuttall et al., Mol Immunol. 38:313-26, 2001) and Camelidae (Hamers-Casterman et al., Nature 363: 446-8, 1993; Nguyen et al., J. Mol. Biol. 275: 413, 1998), such as camels, dromedaries, alpacas and llamas. The antigen-binding site is reduced to a single domain, the VHH
domain, in these animals. These antibodies form antigen-binding regions using only heavy chain variable region, i.e., these functional antibodies are homodimers of heavy chains only having the structure H2L2 (referred to as "heavy-chain antibodies" or "HCAbs"). Camelized VHH
reportedly recombines with IgG2 and IgG3 constant regions that contain hinge, CH2, and CH3 domains and lack a CH1 domain (Hamers-Casterman et al., supra). For example, llama IgG1 is a conventional (H2L2) antibody isotype in which VH recombines with a constant region that contains hinge, CH1, CH2 and CH3 domains, whereas the llama IgG2 and IgG3 are heavy chain-only isotypes that lack CH1 domains and that contain no light chains.
Classical VH-only fragments are difficult to produce in soluble form, but improvements in solubility and specific binding can be obtained when framework residues are altered to be more VHH-like. (See, e.g., Reichman, etal.) [000129] J Immunol Methods 1999, 231:25-38.) Camelized VHH domains have been found to bind to antigen with high affinity (Desmyter et al., J. Biol. Chem.
276:26285-90, 2001) and possess high stability in solution (Ewert et al., Biochemistry 41:3628-36, 2002). Methods for generating antibodies having camelized heavy chains are described in, for example, in U.S.
Patent Publication Nos. 20050136049 and 20050037421.

[000130] Because the variable domain of the heavy-chain antibodies is the smallest fully functional antigen-binding fragment with a molecular mass of only 15 kDa, this entity is referred to as a nanobody (Cortez-Retamozo et al., Cancer Research 64:2853-57, 2004). A
nanobody library may be generated from an immunized dromedary as described in Conrath et al., (Antimicrob Agents Chemother 45: 2807-12, 2001) or using recombinant methods as described in [000131] Production of bispecific Fab-scFv ("bibody") and trispecific Fab-(scFv)(2) ("tribody") are described in Schoonjans et al. (J Immunol. 165:7050-57, 2000) and Willems et al. (J Chromatogr B Analyt Technol Biomed Life Sci. 786:161-76, 2003). For bibodies or tribodies, a scFv molecule is fused to one or both of the VL-CL (L) and VH-CH1 (Fd) chains, e.g., to produce a tribody two scFvs are fused to C-term of Fab while in a bibody one scFv is fused to C-term of Fab.

[000132] Intrabodies are single chain antibodies which demonstrate intracellular expression and can manipulate intracellular protein function (Biocca, et al., EMBO J.
9:101-108, 1990;
Colby et al., Proc Natl Acad Sci U S A. 101:17616-21, 2004). Intrabodies, which comprise cell signal sequences which retain the antibody contruct in intracellular regions, may be produced as described in Mhashilkar et al (EMBO J 14:1542-51, 1995) and Wheeler et al.
(FASEB J. 17:1733-5. 2003). Transbodies are cell-permeable antibodies in which a protein transduction domains (PTD) is fused with single chain variable fragment (scFv) antibodies Heng et al., (Med Hypotheses. 64:1105-8, 2005).

[000133] Further contemplated are antibodies that are SMIPs or binding domain immunoglobulin fusion proteins specific for target protein. These constructs are single-chain polypeptides comprising antigen binding domains fused to immunoglobulin domains necessary to carry out antibody effector functions. See e.g., W003/041600, U.S. Patent publication 20030133939 and US Patent Publication 20030118592.

[000134] Multispecific antibodies [000135] In some embodiments, it may be desirable to generate a multispecific (e.g.
bispecific, trispecific, etc.) monoclonal antibody having binding specificities for at least two different epitopes. Exemplary bispecific antibodies may bind to two different epitopes of FZD10. Alternatively, an anti-FZD10 arm may be combined with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g., CD2 or CD3), or Fc receptors for IgG (FcyR), such as FcyRI (CD64), FcyRII (CD32) and FcyRIII
(CD16) so as to focus cellular defense mechanisms to the FZD10-expressing cell.

[000136] In another example, one of the binding specificities is for a FZD10 polypeptide or a fragment thereof, the other one is for any other antigen, e.g., a cell-surface protein or receptor or receptor subunit, preferably one that is tumor specificor tissue specific. Examples include kidney cancer specific antigen, an ovarian cancer specific antigen, a colon cancer specific antigen, a lung cancer specific antigen, or a uterine cancer specific antigen.

[000137] Bispecific antibodies may also be used to localize cytotoxic agents to cells which express FZD10. These antibodies possess a FZD10-binding arm and an arm which binds the cytotoxic agent (e.g., saporin, anti-interferon-60, vinca alkaloid, ricin A
chain, methotrexate or radioactive isotope hapten). Bispecific antibodies can be prepared as full length antibodies or antibody fragments.

[000138] According to another approach for making bispecific antibodies, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture. The preferred interface comprises at least a part of the CH3 domain of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). Compensatory "cavities" of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers. See W096/27011 published Sep.
6, 1996.

[000139] Bispecific antibodies include cross-linked or "heteroconjugate"
antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin, the other to biotin. Heteroconjugate antibodies may be made using any convenient cross-linking methods.
Suitable cross-linking agents are well known in the art, and are disclosed in U.S. Pat. No.
4,676,980, along with a number of cross-linking techniques.

[000140] Techniques for generating bispecific antibodies from antibody fragments have also been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science 229:81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab')2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB
derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes. In yet a further embodiment, Fab'-SH fragments directly recovered from E. coli can be chemically coupled in vitro to form bispecific antibodies. (Shalaby et al., J. Exp. Med.
175:217-225 (1992)) [000141] Shalaby et al., J. Exp. Med. 175:217-225 (1992) describe the production of a fully humanized bispecific antibody F(ab')2 molecule. Each Fab' fragment was separately secreted from E.coli and subjected to directed chemical coupling in vitro to form the bispecfic antibody. The bispecific antibody thus formed was able to bind to cells overexpressing the HER2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.

[000142] Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. (Kostelny et al., J. Immunol.
148(5):1547-1553 (1992)) The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers.
This method can also be utilized for the production of antibody homodimers.
The "diabody"
technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments.

[000143] The fragments comprise a heavy chain variable region (VH) connected to a light-chain variable region (VL) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See Gruber et al., J. Immunol. 152:
5368 (1994).

[000144] Alternatively, the bispecific antibody may be a "linear antibody"
produced as described in Zapata et al. Protein Eng. 8(10):1057-1062 (1995). Briefly, these antibodies comprise a pair of tandem Fd segments (VH -CH1-VH -CH1) which form a pair of antigen binding regions. Linear antibodies can be bispecific or monospecific.

[000145] Antibodies with more than two valencies are also contemplated. For example, trispecific antibodies can be prepared. (Tutt et al., J. Immunol. 147:60 (1991))A "chelating recombinant antibody" is a bispecific antibody that recognizes adjacent and non-overlapping epitopes of the target antigen, and is flexible enough to bind to both epitopes simultaneously (Neri et al., J Mol Biol. 246:367-73, 1995).

[000146] In another embodiment, the antibodies to FZD10 are capable of reducing or eliminating the biological function of FZD10, as is described herein. That is, the addition of anti-FZD10 antibodies (either polyclonal or preferably monoclonal) may reduce or eliminate FZD10 activity. Generally, at least a 25% decrease in activity is detected, with at least about 50% and about a 95-100% decrease being exemplary embodiments.

[000147] Recombinant Production of Antibodies [000148] For recombinant production of the antibody, the nucleic acid encoding it is isolated and inserted into a replicable vector for further cloning (amplification of the DNA) or for expression. DNA encoding the monoclonal antibody is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody).
Many vectors are available. The vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more selective marker genes, an enhancer element, a promoter, and a transcription termination sequence, examples of which are well known in the art.

[000149] A variety of heterologous systems is available for functional expression of recombinant polypeptides that are well known to those skilled in the art. Such systems include bacteria (Strosberg, et al., Trends in Pharmacological Sciences (1992) 13:95-98), yeast (Pausch, Trends in Biotechnology (1997) 15:487-494), several kinds of insect cells (Vanden Broeck, Int. Rev. Cytology (1996) 164:189-268), amphibian cells (Jayawickreme et al., Current Opinion in Biotechnology (1997) 8: 629-634) and several mammalian cell lines (CHO, HEK293, COS, etc.; see Gerhardt, et al., Eur. J. Pharmacology (1997) 334:1-23).
These examples do not preclude the use of other possible cell expression systems, including cell lines obtained from nematodes (PCT application WO 98/37177).

[000150] Chimeric, humanized, Human EngineeredTM and human antibodies [000151] A rodent antibody on repeated in vivo administration in man either alone or as a conjugate will bring about an immune response in the recipient against the rodent antibody;
the so-called HAMA response (Human Anti Mouse Antibody). The HAMA response may limit the effectiveness of the pharmaceutical if repeated dosing is required.
The immunogenicity of the antibody may be reduced by chemical modification of the antibody with a hydrophilic polymer such as polyethylene glycol or by using the methods of genetic engineering to make the antibody binding structure more human like.

[000152] In another embodiment the antibodies to FZD10 are humanized or chimeric antibodies. "Humanized" antibodies refer to a molecule having an antigen binding site that is substantially derived from an immunoglobulin from a non-human species and the remaining immunoglobulin structure of the molecule based upon the structure and/or sequence of a human immunoglobulin. The antigen binding site may comprise either complete variable domains fused onto constant domains or only the complementarity determining regions (CDRs) grafted onto appropriate framework regions in the variable domains.
Antigen binding sites may be wild type or modified by one or more amino acid substitutions, e.g., modified to resemble human immunoglobulin more closely. Alternatively, a humanized antibody may be derived from a chimeric antibody that retains or substantially retains the antigen-binding properties of the parental, non-human, antibody but which exhibits diminished immunogenicity as compared to the parental antibody when administered to humans. The phrase "chimeric antibody," as used herein, refers to an antibody containing sequence derived from two different antibodies (see, e.g., U.S. Patent No. 4,816,567) that typically originate from different species. Typically, in these chimeric antibodies, the variable region of both light and heavy chains mimics the variable regions of antibodies derived from one species of mammals, while the constant portions are homologous to the sequences in antibodies derived from another. Most typically, chimeric antibodies comprise human and murine antibody fragments, generally human constant and mouse variable regions. Chimeric monoclonal antibodies, in which the variable Ig domains of a rodent monoclonal antibody are fused to human constant Ig domains, can be generated using standard procedures known in the art (See Morrison, S. L., et al. (1984) Chimeric Human Antibody Molecules; Mouse Antigen Binding Domains with Human Constant Region Domains, Proc. Natl. Acad. Sci. USA 81, 6841-6855;
and, Boulianne, G. L., et al, Nature 312, 643-646 . (1984)). Although some chimeric monoclonal antibodies have proved less immunogenic in humans, the mouse variable Ig domains can still lead to a significant human anti-mouse response.

[000153] Humanized antibodies are made by replacing the complementarity determining regions (CDRs) of a human antibody (acceptor antibody) with those from a non-human antibody (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human "acceptor"
antibody are replaced by corresponding non human residues from the "donor" antibody.
Humanized antibodies may also comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non human immunoglobulin and all or substantially all of the framework residues (FR) regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., Nature, 321:522 525 (1986); Riechmann et al., Nature, 332:323 329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593 596 (1992)). One clear advantage to such chimeric forms is that, for example, the variable regions can conveniently be derived from presently known sources using readily available hybridomas or B cells from non human host organisms in combination with constant regions derived from, for example, human cell preparations. While the variable region has the advantage of ease of preparation, and the specificity is not affected by its source, the constant region being human, is less likely to elicit an immune response from a human subject when the antibodies are injected than would the constant region from a non-human source. However, the definition is not limited to this particular example.

[000154] Because humanized antibodies are far less immunogenic in humans than the parental mouse monoclonal antibodies, they can be used for the treatment of humans with far less risk of anaphylaxis. Thus, these antibodies may be preferred in therapeutic applications that involve in vivo administration to a human such as, e.g., use as radiation sensitizers for the treatment of neoplastic disease or use in methods to reduce the side effects of, e.g., cancer therapy. Methods for humanizing non human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source that is non human. These non human amino acid residues are often referred to as import residues, which are typically taken from an import variable domain.
Humanization can be essentially performed following the method of Winter and co workers (Jones et al., Nature 321:522 525 (1986); Riechmann et al., Nature 332:323 327 (1988); Verhoeyen et al., Science 239:1534 1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such humanized antibodies are chimeric antibodies (U.S. Patent No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non human species. In practice, humanized antibodies are typically human antibodies in which some CDR
residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

[000155] A disadvantage of CDR grafting, however, is that it can result in a humanized antibody that has a substantially lower binding affinity than the original mouse antibody, because amino acids of the framework regions can contribute to antigen binding, and because amino acids of the CDR loops can influence the association of the two variable Ig domains.
To maintain the affinity of the humanized monoclonal antibody, the CDR
grafting technique can be improved by choosing human framework regions that most closely resemble the framework regions of the original mouse antibody, and by site-directed mutagenesis of single amino acids within the framework or CDRs aided by computer modeling of the antigen binding site (e.g., Co, M. S., et al. (1994), J. Immunol. 152, 2968-2976).

[000156] One method of humanizing antibodies comprises aligning the non-human heavy and light chain sequences to human heavy and light chain sequences, selecting and replacing the non-human framework with a human framework based on such alignment, molecular modeling to predict the conformation of the humanized sequence and comparing to the conformation of the parent antibody. This process is followed by repeated back mutation of residues in the CDR region which disturb the structure of the CDRs until the predicted conformation of the humanized sequence model closely approximates the conformation of the non-human CDRs of the parent non-human antibody.

[000157] A number of "humanized" antibody molecules comprising an antigen-binding site derived from a non-human immunoglobulin have been described, including chimeric antibodies having rodent V regions and their associated CDRs fused to human constant domains (Winter et al. (1991) Nature 349:293-299; Lobuglio et al. (1989) Proc.
Nat. Acad.
Sci. USA 86:4220-4224; Shaw et al. (1987) J Immunol. 138:4534-4538; and Brown et al.
(1987) Cancer Res. 47:3577-3583), rodent CDRs grafted into a human supporting FR prior to fusion with an appropriate human antibody constant domain (Riechmann et al.
(1988) Nature 332:323-327; Verhoeyen et al. (1988) Science 239:1534-1536; and Jones et al.
(1986) Nature 321:522-525), and rodent CDRs supported by recombinantly veneered rodent FRs (European Patent Publication No. 519,596, published Dec. 23, 1992).

[000158] Humanized antibodies may be achieved by a variety of methods including, for example: (1) grafting the non-human complementarity determining regions (CDRs) onto a human framework and constant region (a process referred to in the art as "humanizing"), or, alternatively, (2) transplanting the entire non-human variable domains, but "cloaking" them with a human-like surface by replacement of surface residues (a process referred to in the art as "veneering"). In the present invention, humanized antibodies will include both "humanized" and "veneered" antibodies. Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated.
Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Patent Nos. 5,545,807; 5,545,806;
5,569,825;
5,625,126; 5,633,425; 5,661,016, and in the following scientific publications:
Marks et al., Bio/Technology 10, 779 783 (1992); Lonberg et al., Nature 368 856 859 (1994);
Morrison, Nature 368, 812 13 (1994); Fishwild et al., Nature Biotechnology 14, 845 51 (1996);
Neuberger, Nature Biotechnology 14, 826 (1996); Lonberg and Huszar, Intern.
Rev. Immunol.
13 65 93 (1995); Jones et al., Nature 321:522-525 (1986); Morrison et al., Proc. Natl. Acad.
Sci, US.A., 81:6851-6855 (1984); Morrison and Oi, Adv. Immunol., 44:65-92 (1988);
Verhoeyer et al., Science 239:1534-1536 (1988); Padlan, Molec. Immun. 28:489-498 (1991);
Padlan, Molec. Immunol. 31(3):169-217 (1994); and Kettleborough, C.A. et al., Protein Eng.
4(7):773-83 (1991) each of which is incorporated herein by reference.The phrase "complementarity determining region" refers to amino acid sequences which together define the binding affinity and specificity of the natural Fv region of a native immunoglobulin binding site. See, e.g., Chothia et al., J. Mol. Biol. 196:901-917 (1987);
Kabat et al., U.S.
Dept. of Health and Human Services NIH Publication No. 91-3242 (1991). The phrase "constant region" refers to the portion of the antibody molecule that confers effector functions. In the present invention, mouse constant regions are substituted by human constant regions. The constant regions of the subject humanized antibodies are derived from human immunoglobulins. The heavy chain constant region can be selected from any of the five isotypes: alpha, delta, epsilon, gamma or mu. One method of humanizing antibodies comprises aligning the non-human heavy and light chain sequences to human heavy and light chain sequences, selecting and replacing the non-human framework with a human framework based on such alignment, molecular modeling to predict the conformation of the humanized sequence and comparing to the conformation of the parent antibody. This process is followed by repeated back mutation of residues in the CDR region that disturb the structure of the CDRs until the predicted conformation of the humanized sequence model closely approximates the conformation of the non-human CDRs of the parent non-human antibody.
Such humanized antibodies may be further derivatized to facilitate uptake and clearance, e.g, via Ashwell receptors. See, e.g., U.S. Patent Nos. 5,530,101 and 5,585,089 which are incorporated herein by reference.

[000159] Human EngineeringTM

[000160] Human EngineeringTm of antibody variable domains has been described by Studnicka [See, e.g., Studnicka et al. U.S. Patent No. 5,766,886; Studnicka et al. Protein Engineering 7: 805-814 (1994)] as a method for reducing immunogenicity while maintaining binding activity of antibody molecules. According to the method, each variable region amino acid has been assigned a risk of substitution. Amino acid substitutions are distinguished by one of three risk categories : (1) low risk changes are those that have the greatest potential for reducing immunogenicity with the least chance of disrupting antigen binding;
(2) moderate risk changes are those that would further reduce immunogenicity, but have a greater chance of affecting antigen binding or protein folding; (3) high risk residues are those that are important for binding or for maintaining antibody structure and carry the highest risk that antigen binding or protein folding will be affected. Due to the three-dimensional structural role of prolines, modifications at prolines are generally considered to be at least moderate risk changes, even if the position is typically a low risk position.

[000161] Variable regions of the light and heavy chains of a rodent antibody are Human EngineeredTm as follows to substitute human amino acids at positions determined to be unlikely to adversely effect either antigen binding or protein folding, but likely to reduce immunogenicity in a human environment. Amino acid residues that are at "low risk"
positions and that are candidates for modification according to the method are identified by aligning the amino acid sequences of the rodent variable regions with a human variable region sequence. Any human variable region can be used, including an individual VH or VL
sequence or a human consensus VH or VL sequence or an individual or consensus human germline sequence. The amino acid residues at any number of the low risk positions, or at all of the low risk positions, can be changed. For example, at each low risk position where the aligned murine and human amino acid residues differ, an amino acid modification is introduced that replaces the rodent residue with the human residue.
Alternatively, the amino acid residues at all of the low risk positions and at any number of the moderate risk positions can be changed. Ideally, to achieve the least immunogenicity all of the low and moderate risk positions are changed from rodent to human sequence.

[000162] Synthetic genes containing modified heavy and/or light chain variable regions are constructed and linked to human y heavy chain and/or kappa light chain constant regions.
Any human heavy chain and light chain constant regions may be used in combination with the Human EngineeredTm antibody variable regions, including IgA (of any subclass, such as IgAl or IgA2), IgD, IgE, IgG (of any subclass, such as IgG1, IgG2, IgG3, or IgG4), or IgM. The human heavy and light chain genes are introduced into host cells, such as mammalian cells, and the resultant recombinant immunoglobulin products are obtained and characterized.
Human antibodies can also be produced using various techniques known in the art, including phage display libraries [Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991);
Marks et al., J. Mol. Biol., 222:581 (1991)]. The techniques of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies [Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J. Immunol., 147(1):86 95 (1991)]. Human antibodies to FZD10 polypeptides can also be produced using transgenic animals that are engineered to contain human immunoglobulin loci.
For example, WO 98/24893 discloses transgenic animals having a human Ig locus wherein the animals do not produce functional endogenous immunoglobulins due to the inactivation of endogenous heavy and light chain loci. WO 91/10741 also discloses transgenic non-primate mammalian hosts capable of mounting an immune response to an immunogen, wherein the antibodies have primate constant and/or variable regions, and wherein the endogenous immunoglobulin-encoding loci are substituted or inactivated. WO 96/30498 discloses the use of the Cre/Lox system to modify the immunoglobulin locus in a mammal, such as to replace all or a portion of the constant or variable region to form a modified antibody molecule. WO

discloses non-human mammalian hosts having inactivated endogenous Ig loci and functional human Ig loci. U.S. Patent No. 5,939,598 discloses methods of making transgenic mice in which the mice lack endogenous heavy chains, and express an exogenous immunoglobulin locus comprising one or more xenogeneic constant regions.

[000163] Using a transgenic animal described above, an immune response can be produced to a selected antigenic molecule, and antibody-producing cells can be removed from the animal and used to produce hybridomas that secrete human monoclonal antibodies.
Immunization protocols, adjuvants, and the like are known in the art, and are used in immunization of, for example, a transgenic mouse as described in WO 96/33735.
The monoclonal antibodies can be tested for the ability to inhibit or neutralize the biological activity or physiological effect of the corresponding protein. See also Jakobovits et al., Proc.
Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993);
Bruggermann et al., Year in Immuno., 7:33 (1993); and U.S. Pat. No. 5,591,669, U.S. Patent No. 5,589,369, U.S. Patent No. 5,545,807; and U.S Patent Application No.
20020199213, WO
96/34096 and U.S. patent application no. 20030194404; and U.S. patent application no.
20030031667. U.S. Patent Application No. 20030092125 describes methods for biasing the immune response of an animal to the desired epitope. Human antibodies may also be generated by in vitro activated B cells (see U.S. Pat. Nos. 5,567,610 and 5,229,275).

[000164] Additional transgenic animals useful to make monoclonal antibodies include the Medarex HuMAb-MOUSE , described in U.S. Pat. No. 5,770,429 and Fishwild, et al. (Nat.
Biotechnol. 14:845-851, 1996), which contains gene sequences from unrearranged human antibody genes that code for the heavy and light chains of human antibodies.
Immunization of a HuMAb-MOUSE enables the production of monoclonal antibodies to the target protein.

[000165] Also, Ishida et al. (Cloning Stem Cells. 4:91-102, 2002) describes the TransChromo Mouse (TCMOUSETM) which comprises megabase-sized segments of human DNA and which incorporates the entire human immunoglobulin (hlg) loci. The TCMOUSE
has a fully diverse repertoire of hlgs, including all the subclasses of IgGs (IgGI-G4).
Immunization of the TC Mouse with various human antigens produces antibody responses comprising human antibodies.

[000166] The development of technologies for making repertoires of recombinant human antibody genes, and the display of the encoded antibody fragments on the surface of filamentous bacteriophage, has provided a means for making human antibodies directly. The antibodies produced by phage technology are produced as antigen binding fragments-usually Fv or Fab fragments-in bacteria and thus lack effector functions. Effector functions can be introduced by one of two strategies: The fragments can be engineered either into complete antibodies for expression in mammalian cells, or into bispecific antibody fragments with a second binding site capable of triggering an effector function.

[000167] Typically, the Fd fragment (VH-CH1) and light chain (VL-CL) of antibodies are separately cloned by PCR and recombined randomly in combinatorial phage display libraries, which can then be selected for binding to a particular antigen. The Fab fragments are expressed on the phage surface, i.e., physically linked to the genes that encode them. Thus, selection of Fab by antigen binding co-selects for the Fab encoding sequences, which can be amplified subsequently. By several rounds of antigen binding and re-amplification, a procedure termed panning, Fab specific for the antigen are enriched and finally isolated.

[000168] In 1994, an approach for the humanization of antibodies, called "guided selection", was described. Guided selection utilizes the power of the phage display technique for the humanization of mouse monoclonal antibody (See Jespers, L. S., et al., Bio/Technology 12, 899-903 (1994)). For this, the Fd fragment of the mouse monoclonal antibody can be displayed in combination with a human light chain library, and the resulting hybrid Fab library may then be selected with antigen. The mouse Fd fragment thereby provides a template to guide the selection. Subsequently, the selected human light chains are combined with a human Fd fragment library. Selection of the resulting library yields entirely human Fab.

[000169] A variety of procedures have been described for deriving human antibodies from phage-display libraries (See, for example, Hoogenboom et al., J. Mol. Biol., 227:381 (1991);
Marks et al., J. Mol. Biol, 222:581-597 (1991); U.S. Pat. Nos. 5,565,332 and 5,573,905;
Clackson, T., and Wells, J. A., TIBTECH 12, 173-184 (1994)). In particular, in vitro selection and evolution of antibodies derived from phage display libraries has become a powerful tool (See Burton, D. R., and Barbas III, C. F., Adv. Immunol. 57, 191-280 (1994);
and, Winter, G., et al., Annu. Rev. Immunol. 12, 433-455 (1994); U.S. patent application no.
20020004215 and W092/01047; U.S. patent application no. 20030190317 published October 9, 2003 and U.S.
Patent No. 6,054,287; U.S. Patent No. 5,877,293.

[000170] Watkins, "Screening of Phage-Expressed Antibody Libraries by Capture Lift,"
Methods in Molecular Biology, Antibody Phage Display: Methods and Protocols 178: 187-193, and U.S. patent application no. 200120030044772 published March 6, 2003 describe methods for screening phage-expressed antibody libraries or other binding molecules by capture lift, a method involving immobilization of the candidate binding molecules on a solid support.

[000171] The antibody products may be screened for activity and for suitability in the treatment methods of the invention using assays as described in the section entitled "Screening Methods" herein or using any suitable assays known in the art.

[000172] In the present invention, FZD10 polypeptides as recited above and variants thereof may be used to immunize a transgenic animal as described above. Monoclonal antibodies are made using methods known in the art, and the specificity of the antibodies is tested using isolated FZD 10 polypeptides. Methods for preparation of the human or primate FZD 10 or an epitope thereof include, but are not limited to chemical synthesis, recombinant DNA
techniques or isolation from biological samples. Chemical synthesis of a peptide can be performed, for example, by the classical Merrifeld method of solid phase peptide synthesis (Merrifeld, J. Am. Chem. Soc. 85:2149, 1963 which is incorporated by reference) or the FMOC strategy on a Rapid Automated Multiple Peptide Synthesis system (E. I. du Pont de Nemours Company, Wilmington, DE) (Caprino and Han, J. Org. Chem. 37:3404, 1972 which is incorporated by reference).

[000173] Polyclonal antibodies can be prepared by immunizing rabbits or other animals by injecting antigen followed by subsequent boosts at appropriate intervals.
Alternative animals include mice, rats, chickens, guinea pigs, sheep, horses, monkeys, camels and sharks. The animals are bled and sera assayed against purified FZD10 proteins usually by ELISA or by bioassay based upon the ability to block the action of FZD10. When using avian species, e.g., chicken, turkey and the like, the antibody can be isolated from the yolk of the egg.
Monoclonal antibodies can be prepared after the method of Milstein and Kohler by fusing splenocytes from immunized mice with continuously replicating tumor cells such as myeloma or lymphoma cells. (Milstein and Kohler, Nature 256:495-497, 1975; Gulfre and Milstein, Methods in Enzymology: Immunochemical Techniques 73:1-46, Langone and Banatis eds., Academic Press, 1981 which are incorporated by reference). The hybridoma cells so formed are then cloned by limiting dilution methods and supernates assayed for antibody production by ELISA, RIA or bioassay.

[000174] The unique ability of antibodies to recognize and specifically bind to target proteins provides an approach for treating an overexpression of the protein.
Thus, another aspect of the present invention provides for a method for preventing or treating diseases involving overexpression of a FZD10 polypeptide by treatment of a patient with specific antibodies to the FZD10.

[000175] Specific antibodies, either polyclonal or monoclonal, to FZD10 can be produced by any suitable method known in the art as discussed above. For example, murine or human monoclonal antibodies can be produced by hybridoma technology or, alternatively, the FZD10 proteins, or an immunologically active fragment thereof, or an anti-idiotypic antibody, or fragment thereof can be administered to an animal to elicit the production of antibodies capable of recognizing and binding to the FZD10. Such antibodies can be from any class of antibodies including, but not limited to IgG, IgA, IgM, IgD, and IgE or in the case of avian species, IgY and from any subclass of antibodies.

[000176] Amino acid sequence variants [000177] A useful method for identification of certain residues or regions of the antibody that are preferred locations for mutagenesis is called "alanine scanning mutagenesis," as described by Cunningham and Wells Science, 244:1081-1085 (1989). Here, a residue or group of target residues are identified (e.g., charged residues such as arg, asp, his, lys, and glu) and replaced by a neutral or negatively charged amino acid (most preferably alanine or polyalanine) to affect the interaction of the amino acids with antigen. Those amino acid locations demonstrating functional sensitivity to the substitutions then are refined by introducing further or other variants at, or for, the sites of substitution.
Thus, while the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation per se need not be predetermined. For example, to analyze the performance of a mutation at a given site, ala scanning or random mutagenesis is conducted at the target codon or region and the expressed antibody variants are screened for the desired activity.

[000178] Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intra-sequence insertions of single or multiple amino acid residues.
Examples of terminal insertions include an antibody with an N-terminal methionyl residue or the antibody (including antibody fragment) fused to an epitope tag or a salvage receptor epitope. Other insertional variants of the antibody molecule include the fusion to a polypeptide which increases the serum half-life of the antibody, e.g. at the N-terminus or C-terminus.

[000179] The term "epitope tagged" refers to the antibody fused to an epitope tag. The epitope tag polypeptide has enough residues to provide an epitope against which an antibody there against can be made, yet is short enough such that it does not interfere with activity of the antibody. The epitope tag preferably is sufficiently unique so that the antibody there against does not substantially cross-react with other epitopes. Suitable tag polypeptides generally have at least 6 amino acid residues and usually between about 8-50 amino acid residues (preferably between about 9-30 residues). Examples include the flu HA
tag polypeptide and its antibody 12CA5 [Field et al., Mol. Cell. Biol. 8: 2159-2165 (1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto [Evan et al., Mol. Cell.
Biol. 5(12): 3610-3616 (1985)]; and the Herpes Simplex virus glycoprotein D
(gD) tag and its antibody [Paborsky et al., Protein Engineering 3(6): 547-553 (1990)]. Other exemplary tags are a poly-histidine sequence, generally around six histidine residues, that permits isolation of a compound so labeled using nickel chelation. Other labels and tags, such as the FLAG tag (Eastman Kodak, Rochester, NY), well known and routinely used in the art, are embraced by the invention.

[000180] Another type of variant is an amino acid substitution variant. These variants have at least one amino acid residue in the antibody molecule removed and a different residue inserted in its place. Substitutional mutagenesis within any of the hypervariable or CDR
regions or framework regions is contemplated. Conservative substitutions are shown in Table 1. The most conservative substitution is found under the heading of "preferred substitutions".
If such substitutions result in no change in biological activity, then more substantial changes, denominated "exemplary substitutions" in Table 1, or as further described below in reference to amino acid classes, may be introduced and the products screened.

Original Exemplary Preferred Residue Substitutions Ala (A) val; leu; ile val Arg (R) lys; gln; asn lys Asn (N) gln; his; asp, lys; gln arg Asp (D) glu; asn glu Cys (C) ser; ala ser Gln (Q) asn; glu asn Glu (E) asp; gln asp Gly (G) ala His (H) asn; gln; lys; arg Ile (I) leu; val; met; ala; leu phe; norleucine Leu (L) norleucine; ile; val; ile met; ala; phe Lys (K) arg; gln; asn arg Met (M) leu; phe; ile leu Phe (F) leu; val; ile; ala; tyr Pro (P) ala Ser(S) thr Thr (T) ser ser Trp (W) tyr; phe tyr Tyr (Y) trp; phe; thr; ser phe Val (V) ile; leu; met; phe; leu ala; norleucine [000181] Substantial modifications in the biological properties of the antibody are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Naturally occurring residues are divided into groups based on common side-chain properties:

[000182] (1) hydrophobic: norleucine, met, ala, val, leu, ile;

[000183] (2) neutral hydrophilic: cys, ser, thr;

[000184] (3) acidic: asp, glu;

[000185] (4) basic: asn, gln, his, lys, arg;

[000186] (5) residues that influence chain orientation: gly, pro; and [000187] (6) aromatic: trp, tyr, phe.

[000188] Conservative substitutions involve replacing an amino acid with another member of its class. Non-conservative substitutions involve replacing a member of one of these classes with a member of another class.

[000189] Any cysteine residue not involved in maintaining the proper conformation of the monoclonal, human, humanized, or variant antibody also may be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking.
Conversely, cysteine bond(s) may be added to the antibody to improve its stability (particularly where the antibody is an antibody fragment such as an Fv fragment).

[000190] Affinity maturation involves preparing and screening antibody variants that have substitutions within the CDRs of a parent antibody and selecting variants that have improved biological properties such as binding affinity relative to the parent antibody. A convenient way for generating such substitutional variants is affinity maturation using phage display.
Briefly, several hypervariable region sites (e.g. 6-7 sites) are mutated to generate all possible amino substitutions at each site. The antibody variants thus generated are displayed in a monovalent fashion from filamentous phage particles as fusions to the gene III
product of M13 packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g. binding affinity).

[000191] Alanine scanning mutagenesis can be performed to identify hypervariable region residues that contribute significantly to antigen binding. Alternatively, or in addition, it may be beneficial to analyze a crystal structure of the antigen-antibody complex to identify contact points between the antibody and antigen. Such contact residues and neighboring residues are candidates for substitution according to the techniques elaborated herein.
Once such variants are generated, the panel of variants is subjected to screening as described herein and antibodies with superior properties in one or more relevant assays may be selected for further development.

[000192] Antibody variants can also be produced that have a modified glycosylation pattern relative to the parent antibody, for example, deleting one or more carbohydrate moieties found in the antibody, and/or adding one or more glycosylation sites that are not present in the antibody.

[000193] Glycosylation of antibodies is typically either N-linked or 0-linked.
N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue.
The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. The presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. Thus, N-linked glycosylation sites may be added to an antibody by altering the amino acid sequence such that it contains one or more of these tripeptide sequences. 0-linked glycosylation refers to the attachment of one of the sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used. 0-linked glycosylation sites may be added to an antibody by inserting or substituting one or more serine or threonine residues to the sequence of the original antibody.

[000194] Altered effector function [000195] Other modifications of the antibody are contemplated. For example, it may be desirable to modify the antibody of the invention with respect to effector function, e.g., half-life, CDC or ADCC activity, so as to enhance the effectiveness of the antibody in treating cancer, for example. For example cysteine residue(s) may be introduced in the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated may have improved internalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al., J. Exp Med. 176: 1191-1195 (1992) and Shopes, B. J. Immunol.
148: 2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumor activity may also be prepared using heterobifunctional cross-linkers as described in Wolff et al., Cancer Research 53: 2560-2565 (1993). Alternatively, an antibody can be engineered which has dual Fc regions and may thereby have enhanced complement lysis and ADCC capabilities. See Stevenson et al., Anti-Cancer Drug Design 3: 219-230 (1989). In addition, it has been shown that sequences within the CDR can cause an antibody to bind to MHC Class II and trigger an unwanted helper T-cell response. A conservative substitution can allow the antibody to retain binding activity yet lose its ability to trigger an unwanted T-cell response. Also see Steplewski et al., Proc Natl Acad Sci U S A. 1988;85(13):4852-6, incorporated herein by reference in its entirety, which described chimeric antibodies wherein a murine variable region was joined with human gamma 1, gamma 2, gamma 3, and gamma 4 constant regions. Also see Presta et al., Biochem Soc Trans. 2002;30(4):487-90, incorporated herein by reference in its entirety, which described several positions in the Fc region of IgG1 were found which improved binding only to specific Fc gamma receptors (R) or simultaneously improved binding to one type of Fc gamma R and reduced binding to another type. Selected IgG1 variants with improved binding to Fc gamma RIIIa were then tested in an in vitro antibody-dependent cellular cytotoxicity (ADCC) assay and showed an enhancement in ADCC when either peripheral blood mononuclear cells or natural killer cells were used.

[000196] In certain embodiments of the invention, it may be desirable to use an antibody fragment, rather than an intact antibody, to increase tumor penetration, for example. In this case, it may be desirable to modify the antibody fragment in order to increase its serum half-life, for example, adding molecules such as PEG or other water soluble polymers, including polysaccharide polymers, to antibody fragments to increase the half-life. This may also be achieved, for example, by incorporation of a salvage receptor binding epitope into the antibody fragment (e.g., by mutation of the appropriate region in the antibody fragment or by incorporating the epitope into a peptide tag that is then fused to the antibody fragment at either end or in the middle, e.g., by DNA or peptide synthesis) (see, e.g., W096/32478).

[000197] Additionally, antibodies can be chemically modified by covalent conjugation to a polymer to increase their circulating half-life, for example. Each antibody molecule may be attached to one or more (i.e. 1, 2, 3, 4, 5 or more) polymer molecules.
Polymer molecules are preferably attached to antibodies by linker molecules. The polymer may, in general, be a synthetic or naturally occurring polymer, for example an optionally substituted straight or branched chain polyalkene, polyalkenylene or polyoxyalkylene polymer or a branched or unbranched polysaccharide, e.g. homo- or hetero-polysaccharide. Preferred polymers are polyoxyethylene polyols and polyethylene glycol (PEG). PEG is soluble in water at room temperature and has the general formula: R(O--CH2--CH2) n O--R where R can be hydrogen, or a protective group such as an alkyl or alkanol group. Preferably, the protective group has between 1 and 8 carbons, more preferably it is methyl. The symbol n is a positive integer, preferably between 1 and 1,000, more preferably between 2 and 500. The PEG has a preferred average molecular weight between 1000 and 40,000, more preferably between 2000 and 20,000, most preferably between 3,000 and 12, 000. Preferably, PEG has at least one hydroxy group, more preferably it is a terminal hydroxy group. It is this hydroxy group which is preferably activated to react with a free amino group on the inhibitor.
However, it will be understood that the type and amount of the reactive groups may be varied to achieve a covalently conjugated PEG/antibody of the present invention. Preferred polymers, and methods to attach them to peptides, are shown in U. S. Pat. Nos. 4,766, 106;
4,179, 337;
4,495, 285; and 4,609, 546 which are all hereby incorporated by reference in their entireties.

[000198] As used herein, the term "salvage receptor binding epitope" refers to an epitope of the Fc region of an IgG molecule (e.g., IgG1, IgG2, IgG3, or IgG4) that is responsible for increasing the in vivo serum half-life of the IgG molecule.

[000199] The salvage receptor binding epitope preferably constitutes a region wherein any one or more amino acid residues from one or two loops of a Fc domain are transferred to an analogous position of the antibody fragment. Even more preferably, three or more residues from one or two loops of the Fc domain are transferred. Still more preferred, the epitope is taken from the CH2 domain of the Fc region (e.g., of an IgG) and transferred to the CH1, CH3, or VH region, or more than one such region, of the antibody.
Alternatively, the epitope is taken from the CH2 domain of the Fc region and transferred to the CL region or VL region, or both, of the antibody fragment. See also International applications WO
97/34631 and WO
96/32478 which describe Fc variants and their interaction with the salvage receptor.

[000200] Thus, antibodies of the invention may comprise a human Fc portion, a human consensus Fc portion, or a variant thereof that retains the ability to interact with the Fc salvage receptor, including variants in which cysteines involved in disulfide bonding are modified or removed, and/or in which the a met is added at the N-terminus and/or one or more of the N-terminal 20 amino acids are removed, and/or regions that interact with complement, such as the C1q binding site, are altered or removed, and/or the ADCC site is altered or removed [see, e.g., Molec. Immunol. 29 (5): 633-9 (1992)].

[000201] Previous studies mapped the binding site on human and murine IgG for FcR
primarily to the lower hinge region composed of IgG residues 233-239. Other studies proposed additional broad segments, e.g. G1y316-Lys338 for human Fc receptor I, Lys274-Arg301 and Tyr407-Arg416 for human Fc receptor III, or found a few specific residues outside the lower hinge, e.g. Asn297 and G1u318 for murine IgG2b interacting with murine Fc receptor II. The report of the 3.2-A crystal structure of the human IgGI Fc fragment with human Fc receptor IIIA delineated IgGI residues Leu234-Ser239, Asp265-G1u269, Asn297-Thr299, and A1a327-Ile332 as involved in binding to Fc receptor IIIA. It has been suggested based on crystal structure that in addition to the lower hinge (Leu234-G1y237), residues in IgG
CH2 domain loops FG (residues 326-330) and BC (residues 265-271) might play a role in binding to Fc receptor IIA. See Shields et al., J. Biol. Chem., 276(9):6591-6604 (2001), incorporated by reference herein in its entirety. Mutation of residues within Fc receptor binding sites can result in altered effector function, such as altered ADCC or CDC activity, or altered half-life. As described above, potential mutations include insertion, deletion or substitution of one or more residues, including substitution with alanine, a conservative substitution, a non-conservative substitution, or replacement with a corresponding amino acid residue at the same position from a different IgG subclass (e.g. replacing an IgGI residue with a corresponding IgG2 residue at that position).

[000202] Shields et al. reported that IgGI residues involved in binding to all human Fc receptors are located in the CH2 domain proximal to the hinge and fall into two categories as follows: 1) positions that may interact directly with all FcR include Leu234-Pro238, AIa327, and Pro329 (and possibly Asp265); 2) positions that influence carbohydrate nature or position include Asp265 and Asn297. The additional IgGI residues that affected binding to Fc receptor II are as follows: (largest effect) Arg255, Thr256, G1u258, Ser267, Asp270, GIu272, Asp280, Arg292, Ser298, and (less effect) His268, Asn276, His285, Asn286, Lys290, GIn295, Arg301, Thr307, Leu309, Asn315, Lys322, Lys326, Pro331, Ser337, A1a339, A1a378, and Lys414. A327Q, A327S, P329A, D265A and D270A reduced binding. In addition to the residues identified above for all FcR, additional IgG1 residues that reduced binding to Fc receptor IIIA by 40% or more are as follows: Ser239, Ser267 (Gly only), His268, GIu293, G1n295, Tyr296, Arg301, Va1303, Lys338, and Asp376. Variants that improved binding to FcRIIIA include T256A, K290A, S298A, E333A, K334A, and A339T. Lys414 showed a 40% reduction in binding for FcRIIA and FcRIIB, Arg416 a 30% reduction for FcRIIA and FcRIIIA, G1n419 a 30% reduction to FcRIIA and a 40% reduction to FcRIIB, and Lys360 a 23% improvement to FcRIIIA. See also Presta et al., Biochem. Soc. Trans.
(2001) 30, 487-490.

[000203] For example, United States Patent No. 6,194,551, incorporated herein by reference in its entirety, describes variants with altered effector function containing mutations in the human IgG Fc region, at amino acid position 329, 331 or 322 (using Kabat numbering), some of which display reduced Clq binding or CDC activity. As another example, United States Patent No. 6,737,056, incorporated herein by reference in its entirety, describes variants with altered effector or Fc-gamma-receptor binding containing mutations in the human IgG Fc region, at amino acid position 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 294, 295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or 439 (using Kabat numbering), some of which display receptor binding profiles associated with reduced ADCC
or CDC activity. Of these, a mutation at amino acid position 238, 265, 269, 270, 327 or 329 are stated to reduce binding to FcRI, a mutation at amino acid position 238, 265, 269, 270, 292, 294, 295, 298, 303, 324, 327, 329, 333, 335, 338, 373, 376, 414, 416, 419, 435, 438 or 439 are stated to reduce binding to FcRII, and a mutation at amino acid position 238, 239, 248, 249, 252, 254, 265, 268, 269, 270, 272, 278, 289, 293, 294, 295, 296, 301, 303, 322, 327, 329, 338, 340, 373, 376, 382, 388, 389, 416, 434, 435 or 437 is stated to reduce binding to FcRII1.

[000204] United States Patent No. 5,624,821, incorporated by reference herein in its entirety, reports that Clq binding activity of an murine antibody can be altered by mutating amino acid residue 318, 320 or 322 of the heavy chain and that replacing residue 297 (Asn) results in removal of lytic activity.

[000205] United States Application Publication No. 20040132101, incorporated by reference herein in its entirety, describes variants with mutations at amino acid positions 240, 244, 245, 247, 262, 263, 266, 299, 313, 325, 328, or 332 (using Kabat numbering) or positions 234, 235, 239, 240, 241, 243, 244, 245, 247, 262, 263, 264, 265, 266, 267, 269, 296, 297, 298, 299, 313, 325, 327, 328, 329, 330, or 332 (using Kabat numbering), of which mutations at positions 234, 235, 239, 240, 241, 243, 244, 245, 247, 262, 263, 264, 265, 266, 267, 269, 296, 297, 298, 299, 313, 325, 327, 328, 329, 330, or 332 may reduce ADCC activity or reduce binding to an Fc gamma receptor.

[000206] Chappel et al., Proc Natl Acad Sci U S A. 1991;88(20):9036-40, incorporated herein by reference in its entirety, report that cytophilic activity of IgG1 is an intrinsic property of its heavy chain CH2 domain. Single point mutations at any of amino acid residues 234-237 of IgG1 significantly lowered or abolished its activity. Substitution of all of IgGl residues 234-237 (LLGG) into IgG2 and IgG4 were required to restore full binding activity.
An IgG2 antibody containing the entire ELLGGP sequence (residues 233-238) was observed to be more active than wild-type IgG1.

[000207] Isaacs et al., J Immunol. 1998;161(8):3862-9, incorporated herein by reference in its entirety, report that mutations within a motif critical for Fc gammaR
binding (glutamate 233 to proline, leucine/phenylalanine 234 to valine, and leucine 235 to alanine) completely prevented depletion of target cells. The mutation glutamate 318 to alanine eliminated effector function of mouse IgG2b and also reduced the potency of human IgG4.

[000208] Armour et al., Mol Immunol. 2003;40(9):585-93, incorporated by reference herein in its entirety, identified IgG1 variants which react with the activating receptor, FcgammaRlIa, at least 10-fold less efficiently than wildtype IgG1 but whose binding to the inhibitory receptor, FcgammaRIIb, is only four-fold reduced. Mutations were made in the region of amino acids 233-236 and/or at amino acid positions 327, 330 and 331. See also WO
99/58572, incorporated by referehce herein in its entirety.

[000209] Xu et al., J Biol Chem. 1994;269(5):3469-74, incorporated by reference herein in its entirety, report that mutating IgG1 Pro331 to Ser markedly decreased Clq binding and virually eliminated lytic activity. In contrast, the substitution of Pro for Ser331 in IgG4 bestowed partial lytic activity (40%) to the IgG4 Pro331 variant.

[000210] Schuurman et al,. Mol Immunol. 2001;38(1):1-8, incorporated by reference herein in its entirety, report that mutating one of the hinge cysteines involved in the inter-heavy chain bond formation, Cys226, to serine resulted in a more stable inter-heavy chain linkage.
Mutating the IgG4 hinge sequence Cys-Pro-Ser-Cys to the IgG1 hinge sequence Cys-Pro-Pro-Cys also markedly stabilizes the covalent interaction between the heavy chains.

[000211] Angal et al., Mol Immunol. 1993;30(1):105-8, incorporated by reference herein in its entirety, report that mutating the serine at amino acid position 241 in IgG4 to proline (found at that position in IgG1 and IgG2) led to the production of a homogeneous antibody, as well as extending serum half-life and improving tissue distribution compared to the original chimeric IgG4.

[000212] The invention also contemplates production of antibody molecules with altered carbohydrate structure resulting in altered effector activity, including antibody molecules with absent or reduced fucosylation that exhibit improved ADCC activity. A variety of ways are known in the art to accomplish this. For example, ADCC effector activity is mediated by binding of the antibody molecule to the FcyRIII receptor, which has been shown to be dependent on the carbohydrate structure of the N-linked glycosylation at the Asn-297 of the CH2 domain. Non-fucosylated antibodies bind this receptor with increased affinity and trigger FcyRIII-mediated effector functions more efficiently than native, fucosylated antibodies. For example, recombinant production of non-fucosylated antibody in CHO cells in which the alpha-1,6-fucosyl transferase enzyme has been knocked out results in antibody with 100-fold increased ADCC activity [Yamane-Ohnuki et al., Biotechnol Bioeng. 2004 Sep 5;87(5):614-22]. Similar effects can be accomplished through decreasing the activity of this or other enzymes in the fucosylation pathway, e.g., through siRNA or antisense RNA
treatment, engineering cell lines to knockout the enzyme(s), or culturing with selective glycosylation inhibitors [Rothman et al., Mol Immunol. 1989 Dec;26(12):1113-23]. Some host cell strains, e.g. Lec13 or rat hybridoma YB2/0 cell line naturally produce antibodies with lower fucosylation levels. Shields et al., J Biol Chem. 2002 Jul 26;277(30):26733-40;
Shinkawa et al., J Biol Chem. 2003 Jan 31;278(5):3466-73. An increase in the level of bisected carbohydrate, e.g. through recombinantly producing antibody in cells that overexpress GnTIII enzyme, has also been determined to increase ADCC activity.
Umana et al., Nat Biotechnol. 1999 Feb;17(2):176-80. It has been predicted that the absence of only one of the two fucose residues may be sufficient to increase ADCC activity (Ferrara et al., Biotechnol Bioeng. 2006 Apr 5;93(5):851-61).

[000213] Other covalent modifications [000214] Covalent modifications of the antibody are also included within the scope of this invention. They may be made by chemical synthesis or by enzymatic or chemical cleavage of the antibody, if applicable. Other types of covalent modifications of the antibody are introduced into the molecule by reacting targeted amino acid residues of the antibody with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C-terminal residues.

[000215] Cysteinyl residues most commonly are reacted with a-haloacetates (and corresponding amines), such as chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residues also are derivatized by reaction with bromotrifluoroacetone, .alpha.-bromo-(3-(5-imidozoyl)propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.

[000216] Histidyl residues are derivatized by reaction with diethylpyrocarbonate at pH 5.5-7.0 because this agent is relatively specific for the histidyl side chain.
Para-bromophenacyl bromide also is useful; the reaction is preferably performed in 0.1 M sodium cacodylate at pH
6Ø

[000217] Lysinyl and amino-terminal residues are reacted with succinic or other carboxylic acid anhydrides. Derivatization with these agents has the effect of reversing the charge of the lysinyl residues. Other suitable reagents for derivatizing .alpha.-amino-containing residues include imidoesters such as methyl picolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid, 0-methylisourea, 2,4-pentanedione, and transaminase-catalyzed reaction with glyoxylate.

[000218] Arginyl residues are modified by reaction with one or several conventional reagents, among them phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin.
Derivatization of arginine residues requires that the reaction be performed in alkaline conditions because of the high pKa of the guanidine functional group.
Furthermore, these reagents may react with the groups of lysine as well as the arginine epsilon-amino group.

[000219] The specific modification of tyrosyl residues may be made, with particular interest in introducing spectral labels into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane. Most commonly, N-acetylimidizole and tetranitromethane are used to form 0-acetyl tyrosyl species and 3-nitro derivatives, respectively. Tyrosyl residues are iodinated using 125 1 or 131I to prepare labeled proteins for use in radioimmunoassay.

[000220] Carboxyl side groups (aspartyl or glutamyl) are selectively modified by reaction with carbodiimides (R-N=C=N-R'), where R and R' are different alkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl) carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermore, aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.

[000221] Glutaminyl and asparaginyl residues are frequently deamidated to the corresponding glutamyl and aspartyl residues, respectively. These residues are deamidated under neutral or basic conditions. The deamidated form of these residues falls within the scope of this invention.

[000222] Other modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the .alpha.-amino groups of lysine, arginine, and histidine side chains (T. E. Creighton, Proteins:
Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)), acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.

[000223] Another type of covalent modification involves chemically or enzymatically coupling glycosides to the antibody. These procedures are advantageous in that they do not require production of the antibody in a host cell that has glycosylation capabilities for N- or 0-linked glycosylation. Depending on the coupling mode used, the sugar(s) may be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups such as those of cysteine, (d) free hydroxyl groups such as those of serine, threonine, or hydroxyproline, (e) aromatic residues such as those of phenylalanine, tyrosine, or tryptophan, or (f) the amide group of glutamine. These methods are described in W087/05330 published 11 Sep. 1987, and in Aplin and Wriston, CRC Crit. Rev. Biochem., pp. 259-306 (1981).

[000224] Removal of any carbohydrate moieties present on the antibody may be accomplished chemically or enzymatically. Chemical deglycosylation requires exposure of the antibody to the compound trifluoromethanesulfonic acid, or an equivalent compound. This treatment results in the cleavage of most or all sugars except the linking sugar (N-acetylglucosamine or N-acetylgalactosamine), while leaving the antibody intact. Chemical deglycosylation is described by Hakimuddin, et al. Arch. Biochem. Biophys.
259: 52 (1987) and by Edge et al. Anal. Biochem., 118: 131 (1981). Enzymatic cleavage of carbohydrate moieties on antibodies can be achieved by the use of a variety of endo- and exo-glycosidases as described by Thotakura et al. Meth. Enzymol. 138: 350 (1987).

[000225] Another type of covalent modification of the antibody comprises linking the antibody to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol, polypropylene glycol, polyoxyethylated polyols, polyoxyethylated sorbitol, polyoxyethylated glucose, polyoxyethylated glycerol, polyoxyalkylenes, or polysaccharide polymers such as dextran. Such methods are known in the art, see, e.g. U.S. Patent Nos.
4,640,835; 4,496,689;
4,301,144; 4,670,417; 4,791,192, 4,179,337, 4,766,106, 4,179,337, 4,495,285, 4,609,546or EP
315 456.

[000226] Gene Therapy [000227] Delivery of a therapeutic antibody to appropriate cells can be effected via gene therapy ex vivo, in situ, or in vivo by use of any suitable approach known in the art, including by use of physical DNA transfer methods (e.g., liposomes or chemical treatments) or by use of viral vectors (e.g., adenovirus, adeno-associated virus, or a retrovirus). For example, for in vivo therapy, a nucleic acid encoding the desired antibody, either alone or in conjunction with a vector, liposome, or precipitate may be injected directly into the subject, and in some embodiments, may be injected at the site where the expression of the antibody compound is desired. For ex vivo treatment, the subject's cells are removed, the nucleic acid is introduced into these cells, and the modified cells are returned to the subject either directly or, for example, encapsulated within porous membranes which are implanted into the patient. See, e.g. U.S. Pat. Nos. 4,892,538 and 5,283,187. There are a variety of techniques available for introducing nucleic acids into viable cells. The techniques vary depending upon whether the nucleic acid is transferred into cultured cells in vitro, or in vivo in the cells of the intended host. Techniques suitable for the transfer of nucleic acid into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, DEAE-dextran, and calcium phosphate precipitation. A commonly used vector for ex vivo delivery of a nucleic acid is a retrovirus.

[000228] Other in vivo nucleic acid transfer techniques include transfection with viral vectors (such as adenovirus, Herpes simplex I virus, or adeno-associated virus) and lipid-based systems. The nucleic acid and transfection agent are optionally associated with a microparticle. Exemplary transfection agents include calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, quaternary ammonium amphiphile DOTMA ((dioleoyloxypropyl) trimethylammonium bromide, commercialized as Lipofectin by GIBCO-BRL))(Felgner et al, (1987) Proc. Natl. Acad. Sci. USA 84, 7413-7417;
Malone et al. (1989) Proc. Natl Acad. Sci. USA 86 6077-6081); lipophilic glutamate diesters with pendent trimethylammonium heads (Ito et al. (1990) Biochem. Biophys. Acta 1023, 124-132);
the metabolizable parent lipids such as the cationic lipid dioctadecylamido glycylspermine (DOGS, Transfectam, Promega) and dipalmitoylphosphatidyl ethanolamylspermine (DPPES)(J. P. Behr (1986) Tetrahedron Lett. 27, 5861-5864; J. P. Behr et al.
(1989) Proc.
Natl. Acad. Sci. USA 86, 6982-6986); metabolizable quatemary ammonium salts (DOTB, N-(1-[2,3-dioleoyloxy]propyl)-N,N,N-trimethylammonium methylsulfate (DOTAP)(Boehringer Mannheim), polyethyleneimine (PEI), dioleoyl esters, ChoTB, ChoSC, DOSC)(Leventis et al.

(1990) Biochim. Inter. 22, 235-241); 3beta[N-(N', N'-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol), dioleoylphosphatidyl ethanolamine (DOPE)/3beta[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterolDC-Chol in one to one mixtures (Gao et al., (1991) Biochim. Biophys. Acta 1065, 8-14), spermine, spermidine, lipopolyamines (Behr et al., Bioconjugate Chem, 1994, 5: 382-389), lipophilic polylysines (LPLL) (Zhou et al., (1991) Biochim. Biophys. Acta 939, 8-18), [[(1,1,3,3-tetramethylbutyl)cre-soxy] ethoxy] ethyl] dimethylbe nzylammonium hydroxide (DEBDA hydroxide) with excess phosphatidylcholine/cholesterol (Ballas et al., (1988) Biochim. Biophys. Acta 939, 8-18), cetyltrimethylammonium bromide (CTAB)/DOPE mixtures (Pinnaduwage et al, (1989) Biochim. Biophys. Acta 985, 33-37), lipophilic diester of glutamic acid (TMAG) with DOPE, CTAB, DEBDA, didodecylammonium bromide (DDAB), and stearylamine in admixture with phosphatidylethanolamine (Rose et al., (1991) Biotechnique 10, 520-525), DDAB/DOPE
(TransfectACE, GIBCO BRL), and oligogalactose bearing lipids. Exemplary transfection enhancer agents that increase the efficiency of transfer include, for example, DEAE-dextran, polybrene, lysosome-disruptive peptide (Ohmori N I et al, Biochem Biophys Res Commun Jun. 27, 1997;235(3):726-9), chondroitan-based proteoglycans, sulfated proteoglycans, polyethylenimine, polylysine (Pollard H et al. J Biol Chem, 1998 273 (13):7507-11), integrin-binding peptide CYGGRGDTP, linear dextran nonasaccharide, glycerol, cholesteryl groups tethered at the 3'-terminal internucleoside link of an oligonucleotide (Letsinger, R. L. 1989 Proc Natl Acad Sci USA 86: (17):6553-6), lysophosphatide, lysophosphatidylcholine, lysophosphatidylethanolamine, and 1-oleoyl lysophosphatidylcholine.

[000229] In some situations it may be desirable to deliver the nucleic acid with an agent that directs the nucleic acid-containing vector to target cells. Such "targeting"
molecules include antibodies specific for a cell-surface membrane protein on the target cell, or a ligand for a receptor on the target cell. Where liposomes are employed, proteins which bind to a cell-surface membrane protein associated with endocytosis may be used for targeting and/or to facilitate uptake. Examples of such proteins include capsid proteins and fragments thereof tropic for a particular cell type, antibodies for proteins which undergo internalization in cycling, and proteins that target intracellular localization and enhance intracellular half-life. In other embodiments, receptor-mediated endocytosis can be used. Such methods are described, for example, in Wu et al., 1987 or Wagner et al., 1990. For review of the currently known gene marking and gene therapy protocols, see Anderson 1992. See also WO
93/25673 and the references cited therein. For additional reviews of gene therapy technology, see Friedmann, Science, 244: 1275-1281 (1989); Anderson, Nature, supplement to vol. 392, no 6679, pp. 25-30 (1998); Verma, Scientific American: 68-84 (1990); and Miller, Nature, 357:

(1992).

[000230] Screening Methods [000231] Effective therapeutics depend on identifying efficacious agents devoid of significant toxicity. Antibodies may be screened for binding affinity by methods known in the art. For example, gel-shift assays, Western blots, radiolabeled competition assay, co-fractionation by chromatography, co-precipitation, cross linking, ELISA, and the like may be used, which are described in, for example, Current Protocols in Molecular Biology (1999) John Wiley & Sons, NY, which is incorporated herein by reference in its entirety.

[000232] To initially screen for antibodies which bind to the desired epitope on FZD10 (e.g., the extracellular domain of FZD10), a routine cross-blocking assay such as that described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988), can be performed. Routine competitive binding assays may also be used, in which the unknown antibody is characterized by its ability to inhibit binding of FZD10 to a FZD10 specific antibody of the invention. Epitope mapping is described in Champe et al., J.
Biol. Chem. 270: 1388-1394 (1995).

[000233] In one variation of an in vitro binding assay, the invention provides a method comprising the steps of (a) contacting an immobilized FZD10 with a candidate antibody and (b) detecting binding of the candidate antibody to the FZD10. In an alternative embodiment, the candidate antibody is immobilized and binding of FZD10 is detected.
Immobilization is accomplished using any of the methods well known in the art, including covalent bonding to a support, a bead, or a chromatographic resin, as well as non-covalent, high affinity interaction such as antibody binding, or use of streptavidin/biotin binding wherein the immobilized compound includes a biotin moiety. Detection of binding can be accomplished (i) using a radioactive label on the compound that is not immobilized, (ii) using a fluorescent label on the non-immobilized compound, (iii) using an antibody immunospecific for the non-immobilized compound, (iv) using a label on the non-immobilized compound that excites a fluorescent support to which the immobilized compound is attached, as well as other techniques well known and routinely practiced in the art.

[000234] Antibodies that modulate (i.e., increase, decrease, or block) the activity or expression of FZD10 may be identified by incubating a putative modulator with a cell expressing a FZD10 and determining the effect of the putative modulator on the activity or expression of the FZD10. The selectivity of an antibody that modulates the activity of a FZD10 polypeptide or polynucleotide can be evaluated by comparing its effects on the FZD10 polypeptide or polynucleotide to its effect on other related compounds.
Selective modulators may include, for example, antibodies and other proteins, peptides, or organic molecules which specifically bind to FZD10 polypeptides or to a nucleic acid encoding a FZD10 polypeptide.
Modulators of FZD10 activity will be therapeutically useful in treatment of diseases and physiological conditions in which normal or aberrant activity of FZD10 polypeptide is involved.

[000235] Compounds potentially useful in preventing or treating cancer may be screened using various assays. For instance, a candidate antagonist may first be characterized in a cultured cell system to determine its ability to decrease phosphorylation of Dishevelled, decrease phosphorylation of c-Jun, and increase phosphorylation of 0-catenin.

[000236] The anti-tumor activity of a particular FZD10 antibody, or combination of FZD10 antibodies, may be evaluated in vivo using a suitable animal model (Loukopoulos et al., Pancreas, 29(3):193-203 (2004)). In addition, the anti-tumor activity of a particular FZD10 antibody may be evaluated by assaying, e.g., phorsphorylation state of Dishevelled-1, Dishevelled-2, Dishevelled-3, LRP5, LRP6 and (3-catenin, as well as level of expression of (3-catenin regulated genes, such as c-myc (He TC et al., Science. 1998 Sep 4;281(5382):1509-12) and cyclinDl (Tetsu O& McCormick F Nature. 1999 Apr 1;398(6726):422-6).
Phosphorylation state, such as serine 63 and serine 73 of c-JUN can also be used to monitor non-p-catenin or non-canonical signaling activities of FZD10. Additionally, cellular assays including proliferation assays, soft agar assays, and/or cytotoxicity assays as described herein may be used to evaluate a particular FZD10 antibody. In one embodiment, the phosphorylation state of LRP5, a known co-receptor of WNT-7b (Wang, Z. et al., Mol Cell Bio125 (12): 5022-30, 2005), is assessed to identify a cell population that would respond well to an anti-FZD10 antibody. In another embodiment, the phosphorylation state of LRP5 is optionally monitored during treatment with an anti-FZD 10 antibody.

[000237] The invention also comprehends high throughput screening (HTS) assays to identify antibodies that interact with or inhibit biological activity (i.e., inhibit enzymatic activity, binding activity, etc.) of a FZD10 polypeptide. HTS assays permit screening of large numbers of compounds in an efficient manner. Cell-based HTS systems are contemplated to investigate the interaction between FZD10 polypeptides and their binding partners. HTS
assays are designed to identify "hits" or "lead compounds" having the desired property, from which modifications can be designed to improve the desired property. Chemical modification of the "hit" or "lead compound" is often based on an identifiable structure/activity relationship between the "hit" and FZD10 polypeptides.

[000238] Another aspect of the present invention is directed to methods of identifying antibodies which modulate (i.e., decrease) activity of a FZD10 comprising contacting a FZD10 with an antibody, and determining whether the antibody modifies activity of the FZD10. The activity in the presence of the test antibody is compared to the activity in the absence of the test antibody. Where the activity of the sample containing the test antibody is lower than the activity in the sample lacking the test antibody, the antibody will have inhibited activity.

[000239] Antibody conjugates [000240] Anti-FZD10 antibodies may be administered in their "naked" or unconjugated form, or may be conjugated directly to other therapeutic or diagnostic agents, or may be conjugated indirectly to carrier polymers comprising such other therapeutic or diagnostic agents.

[000241] Antibodies can be detectably labeled through the use of radioisotopes, affinity labels (such as biotin, avidin, etc.), enzymatic labels (such as horseradish peroxidase, alkaline phosphatase, etc.) fluorescent or luminescent or bioluminescent labels (such as FITC or rhodamine, etc.), paramagnetic atoms, and the like. Procedures for accomplishing such labeling are well known in the art; for example, see (Sternberger, L.A. et al., J. Histochem.
Cytochem. 18:315 (1970); Bayer, E.A. et al., Meth. Enzym. 62:308 (1979);
Engval, E. et al., Immunol. 109:129 (1972); Goding, J.W. J. Immunol. Meth. 13:215 (1976)).

[000242] Conjugation of antibody moieties is described in U.S. Patent No.
6,306,393.
General techniques are also described in Shih et al., Int. J. Cancer 41:832-839 (1988); Shih et al., Int. J. Cancer 46:1101-1106 (1990); and Shih et al., U.S. Pat. No.
5,057,313. This general method involves reacting an antibody component having an oxidized carbohydrate portion with a carrier polymer that has at least one free amine function and that is loaded with a plurality of drug, toxin, chelator, boron addends, or other therapeutic agent.
This reaction results in an initial Schiff base (imine) linkage, which can be stabilized by reduction to a secondary amine to form the final conjugate.

[000243] The carrier polymer may be, for example, an aminodextran or polypeptide of at least 50 amino acid residues. Various techniques for conjugating a drug or other agent to the carrier polymer are known in the art. A polypeptide carrier can be used instead of aminodextran, but the polypeptide carrier should have at least 50 amino acid residues in the chain, preferably 100-5000 amino acid residues. At least some of the amino acids should be lysine residues or glutamate or aspartate residues. The pendant amines of lysine residues and pendant carboxylates of glutamine and aspartate are convenient for attaching a drug, toxin, immunomodulator, chelator, boron addend or other therapeutic agent. Examples of suitable polypeptide carriers include polylysine, polyglutamic acid, polyaspartic acid, co-polymers thereof, and mixed polymers of these amino acids and others, e.g., serines, to confer desirable solubility properties on the resultant loaded carrier and conjugate.

[000244] Alternatively, conjugated antibodies can be prepared by directly conjugating an antibody component with a therapeutic agent. The general procedure is analogous to the indirect method of conjugation except that a therapeutic agent is directly attached to an oxidized antibody component. For example, a carbohydrate moiety of an antibody can be attached to polyethyleneglycol to extend half-life.

[000245] Alternatively, a therapeutic agent can be attached at the hinge region of a reduced antibody component via disulfide bond formation, or using a heterobifunctional cross-linker, such as N-succinyl 3-(2-pyridyldithio)proprionate (SPDP). Yu et al., Int. J.
Cancer56:244 (1994). General techniques for such conjugation are well-known in the art.
See, for example, Wong, Chemistry Of Protein Conjugation and Cross-Linking (CRC Press 1991);
Upeslacis et al., "Modification of Antibodies by Chemical Methods," in Monoclonal Antibodies: Principles and Applications, Birch et al. (eds.), pages 187-230 (Wiley-Liss, Inc. 1995);
Price, "Production and Characterization of Synthetic Peptide-Derived Antibodies," in Monoclonal Antibodies: Production, Enineering and Clinical Application, Ritter et al.
(eds.), pages 60-84 (Cambridge University Press 1995). A variety of bifunctional protein coupling agents are known in the art, such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).

[000246] Finally, fusion proteins can be constructed that comprise one or more anti-FZD10 antibody moieties and another polypeptide. Methods of making antibody fusion proteins are well known in the art. See, e.g., U.S. Patent No. 6,306,393. Antibody fusion proteins comprising an interleukin-2 moiety are described by Boleti et al., Ann. Oncol.
6:945 (1995), Nicolet et al., Cancer Gene Ther. 2:161 (1995), Becker et al., Proc. Nat'l Acad. Sci. USA

93:7826 (1996), Hank et al., Clin. Cancer Res. 2:1951 (1996), and Hu et al., Cancer Res.
56:4998 (1996).

[000247] Immunoconjugates [000248] The invention also pertains to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g.
an enzymatically active toxin or an enzymatically active toxin of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof), prodrug or to another agent such as an immunomodulator, a hormone or hormone antagonist, and enzyme or enzyme inhibitor, a photoactive therapeutic agent such as a chromagen or dye, an angiogenesis inhibitor, an alternate antibody or fragment thereof, or a radioactive isotope (i.e., a radioconjugate) (for review, see Schrama et al, (2006) Nature Reviews 5: 147- 159).

[000249] Chemotherapeutic agents useful in the generation of such immunoconjugates have been described above. Conjugates of an antibody and one or more small molecule toxins, such as a calicheamicin, a maytansine (U.S. Pat. No. 5,208,020), a trichothene, the duocarmycins (also known as `Ultra Potent Toxins'; see generally Lillo et al, (2004) Chemistry and Biology 11; p. 897- 906) and CC-1065 are also contemplated herein.

[000250] In one preferred embodiment of the invention, the antibody is conjugated to one or more maytansine molecules (e.g. about 1 to about 10 maytansine molecules per antibody molecule). Maytansine may, for example, be converted to May-SS-Me which may be reduced to May-SH3 and reacted with modified antibody (Chari et al. Cancer Research 52: 127-131 (1992)) to generate a maytansinoid-antibody immunoconjugate. Alternately, the drug selected may be the highly potent maytansine derivative DMI (N2'-deacetyl-N2'-(3-mercapto-l-oxopropyl)-maytansine) (see for example W002/098883 published Dec. 12, 2002) which has an IC50 of approximately 10-11 M (review, see Payne (2003) Cancer Cell 3:207-212) or DM4 (N2'-deacetyl-N2'(4-methyl- -4-mercapto-l-oxopentyl)-maytansine) (see for example W02004/103272 published Dec. 2, 2004) [000251] Another immunoconjugate of interest comprises an anti-tumor cell antigen antibody conjugated to one or more calicheamicin molecules. The calicheamicin family of antibiotics are capable of producing double-stranded DNA breaks at sub-picomolar concentrations. Structural analogues of calicheamicin which may be used include, but are not limited to, ylI, a2I, a3I, N-acetyl- yll, PSAG and AIl (Hinman et al. Cancer Research 53:
3336-3342 (1993) and Lode et al. Cancer Research 58: 2925-2928 (1998)). See, also, U.S. Pat.

Nos. 5,714,586; 5,712,374; 5,264,586; and 5,773,001 expressly incorporated herein by reference.

[000252] Still another approach involves conjugating the tumor cell antigen antibody to a prodrug, capable of being release in its active form by enzymes overproduced in many cancers. For example, antibody conjugates can be made with a prodrug form of doxorubicin wherein the active component is released from the conjugate by plasmin.
Plasmin is known to be over produced in many cancerous tissues (see Decy et al, (2004) FASEB
Journal 18(3):
565-567).

[000253] Enzymatically active toxins and fragments thereof which can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa),Pseudomonas endotoxin, ricin A chain, abrin A chain, modeccin A
chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), Ribonuclease (Rnase), Deoxyribonuclease (Dnase), pokeweed antiviral protein, momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, neomycin and the tricothecenes. See, for example, WO 93/21232 published Oct. 28, 1993. Particularly preferred are toxins that have low intrinsic immunogenicity and a mechanism of action (e.g. a cytotoxic mechanism versus a cytostatic mechanism) that reduces the opportunity for the cancerous cells to become resistant to the toxin.

[000254] Conjugates made between the antibodies of the invention and immunomodulators are contemplated. For example, immunostimulatory oligonucleotides can be used.
These molecules are potent immunogens that can elicit antigen-specific antibody responses (see Datta et al, (2003) Ann N.Y. Acad. Sci 1002: 105-111). Additional immunomodulatory compounds can include stem cell growth factor such as "S1 factor", lymphotoxins such as tumor necrosis factor (TNF), hematopoietic factor such as an interleukin, colony stimulating factor (CSF) such as granulocyte-colony stimulating factor (G-CSF) or granulocyte macrophage-stimulating factor (GM-CSF), interferon (IFN) such as interferon alpha, beta or gamma, erythropoietin, and thrombopoietin.

[000255] A variety of radioactive isotopes are available for the production of radioconjugated anti-tumor cell antigen antibodies. Therapeutic radioconjugates can be made using 32P, 33P, 47Sc, 59Fe, 64Cu, 67Cu, 75Se, 77As, 89Sr, 90Y, 99MC, 105Rh, lo9Pd, Ag-Il 1 , 125I
, 131I 142Pr 143Pr 149Pm 153Sm 161.Lh 166HC 169Er 177Lu 186Re 188Re 189Re 194~, 198Au 199AU
> > > > > > > > > > > > > > >
211Pb 212Pb, and213Bi SsCo 67Ga soBrm 99Tcm, 103Rhm 109Pt, In-ill, t i9Sb, 1-125, 161 Ho, 189 Osm, 192 ir, 152 D y211At 212Bi 223Ra 219Rn 215PQ 211Bi 225AC 221Fr 217At 213Bi 255Fm and , , , , > , , , , , , combinations thereof. Additionally, boron, gadolinium or uranium atoms can be used, wherein the boron atom is preferably 10B, the gadolinium atom is 157 Gd and the uranium atom is 235U.

[000256] Preferably, the therapeutic radionuclide conjugate has a radionuclide with an energy between 20 and 10,000 keV. The radionuclide can be an Auger emitter, with an energy of less than 1000 keV, a P emitter with an energy between 20 and 5000 keV, or an alpha or `a' emitter with an energy between 2000 and 10,000 keV.

[000257] Diagnostic radioconjugates may contain a radionuclide that is a gamma-beta- or positron-emitting isotope, where the radionuclide has an energy between 20 and 10,000 keV.
The radionuclide may be selected from the group of iaF 51Mn, 52mMn, 52Fe, SSCo, 62Cu, 64Cu, 68Ga , 72As >75Br > 76 Br, 82mRb> 83Sr> 86 y, 89 Zr, 94mTc, IIn> 120 i, 124 i, 51Cr> 57Co> 58Co> 59Fe, 67CU
, 67Ga, 75Se, 97Ru, 99mTc, lllIn, 114mln 123i 125i 131i 169 197Hg, and 21,TI.

[000258] Additional types of diagnostic immunoconjugates are contemplated. The antibody or fragments of the invention may be linked to diagnostic agents that are photoactive or contrast agents. Photoactive compounds can comprise compounds such as chromagens or dyes. Contrast agents may be for example a paramagnetic ion, wherein the ion comprises a metal selected from the group of chromium (III), manganese (II), iron (III), iron (11), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) and erbium (IIl). The contrast agent may also be a radio-opaque compound used in X-ray techniques or computed tomography, such as an iodine, iridium, barium, gallium and thallium compound.
Radio-opaque compounds may be selected from the group of barium, diatrizoate, ethiodized oil, gallium citrate, iocarmic acid, iocetamic acid, iodamide, iodipamide, iodoxamic acid, iogulamide, iohexol, iopamidol, iopanoic acid, ioprocemic acid, iosefamic acid, ioseric acid, iosulamide meglumine, iosemetic acid, iotasul, iotetric acid, iothalamic acid, iotroxic acid, ioxaglic acid, ioxotrizoic acid, ipodate, meglumine, metrizamide, metrizoate, propyliodone, and thallous chloride. Alternatively, the diagnostic immunoconjugates may contain ultrasound-enhancing agents such as a gas filled liposome that is conjugated to an antibody of the invention. Diagnostic immunoconjugates may be used for a variety of procedures including, but not limited to, intraoperative, endoscopic or intravascular methods of tumor or cancer diagnosis and detection.

[000259] Conjugates of the antibody and cytotoxic agent may be made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl) cyclohexane-l-carboxylate, iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al. Science 238: 1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See W094/11026. The linker may be a "cleavable linker" facilitating release of the cytotoxic drug in the cell. For example, an acid-labile linker, peptidase-sensitive linker, dimethyl linker or disulfide-containing linker (Chari et al. Cancer Research 52: 127-131 (1992)) may be used.
Agents may be additionally be linked to the antibodies of the invention through a carbohydrate moiety.

[000260] Alternatively, a fusion protein comprising the anti-tumor cell antigen antibody and cytotoxic agent may be made, e.g. by recombinant techniques or peptide synthesis.

[000261] In yet another embodiment, the antibody may be conjugated to a "receptor" (such as streptavidin) for utilization in tumor pretargeting wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a "ligand" (e.g. avidin) which is conjugated to a cytotoxic agent (e.g. a radionucleotide).

[000262] The anti-tumor cell antigen antibodies may additionally be conjugated to a cytotoxic molecule which is only released inside a target cell lysozome. For example, the drug monomethyl auristatin E (MMAE) can be conjugated via a valine-citrulline linkage which will be cleaved by the proteolytic lysozomal enzyme cathepsin B
following internalization of the antibody conjugate (see for example W003/026577 published April 3, 2003). Alternatively, the MMAE can be attached to the antibody using an acid-labile linker containing a hydrazone functionality as the cleavable moiety (see for example published Nov. 11, 2002).

[000263] While it is desireable for an aforementioned antibody conjugate to internalize rapidly, a slow-internalizing antyibody would be desirable where the anti-tumor activity is mediated primarily through ADCC. By "slow-internalizing antibody" it is meant an antibody that remains disposed on the cell surface for a significant period of time. As the skilled person will be aware, this property contrasts with properties deemed advantageous for many therapeutic applications that actually require internalization of antibody-receptor complex in order for the therapy to be efficacious. In this context, a significant period of time generally exceeds 3 hours, preferably 6 hours, more preferably 12 hours, more preferably 24 hours, 36 hours, 48 hours, 72 hours, 96 hours, 120 hours, 144 hours, 168 hours or more.

[000264] Internalization of antibodies can be assessed by various assays. For example, cell lines transfected with FZD10 or a cell line that expresses FZD10 on the surface (e.g., the NCI-H460 cells) can be used to evaluate the effect of a candidate antibody binding on internalization. Cells are incubated with human IgG1 (control antibody) or the candidate antibody on ice (with 0.1% sodium azide to block internalization) or 37 C
(without sodium azide) for a period of time, suitably 3 hours. After a wash with cold staining buffer (e.g.
PBS+1%BSA+0.1% sodium azide), cells are stained, for example with goat anti-human IgG-FITC for 30 minutes on ice. The degree of staining can then be assessed; in this example, geometric mean fluorescent intensity (MFI) could be recorded, such as by FACS
Calibur.
Other suitable assays will be known to those of skill in the art.

[000265] Combination Therapy [000266] Having identified more than one FZD10 antibody that is effective in an animal model, it may be further advantageous to mix two or more such FZD10 antibodies together to provide still improved efficacy against cancer. Compositions comprising one or more FZD10 antibody may be administered to persons or mammals suffering from, or predisposed to suffer from, cancer. A FZD10 antibody may also be administered with another therapeutic agent, such as a cytotoxic agent, or traditional cancer chemotherapeutic. Concurrent administration of two therapeutic agents does not require that the agents be administered at the same time or by the same route, as long as there is an overlap in the time period during which the agents are exerting their therapeutic effect. Simultaneous or sequential administration is contemplated, as is administration on different days or weeks.

[000267] The method of the invention contemplate the administration of single anti-FZD10 antibodies, as well as combinations, or "cocktails", of different antibodies.
Such antibody cocktails may have certain advantages inasmuch as they contain antibodies which exploit different effector mechanisms or combine directly cytotoxic antibodies with antibodies that rely on immune effector functionality. Such antibodies in combination may exhibit synergistic therapeutic effects.

[000268] A cytotoxic agent refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells. The term is intended to include radioactive isotopes (e.g., 1131, 1125, Y90 and Re186), chemotherapeutic agents, and toxins such as enzymatically active toxins of bacterial, fungal, plant or animal origin or synthetic toxins, or fragments thereof. A non-cytotoxic agent refers to a substance that does not inhibit or prevent the function of cells and/or does not cause destruction of cells. A non-cytotoxic agent may include an agent that can be activated to be cytotoxic. A non-cytotoxic agent may include a bead, liposome, matrix or particle (see, e.g., U.S. Patent Publications 2003/0028071 and 2003/0032995 which are incorporated by reference herein). Such agents may be conjugated, coupled, linked or associated with an antibody according to the invention.

[000269] Traditional cancer chemotherapeutic agents include, without limitation, alkylating agents, such as carboplatin and cisplatin; nitrogen mustard alkylating agents;
nitrosourea alkylating agents, such as carmustine (BCNU); antimetabolites, such as methotrexate; folinic acid; purine analog antimetabolites, mercaptopurine; pyrimidine analog antimetabolites, such as fluorouracil (5-FU) and gemcitabine (Gemzar ); hormonal antineoplastics, such as goserelin, leuprolide, and tamoxifen; natural antineoplastics, such as aldesleukin, interleukin-2, docetaxel, etoposide (VP-16), interferon alfa, paclitaxel (Taxol ), and tretinoin (ATRA);
antibiotic natural antineoplastics, such as bleomycin, dactinomycin, daunorubicin, doxorubicin, daunomycin and mitomycins including mitomycin C; and vinca alkaloid natural antineoplastics, such as vinblastine, vincristine, vindesine; hydroxyurea;
aceglatone, adriamycin, ifosfamide, enocitabine, epitiostanol, aclarubicin, ancitabine, nimustine, procarbazine hydrochloride, carboquone, carboplatin, carmofur, chromomycin A3, antitumor polysaccharides, antitumor platelet factors, cyclophosphamide (Cytoxin ), Schizophyllan, cytarabine (cytosine arabinoside), dacarbazine, thioinosine, thiotepa, tegafur, dolastatins, dolastatin analogs such as auristatin, CPT-11 (irinotecan), mitozantrone, vinorelbine, teniposide, aminopterin, carminomycin, esperamicins (See, e.g., U.S. Patent No. 4,675,187), neocarzinostatin, OK-432, bleomycin, furtulon, broxuridine, busulfan, honvan, peplomycin, bestatin (Ubenimex ), interferon- 0, mepitiostane, mitobronitol, melphalan, laminin peptides, lentinan, Coriolus versicolor extract, tegafur/uracil, estramustine (estrogen/mechlorethamine).

[000270] Further, traditional cancer therapeutics include EPO, G-CSF, ganciclovir;
antibiotics, leuprolide; meperidine; zidovudine (AZT); interleukins 1 through 18, including mutants and analogues; interferons or cytokines, such as interferons a, (3, and 7 hormones, such as luteinizing hormone releasing hormone (LHRH) and analogues and, gonadotropin releasing hormone (GnRH); growth factors, such as transforming growth factor-0 (TGF- (3), fibroblast growth factor (FGF), nerve growth factor (NGF), growth hormone releasing factor (GHRF), epidermal growth factor (EGF), fibroblast growth factor homologous factor (FGFHF), hepatocyte growth factor (HGF), and insulin growth factor (IGF);
tumor necrosis factor- a&(3 (TNF- a&(3); invasion inhibiting factor-2 (IIF-2); bone morphogenetic proteins 1-7 (BMP 1-7); somatostatin; thymosin- a -1; y-globulin; superoxide dismutase (SOD);
complement factors; anti-angiogenesis factors; antigenic materials; and pro-drugs.

[000271] Prodrug refers to a precursor or derivative form of a pharmaceutically active substance that is less cytotoxic or non-cytotoxic to tumor cells compared to the parent drug and is capable of being enzymatically activated or converted into an active or the more active parent form. See, e.g., Wilman, "Prodrugs in Cancer Chemotherapy" Biochemical Society Transactions, 14, pp. 375-382, 615th Meeting Belfast (1986) and Stella et al., "Prodrugs: A
Chemical Approach to Targeted Drug Delivery," Directed Drug Delivery, Borchardt et al., (ed.), pp. 247-267, Humana Press (1985). Prodrugs include, but are not limited to, phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, D-amino acid-modified prodrugs, glycosylated prodrugs, ^-lactam-containing prodrugs, optionally substituted phenoxyacetamide-containing prodrugs or optionally substituted phenylacetamide-containing prodrugs, 5-fluorocytosine and other 5-fluorouridine prodrugs which can be converted into the more active cytotoxic free drug.
Examples of cytotoxic drugs that can be derivatized into a prodrug form for use herein include, but are not limited to, those chemotherapeutic agents described above.

[000272] WO2005/044294, incorporated by reference herein in its entirety, discloses that IL-2 may be administered in combination with a n antibody that binds to an antigen expressed on cancerous cells (e.g., CD40) to improve ADCC-mediated anti-tumor activity of the antibody.
Monoclonal antibody ADCC activity may arise from activity from the neutrophils and monocytes/macrophage population. See, for example, Hernandez-Ilizaliturri et al. (2003) Clin.
Cancer Res. 9 (16, Pt 1): 5866-5873; and Uchida et al. (2004) J. Exp. Med. 199 (12): 1659-1669). As IL-2 is known to activate these cell populations, one embodiment of the present invention provides combination therapy of the antagonist anti-FZD10 antibody with IL-2 or biologically active variant thereof may improve ADCC-mediated anti-tumor activity of the antibody.

[000273] Oligonucleotides [000274] In some embodiments, the FZD10 modulator is an oligonucleotide. In some embodiments the oligonucleotide is an antisense or RNAi oligonucleotide. In some embodiments the oligonucleotide is complementary to a nucleotide encoding a region, domain, portion, or segment of FZD10. In some embodiments, the oligonucleotide comprises from about 5 to about 100 nucleotides, from about 10 to about 50 nucleotides, from about 12 to about 35, and from about 18 to about 25 nucleotides. In some embodiments, the oligonucleotide is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%
homologous to a region, portion, domain, or segment of the FZD10 gene. In some embodiments there is substantial sequence homology over at least 15, 20, 25, 30, 35, 40, 50, or 100 consecutive nucleotides of the FZD 10 gene. In some embodiments there is substantial sequence homology over the entire length of the FZD10 gene. In some embodiments, the oligonucleotide binds under moderate or stringent hybridization conditions to a nucleic acid molecule having a nucleotide sequence of SEQ ID NO: 1.

[000275] In some embodiments, the FZD10 modulator is an oligonucleotide having a sequence selected from the group consisting of SEQ ID NOs: 3-22 ~ IN ~ A$ g d VC N 'tli i 8 91 d d [000276] In some embodiments, the FZD10 modulator is a double stranded RNA
(dsRNA) molecule and works via RNAi (RNA interference). In some embodiments, one strand of the dsRNA is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%
homologous to a region, portion, domain, or segment of the FZD10 gene. In some embodiments there is substantial sequence homology over at least 15, 20, 25, 30, 35, 40, 50, 100, 200, 300, 400, 500, or 1000 consecutive nucleotides of the FZD10 gene. In some embodiments there is substantial sequence homology over the entire length of the FZD10 gene.

[000277] In some embodiments oligonucleotides are used in a polymerase chain reaction (PCR). This sequence may be based on (or designed from) a genomic sequence or cDNA
sequence and is used to amplify, confirm, or detect the presence of an identical, similar, or complementary DNA or RNA in a particular cell or tissue. Oligonucleotides may also be used to modulate the expression of a gene. Such oligonucleotide modulators comprise portions of a DNA sequence and have at least about 10 nucleotides and as many as about 500 nucleotides.
In some embodiments the oligonucleotides comprise from about 10 nucleotides to about 50 nucleotides, from about 15 nucleotides to about 30 nucleotides, and from about 20 nucleotides to about 25 nucleotides. Oligonucleotides may be chemically synthesized and can also be used as probes. In some embodiments the oligonucleotide modulators are single stranded. In some embodiments oligonucleotide modulators comprise at least one portion which is double stranded. In some embodiments the oligonucleotides are antisense oligonucleotides (ASO).
In some embodiments the oligonucleotides are RNA interference oligonucleotides (RNAi oligonucleotides).

[000278] Small molecules [000279] In some embodiments, the FZD10 modulator is a small molecule. As used herein, the term "small molecule" refers to an organic or inorganic non-polymer compound that has a molecular weight that is less than about 10 kilodaltons. Examples of small molecules include peptides, oligonucleotides, organic compounds, inorganic compounds, and the like. In some embodiments, the small molecule has a molecular weight that is less than about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, or about 1 kilodalton.

[000280] Mimetics [000281] In some embodiments, the FZD10 modulator is a mimetic. As used herein, the term "mimetic" is used to refer to compounds which mimic the activity of a peptide.
Mimetics are non-peptides but may comprise amino acids linked by non-peptide bonds. U.S.
Patent No. 5,637,677, issued on June 10, 1997, and parent applications thereof, all of which are incorporated herein by reference, contain detailed guidance on the production of mimetics.
Briefly, the three-dimensional structure of the peptides which specifically interacts with the three dimensional structure of the FZD10 receptor is duplicated by a molecule that is not a peptide. In some embodiments the FZD10 mimetic is a mimetic of FZD10 receptor or a mimetic of a ligand of FZD 10 receptor.

[000282] Soluble Receptors [000283] In some embodiments, the FZD10 modulator is a soluble receptor. As used herein, the term "soluble receptor" refers to a Frizzled receptor, preferably a FZD10 receptor, which is essentially free of either a membrane domain or has a disrupted membrane domain.

[000284] Decoy Receptors [000285] In some embodiments, the FZD10 modulator is a decoy receptor comprising at least a portion of an FZD 10 receptor. In some embodiments the decoy receptor competes with natural FZD10 receptors for FZD10 ligands. In some embodiments, the decoy receptor is labeled to facilitate quantification, qualification, and/or visualization. In other embodiments, the decoy receptor further comprises a moiety to facilitate isolation and/or separation of the decoy receptor and or the decoy receptor-FZD10 complex. In some embodiments, the decoy receptor, upon binding with an FZD10 receptor ligand, causes an increased signal (compared to a native FZD10 receptor) to be effected. In some embodiments, the decoy receptor is a non-signaling molecule which functions by capturing FZD10 ligand and preventing it from interacting with the signaling FZD10 receptor. In some embodiments the decoy receptor comprises at least a portion of an FZD10 receptor fused to an antibody or antibody fragment.

[000286] Methods of Treating/Preventing Cancer [000287] The present invention provides methods for treating and/or preventing cancer or symptoms of cancer in a subject comprising administering to the subject a therapeutically effective amount of one or more FZD10 modulators. In some embodiments the cancer is a cancer associated with overexpression of FZD10. In some embodiments, the cancer is colon, ovarian, lung or uterine cancer. In some embodiments the subject has been diagnosed as having a cancer or as being predisposed to cancer.

[000288] Symptoms of cancer are well-known to those of skill in the art and include, without limitation, pain, death, weight loss, weakness, difficulty eating, blood in stool, nausea, vomiting, liver metastases, lung metastases, bone metastases, abdominal fullness, bloating, fluid in peritoneal cavity, vaginal bleeding, constipation, abdominal distension, perforation of colon, acute peritonitis (infection, fever, pain), vomiting blood, difficulty swallowing, and the like.

[000289] In additional embodiments, the prophylactic or therapeutic treatment methods of the invention may also involve methods of identifying a subject likely to be responsive to therapy with an FZD10 modulator, such as an anti-FZD10 antibody or inhibitory polynucleotide, prior to treating the subject with such FZD10 modulator.
Methods of identifying responsive subjects involve detecting the expression level and/or phosphorylation state of LRP5 in tumor cells from the subject, wherein increased expression or phosphorylation of LRP5 may correlate with likelihood of being responsive to antibody or siRNA therapy. Alternatively, or in addition to detecting LRP5 status, responsive subjects can be identified by detecting the expression level of FZD10 in tumor cells from the subject, wherein increased expression of FZD10 may correlate with likelihood of being responsive to FZD10 antibody or siRNA therapy. Expression level of a protein in a tissue sample from a subject can be assayed in any of a number of different ways, including immunohistochemistry using an antibody specific for LRP5 or FZD10, and by PCR
to detect relative levels of mRNA. Immunohistochemistry involves contacting the tissue sample with an antibody (e.g. polyclonal or monoclonal) specific for the protein, and detecting relative binding of antibody to the sample. Nucleic acid based methods involve extracting and amplifying nucleic acid from the sample, contacting the amplified nucleic acid with a probe or primer specific for the gene encoding the protein, and detecting hybridization of the probe or primer to the amplified nucleic acid. In related embodiments, the aforementioned probe or primer is labeled. The phosphorylation state of LRP5 can be detected by phospho-specific antibody against Theorine 1492, Serine 1503 and Theorine 1506 of LRP5 which are the homologous phospho-amino acid residues on Theorine 1479, Serine 1490 and Theorine 1493 of LRP6. These specific residues on LRP6 were shown to be phosphorylated upon WNT
ligand mediated activation of the (3-catenin mediated canonical pathway. (Zeng et al., Nature.
2005 Dec 8;438(7069):873-7; Davidson et al., Nature. 2005 Dec 8;438(7069):867-72.) It may be sufficient to detect phosphorylation state of LRP5 through use of such phospho-specific antibodies that cross-react with LRP6. It may also be desirable to distinguish between LRP5 and LRP6 using an antibody specific for LRP5.

[000290] In related embodiments, treatment of a subject with an FZD10 modulator is monitored at intervals after commencement of treatment, for example, at 1 month, 2 months, 3 months, or monthly or quarterly, semiannually or annually, by detecting the expression level of LRP5 and/or FZD10, or the phosphorylation state of LRP5 in tissue samples from the subject. Such monitoring may optionally be used to predict prognosis, wherein decreased expression of LRP5 and/or FZD10, or decreased phosphorylation of LRP5 correlates with improved prognosis.

[000291] A therapeutically effective amount of the modulating compound can be determined empirically, according to procedures well known to medicinal chemists, and will depend, inter alia, on the age of the patient, severity of the condition, and on the ultimate pharmaceutical formulation desired. Administration of the modulators of the present invention can be carried out, for example, by inhalation or suppository or to mucosal tissue such as by lavage to vaginal, rectal, urethral, buccal and sublingual tissue, orally, topically, intranasally, intraperitoneally, parenterally, intravenously, intralymphatically, intratumorly, intramuscularly, interstitially, intra-arterially, subcutaneously, intraoccularly, intrasynovial, transepithelial, and transdermally. In preferred embodiments, the inhibitors are administered by lavage, orally or inter-arterially. Other suitable methods of introduction can also include rechargeable or biodegradable devices and slow or sustained release polymeric devices. As discussed above, the therapeutic compositions of this invention can also be administered as part of a combinatorial therapy with other known anti-cancer agents or other known anti-bone disease treatment regimen.

[000292] The present invention further provides methods of modulating an FZD10-related biological activity in a patient. The methods comprise administering to the patient an amount of an FZD10 modulator effective to modulate one or more FZD10 biological activities.
Suitable assays for measuring FZD10 biological activities are set forth supra and infra.

[000293] The present invention also provides methods of inhibiting cancer cell growth in a patient in need thereof comprising administering a therapeutically effective amount of one or more FZD10 modulators to the patient. Suitable assays for measuring FZD10 -related cell growth are known to those skilled in the art.

[000294] The present invention further provides methods of inhibiting cancer in a patient in need thereof. The methods comprise determining if the patient is a candidate for FZD10 therapy as described herein and administering a therapeutically effective amount of one or more FZD10 modulators to the patient if the patient is a candidate for FZD10 therapy. If the patient is not a candidate for FZD10 therapy, the patient is treated with conventional cancer treatment.

[000295] The present invention also provides methods for inhibiting the interaction of two or more cells in a patient comprising administering a therapeutically effective amount of an FZD10 modulator to said patient. Suitable assays for measuring FZD10-related cell interaction are known to those skilled in the art.

[000296] The present invention also provides methods of modulating one or more symptoms of cancer in a patient comprising administering to said patient a therapeutically effective amount of the FZD10 compositions described herein.

[000297] The present invention further provides methods for inhibiting anchorage-independent cell growth in a patient in need thereof comprising administering to the patient a therapeutically effective amount of an FZD10 modulator. Suitable assays for measuring FZD10-related anchorage-independent cell growth are set forth in the Examples.

[000298] The present invention also provides methods for inhibiting migration of cancer cells in a patient in need thereof comprising administering to the patient a therapeutically effective amount of an FZD10 modulator. Suitable assays for measuring FZD10 -related cell migration are known to those skilled in the art.

[000299] The present invention further provides methods for inhibiting adhesion of cancer cells in a patient in need thereof comprising administering to the patient a therapeutically effective amount of an FZD10 modulator. Suitable assays for measuring FZD10 -related cell adhesion are known to those skilled in the art.

[000300] The present invention also provides methods to prophylactically treat a patient who is predisposed to develop cancer, a cancer metastasis or who has had a metastasis and is therefore susceptible to a relapse or recurrence. The methods are particularly useful in high-risk individuals who, for example, have a family history of cancer or of metastasizing tumors, or show a genetic predisposition for a cancer metastasis. In some embodiments the tumors are FZD10 -related tumors. Additionally, the methods are useful to prevent patients from having recurrences of FZD10-related tumors who have had FZD10-related tumors removed by surgical resection or treated with a conventional cancer treatment.

[000301] The present invention also provides methods of inhibiting cancer progression and/or causing cancer regression comprising administering to the patient a therapeutically effective amount of an FZD10 modulator.

[000302] In some embodiments, the patient in need of anti-cancer treatment is treated with the antibodies, small molecules, mimetics, soluble receptors, decoy receptors, or oligonucleotide modulators in conjunction with chemotherapy and/or radiation therapy. For example, following administration of the antibodies, small molecules, mimetics, soluble receptors, decoy receptors, or oligonucleotide modulators, the patient may also be treated with a therapeutically effective amount of anti-cancer radiation. In some embodiments chemotherapeutic treatment is provided in combination with the antibodies, small molecules, mimetics, soluble receptors, decoy receptors, or oligonucleotide modulators.
In some embodiments antibodies, small molecules, mimetics, soluble receptors, decoy receptors, or oligonucleotide modulators are administered in combination with chemotherapy and radiation therapy.

[000303] Methods of treatment comprise administering single or multiple doses of one or more FZD10 modulators to the patient. In some embodiments the FZD10 modulators are administered as injectable pharmaceutical compositions that are sterile, pyrogen free and comprise the FZD10 modulators in combination with a pharmaceutically acceptable carrier or diluent.

[000304] In some embodiments, the therapeutic regimens of the present invention are used with traditional treatment regimens for cancer including, without limitation, surgery, radiation therapy, hormone ablation and/or chemotherapy. Administration of the FZD 10 modulators of the present invention may take place prior to, simultaneously with, or after traditional cancer treatment.

[000305] In some embodiments, two or more different FZD10 modulators are administered to the patient.

[000306] In some embodiments the amount of FZD10 modulator administered to the patient is effective to inhibit Dishevelled phosphorylation. In some embodiments the amount of FZD10 modulator administered to the patient is effective to inhibit c-Jun phosphorylation In some embodiments the amount of FZD10 modulator administered to the patient is effective to induce (3-catenin phosphorylation. In some embodiments the amount of FZD10 modulator administered to the patient is effective to inhibit cancer progression and/or cause cancer regression.

[000307] Clinical Aspects [000308] In some embodiments, the methods and compositions of the present invention are particularly useful in colon cancer, ovarian cancer, lung cell cancer, and uterine cancer. In some embodiments, the methods and compositions are useful in treating and/or diagnosing cancer metastasis, including, for example, lung metastases.

[000309] Pharmaceutical Compositions [000310] The present invention also provides pharmaceutical compositions comprising one or more of the FZD 10 modulators described herein and a pharmaceutically acceptable carrier.

[000311] In some embodiments the pharmaceutical compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared. Liposomes are included within the definition of a pharmaceutically acceptable carrier. Pharmaceutically acceptable salts can also be present in the pharmaceutical composition, e.g., mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. A thorough discussion of pharmaceutically acceptable excipients is available in Remington: The Science and Practice of Pharmacy (1995) Alfonso Gennaro, Lippincott, Williams, & Wilkins.

[000312] Methods of Detecting FZD10 [000313] The present invention also provides methods for detecting FZD10. In some embodiments the FZD10 is present in a patient or in a patient sample. In some embodiments the method comprises administering a composition comprising one or more FZD10 modulators to the patient and detecting the localization of the imaging agent in the patient. In some embodiments the patient sample comprises cancer cells. In some embodiments the FZD10 modulator is linked to an imaging agent or is detectably labeled. In some embodiments, the FZD10 modulator is an anti-FZD10 antibody conjugated to an imaging agent and is administered to a patient to detect one or more tumors or to determine susceptibility of the patient to FZD10 therapy. The labeled antibodies will bind to the high density of receptors on cells and thereby accumulate on the tumor cells. Using standard imaging techniques, the site of the tumors can be detected.

[000314] The present invention also provides methods of imaging/detecting cells or tumors expressing or overexpressing FZD10 comprising contacting a composition comprising an FZD10 modulator to a sample and detecting the presence of the FZD10 modulator in the sample. In some embodiments the sample is a patient sample. In some embodiments the patient sample comprises cancer cells. In some embodiments the FZD10 modulator is linked to an imaging agent or is detectably labeled.

[000315] The present invention also provides methods for quantifying the amount of FZD10 present in a patient, cell or sample. The methods comprise administering one or more of antibodies, probes, or small molecules to a patient or sample and detecting the amount of FZD10 present in the sample. In some embodiments the antibodies, probes, or small molecules are linked to an imaging agent or are detectably labeled. Such information indicates, for example, whether or not a tumor is related to FZD10, and, therefore, whether specific treatments should be used or avoided. In some embodiments, using standard techniques well known to the art-skilled, samples believed to include tumor cells are obtained and contacted with labeled antibodies, probes, oligonucleotides, and small molecules. After removing any unbound, labeled antibodies, probes, oligonucleotides or small molecules, the quantity of labeled antibodies, peptides, oligonucleotides or mimetics bound to the cell, or the quantity of antibodies, peptides, oligonucleotides or mimetics removed as unbound is determined. The information directly relates to the amount of FZD 10 present.

[000316] Imaging can be performed using procedures well known to those of ordinary skill in the art. Imaging can be performed, for example, by radioscintigraphy, nuclear magnetic resonance imaging (MRI) or computed tomography (CT scan). The most commonly employed radiolabels for imaging agents include radioactive iodine and indium.
Imaging by CT scan may employ a heavy metal such as an iron chelate. MRI scanning may employ chelates of gadolinium or manganese. Additionally, positron emission tomography (PET) may be possible using positron emitters of oxygen, nitrogen, iron, carbon, or gallium.

[000317] Methods of detection are well known to those of skill in the art. For example, methods of detecting polynucleotides include, but are not limited to PCR, Northern blotting, Southern blotting, RNA protection, and DNA hybridization (including in situ hybridization).
Methods of detecting polypeptides include, but are not limited to, Western blotting, ELISA, enzyme activity assays, slot blotting, peptide mass fingerprinting, electrophoresis, immunochemistry and immunohistochemistry. Other examples of detection methods include, but are not limited to, radioimmunoassay (RIA), chemiluminescence immunoassay, fluoroimmunoassay, time-resolved fluoroimmunoassay (TR-FIA), two color fluorescent microscopy, or immunochromatographic assay (ICA), all well known by those of skill in the art. In some preferred embodiments of the present invention, polynucleotide expression is detected using PCR methodologies and polypeptide production is detected using ELISA
technology.

[000318] Methods for determining susceptibility to FZD10 therapy [000319] The present invention also provides methods for determining susceptibility of a patient to FZD10 therapy. The methods comprise detecting the presence or absence of evidence of FZD10 expression in a patient or patient sample. The presence of evidence of FZD10 expression in the patient or sample is indicative of a patient who is susceptible to FZD10 therapy. The absence of evidence of FZD10 expression in the patient or patient sample is indicative of a patient who is not a candidate for FZD10 therapy.

[000320] In some embodiments the therapeutic methods comprise first identifying patients susceptible to FZD10 therapy comprising administering to the patient in need thereof a composition comprising an FZD10 antibody, probe, primer, or oligonucleotide linked to an imaging agent and detecting the presence or absence of evidence of the gene or gene product in the patient. The presence of evidence of FZD10 expression, especially FZD10 overexpression, in the patient is indicative of a patient who is a candidate for FZD10 therapy and the absence of evidence of FZD10 expression in the patient is indicative of a patient who is not a candidate for FZD10 therapy. In some embodiments, the therapeutic methods further comprise administering one or more FZD10 modulators to the patient if the patient is a candidate for FZD10 therapy and treating the patient with conventional cancer treatment if the patient is not a candidate for FZD10 therapy.

[000321] Methods for Screening [000322] The present invention also provides methods of screening for anti-cancer agents.
The methods comprise contacting a cell expressing FZD10 with a candidate compound and determining whether an FZD10-related biological activity is modulated. In some embodiments, a decrease in phosphorylation of Dishevelled, a decrease in phosphorylation of c-Jun, and an increase in phosphorylation of 0-catenin is indicative of a cancer inhibitor. In some embodiments, inhibition of FZD10 expression is indicative of a cancer inhibitor.

[000323] The present invention further provides methods of identifying a cancer inhibitor.
The methods comprise contacting a cell expressing FZD10 with a candidate compound and an FZD101igand, and determining whether an FZD10-related biological activity is modulated. In some embodiments, a decrease in phosphorylation of Dishevelled, a decrease in phosphorylation of c-Jun, and an increase in phosphorylation of 0-catenin is indicative of a cancer inhibitor. In some embodiments, inhibition of FZD10 expression is indicative of a cancer inhibitor.

[000324] In some embodiments, the invention provides methods of screening for anti-cancer agents, particularly anti-metastatic cancer agents, by, for example, screening putative modulators for an ability to decrease phosphorylation of Dishevelled, decrease phosphorylation of c-Jun, and increase phosphorylation of 0-catenin.

[000325] Kits [000326] In some embodiments, the present invention provides kits for imaging and/or detecting a gene or gene product correlated with FZD10 overexpression. Kits of the invention comprise detectable antibodies, small molecules, oligonucleotides, oligonucleotide modulators, soluble receptors, decoy receptors, mimetics or probes as well as instructions for performing the methods of the invention. Optionally, kits may also contain one or more of the following: controls (positive and/or negative), containers for controls, photographs or depictions of representative examples of positive and/or negative results.

[000327] Each of the patents, patent applications, GenBank accession numbers and publications described herein is hereby incorporated by reference in its entirety.

[000328] Various modifications of the invention, in addition to those described herein, will be apparent to those of skill in the art in view of the foregoing description.
Such modifications are also intended to fall within the scope of the appended claims. The present invention is further demonstrated in the following examples that are for purposes of illustration and are not intended to limit the scope of the present invention.

[000329] EXAMPLES
Example 1: Upregulation of FZD10 in lung, ovary, uterine and colon cancers [000330] A set of nucleotide sequences (a "probe set") that are representative of Frizzled 10 mRNA were selected from nucleotide sequences arrayed on polynucleotide arrays (Affymetrix, Santa Clara, CA) and were used to screen relative expression level of mRNA in various tumor or healthy tissue samples. cDNA fragments from mRNA isolated from cell lines or tissues of interest are prepared and labeled according to conventional methods and hybridized to the polynucleotide arrays according to the manufacturer's instructions. mRNA
from a variety of cancerous tissue samples and normal tissue samples was assayed.

[000331] Figure 1 Depicts a graphical representation of the microarray analysis (affy U133 plus 2 chip) of cancerous and normal tissues analyzed using FZD10. Normal and cancerous tissue types are laid out along the horizontal axis. Cancerous tissues are labeled with a`c', for example, "cbreast_duct" which represents a breast cancer tissue sample and normal tissues are similarly represented with a`n'. The tissue types are further labeled with respect to the type and subtype of the tissue, if known. For example "c_breast_duct" is a cancerous tissue from a breast cancer that was localized in a breast duct. If the subtype was not clear during surgical removal or was unknown, the label says, `ns' for `non-specified'.
Each spot on the vertical axes represents a tissue sample from a single patient, and the height of each spot on the vertical axes (log2 based) represents relative expression level of the probeset. Filled circles represent samples with expression levels in the linear detection range. Open circles represent an upper limit on gene expression in samples where the gene was below the probeset's detection limit. Open squares represent a lower limit on gene expression in samples where the probeset was saturated. (Briefly, before performing an analysis, each probeset was calibrated by analyzing the behavior of its constituent probes across a large, diverse set of samples. This calibration determines the relative sensitivity of each probe, and the range of intensities within which the probeset response is linear between probes.
Intensities below this range are called "undetected" while those above it are called "saturated". Because of variation in the hybridization and labeling efficiency between samples, we normalize each chip after applying the calibrations. This causes the upper and lower limits of the range, in terms of gene expression, to vary somewhat from sample to sample.) [000332] DecisionSite software (Spotfire, Somerville, MA) and in-house developed Pipeline Pilot (SciTegic, San Diego, CA) protocols were used to generate the graphical depictions shown in Figure 1. The results show elevated expression for FZD10 in the highlighted tumor indications marked with arrows as compared to its expression in a variety of normal tissue types. A table denoted the number of patients is indicated, followed by the relative expression between the cancer and the normal samples. The results show elevated expression of FZD10 mRNA in ovarian, lung and uterine cancer tissues as compared to its expression in a variety of normal tissue types.

[000333] In Figure 2, mRNA was isolated from laser capture microdissected (LCM) colon cancer tissues, and the mRNA was compared to either a pool of respective normal tissue (RSM= reference standard mix) or normal cells adjacent to the cancer cells within each tissue sample. Samples were tested by oligonucleotide array analysis on Affymetrix GeneChips U133A & B (Affymetrix, Inc., Santa Clara, CA). Y axis is the relative raw expression level.
A table denoted the number of patients is indicated, followed by the relative expression between the cancer and the normal samples. ("%GE2X" and ">=2X" denote up-regulation by 2-fold; "%LE .5X" and "<=2x" denotes down-regulation by 2-fold). The results demonstrated that FZD10 is up-regulated in a subset of colon cancer patient samples relative to respective normal tissues or adjacent normal cells.

[000334] Example 2: Quatitative RT-PCR of primary colon, lung and ovarian cancer patient samples [000335] RT-PCR analysis of primary tumor samples was divided into 4 major steps: 1) RNA purification from primary normal and tumor tissues; 2) Generation of first strand cDNA
from the purified tissue RNA for Real Time Quantitative PCR; 3) Setup RT-PCR
for gene expression using ABI PRISM 7900HT Sequence Detection System tailored for 384-well reactions; 4) Analyze RT-PCR data by statistical methods to identify genes differentially expressed (up-regulated) in cancer.

[000336] Using Qiagen RNeasy mini Kit CAT#74106, tissue samples typically yielded approximately 30 g of RNA and typically resulted in a final concentration of approximately 200 ng/ l if 150 1 of elution buffer was used. After RNA was extracted, Ribogreen quantitation reagents from Molecular Probes were used to determine yield and concentration of RNA according to manufactur's protocol. The Integrity of extracted RNA was assessed on EtBr stained agarose gel to determine if the 28S and 18S band have equal intensity. Integrity of extracted RNA was also assessed using Agilent 2100 according to manufactur's protocol.
The Agilent Bioanalyzer / "Lab-On-A-Chip" is a micro-fluidics system that generates an electropherogram of a RNA sample. By observing the ratio of the 18S and 28S
bands and the smoothness of the baseline a determination of the level of RNA degradation can be made.
Samples having a 28S:18S ratio below 1 were discarded.

[000337] RNA samples were also examined by RT-PCR to determine level of genomic DNA contamination during extraction. RNA was arrayed for cDNA synthesis. In general, a minimum of 10 normals and 20 tumors were assayed for each tumor type. Primers used were as follows: SGP758 (forward sequence; ctggtcatgcgcagggt; (SEQ ID NO:23);
reverse sequence; ctcaccggcttcgtgctc (SEQ ID NO:24); probe sequence ggcagcatggacgtcaacgcg; (SEQ
ID NO:25)) was used to FZD10.

[000338] The expression level of a target gene in both normal and tumor samples was determined using Quantitative RT-PCR using the ABI PRISM 7900HT Sequence Detection System (Applied Biosystems, California). The method is based on the quantitation of the initial copy number of target template in comparison to that of a reference (normalizer) housekeeper gene (Pre-Developed TaqMan Assay Reagents Gene Expression Quantification Protocol, Applied Biosystems, 2001). Accumulation of DNA product with each PCR
cycle is related to amplicon efficiency and the initial template concentration.
Therefore the amplification efficiency of both the target and the normalizer must be similar. The threshold cycle (CT), which is dependent on the starting template copy number and the DNA
amplification efficiency, is a PCR cycle during which PCR product growth is exponential.
Each assay is performed in quadruplicates; therefore, 4 CT values are obtained for the target gene in a given sample.

[000339] Simultaneously, the expression level of a group of housekeeper genes was also measured in the same fashion. The outlier within the 4 quadruplicates is detected and removed if the standard deviation of the remaining 3 triplicates is 30% or less compared to the standard deviation of the original 4 quadruplicates. The prevalence of the isoform tested for in each tumor type was also determined. Results are shown in Figure 3.
Briefly, the quantitative PCR data on primary tumors (panel of minimum 10 normal & 20 tumor samples) indicated that FZD10 was overexpressed in 42% of lung cancer patient samples, 30% of ovary cancer patient samples, and 36% of colon cancer patient samples.

[000340] Example 3: Surface expression of FZD10 in different cancer cell lines [000341] Surface expression of FZD10 on different cancer cell lines was assayed as follows.
Surface expression of FZD10 on cancer cell lines was determined by analysis of the GUAVA
Express Systems and GUAVA 96-PCA instrument (Hayward, CA). Cancer cell lines were assessed for FZD10 mRNA expression by quantitative RT-PCR using the procedure described on Example 2. Selected colon, lung and ovarian cancer cell lines which are FZD10 mRNA
expressing cells (SW620, NCI-H460, DOV13) and FZD10 mRNA non-expressing cells (HCT116, A549 and SKOV3) were grown to 80-90% confluence and harvested for flow cytometry analysis using GUAVA-96 Express System. 96-well round bottom plates were pre-blocked by 50u1 of Staining buffer (1X PBS, 2% BSA, 0.05% sodium azide) per well for 20 minutes at room temperature. Staining buffer was removed from the plates before cells were aliquoted. Appropriate volume of cells was transferred to 15 ml conical tubes for each cell lines, washed and diluted in staining buffer into 5 X 104 cells/100u1 concentration. For each cell line, 100u1 of diluted cells were aliquoted to each well for triplicate samples to the pre-blocked 96 well round-bottom plates. Cells were stained with 50u1 of polyclonal antibody against FZD10 (10ug/ml concentration) or control antibody such as prelmmune rabbit IgG for 30 minutes on ice and immediately after the primary antibody incubation, stained cells were washed twice with 200u1 of Wash buffer (1X PBS, 0.05% sodium azide). Cells were then stained with secondary antibody, goat (Fab'2) anti-rabbit IgG-PE-conjugated for 30 minutes on ice and immediately after the secondary antibody incubation, stained cells were washed twice with 200u1 of Wash buffer (1X PBS, 0.05% sodium azide). The 96-well plates with the stained cells were then analyzed using the Guava-96 PCA instrument and Guava Express Systems (Hayward, CA) according to manufacture protocols to determine relative surface expression of FZD10 of each cancer cell lines based on fluorescence signals that were emitted by the PE-conjugated secondary antibody.

[000342] The results presented in Figure 4 is a representation of the data output from GUAVA Express Systems. The graphs are ploted with cell counts against fluorescence signal in log scale. Protein surface expression of FZD10 was detected on colon cancer cell line SW620, lung cancer cell line NCI-H460, and some surface expression was detectable on ovarian cancer cell line DOV13, but not on cell lines HCT116, A549 and SKOV3 when comparing the to pre-immune rabbit IgG as negative control. The data is consistent with the level of FZD10 mRNA expression of these cancer cell lines based on quantitative PCR assay.

[000343] Example 4: siRNAs reduce surface expression of FZD10 [000344] Cells from a variety of cell lines that express FZD10 at varying levels (H460 lung cancer, EKVX non-small cell lung cancer, NCI-H2126 lung cancer, SW620 colon and Co1o320DM colon) and two cell lines that did not express FZD10 (MCF7 breast and A549 lung) were assayed for effects of FZD10 siRNA-mediated knockdown as follows.
Different cell lines may have different siRNA transfection conditions. For H460 (Figure 5) cells were seeded at 600,000 cells/well in a 10cm dish in 10 ml of medium the day before transfection.
Plates were incubated at 37 C O/N. The next day the medium was removed and 3m1 of complete medium was added. In an Eppendorf tube, 1.2m1 of OptiMem was mixed with 250pmo1 of siRNA. In a separate tube, 1.2m1 of OptiMem was used to dilute 25u1 of Dhamarfect-1 lipid reagent (Dharmacon, Lafayette, CO). The diluted lipid was mixed with diluted siRNA to form a complex at room temperature for 25 minutes and then added to the cells dropwise. The final concentration of the siRNA in the cell media was 50nM. The cells with the siRNA were incubated from 4 h to overnight at 37 C and replaced with complete media. Cells were harvested at 24-72 hours to monitor RNA/FZD10 surface expression levels according to methods established in Example 3. For EKVX cells, seeding cell density was 1,000,000 cells per 10cm dish and 5u1 of Dharmafect-2 lipid (Dharmacon, Lafayette, CO) was used per transfection. For NCI-H2126, seeding cell density was 750,000 cells per 10 cm dish and 15u1 Dharmafect-1 lipid (Dharmacon, Lafayette, CO) was used per transfection. For SW620, seeding cell density was 1,200,000 cells per 10cm dish and 15u1 of Lipofectamine-RNAiMAX (Invitrogen, Carlsbad, CA) lipid as used per transfection. For Co1o320DM, seeding cell density was 1,000,000 cells per 10cm dish and 15u1 Dharmafect-2 lipid (Dharmacon, Lafayette, CO) was used per transfection. All siRNAs were transfected at 50nM
concentration in all cell types.

[000345] FZD10 specific siRNAs used were: CHIR317.1.4 or FZD10-1:
GCACAAGTGCAAAATGAAC SEQ ID N0:26; CHIR317.2.2 or FZD10-2:
AGCAGGTGTGCAGCCGTAGGTTAAA SEQ ID N0:27; Negative control siRNA, Silencer Negative control #2 (Ambion, Cat#4637) matched the chemistry of Ambion (CHIR317.1.4 or NC-1) was purchased and used for all siRNA experiments. Negative control siRNA, Invitrogen Stealth RNAi Negative control medium GC duplex (Invitrogen, Cat#
12935-300) matched the chemistry of Invitrogen Stealth siRNA (CHIR317.2.2 or NC-2) was purchased and used for all siRNA experiments. CHIR109.2.4 or Eg5-1:
GGGAGCAAAUGAACCUGAUTT SEQ ID N0:28 was used as a positive control with matched chemistry of Ambion in all functional studies. CHIR109.1.2 or Eg5-2:
UCCAAACUGGAUCGUAAGAAGGCAG- SEQ ID N0:29 was used as a positive control with matched chemistry of Invitrogen Stealth RNAi in all functional studies.

[000346] mRNA expression was measured 24 hours post siRNAs transfection by Reverse-transcription-coupled polymerase chain reaction (RT-PCR). mRNA of each of the siRNA
transfected sample was extracted and quantitate by methods described in Example 2. All samples were compared by semi-quantitative RT-PCR (GeneAmp , Applied Biosystems, Foster City, CA). Two primer sets were tested with similar results (data from primer set named "ABTP 500/501" is shown in Fig ure 5). FZD10-1 siRNA knockdown 66% of mRNA compared to Negative Control-I and FZD10-2 siRNA knockdown 77% of FZD10 mRNA compared to Negative Control-2. Relative expression was normalized by HPRT gene expression of all samples. The result demonstrated both siRNAs knockdown FZD10 mRNA
level in H460 at least 60% at the 24 hour timepoint.

[000347] Surface expression of FZD10 post-siRNA transfection on NCI-H460 cells was measured as described in Example 3 above. Figure 5 is a graphical representation of the percentage of surface stained cells relative to the prelmmune rabbit IgG
antibody negative control. Transfected cells were collected at three different timepoints (24, 48 and 72 hrs post-transfection) to monitor kinetics of siRNA mediated knockdown of the surface expression of FZD10 on NCI-H460. The percent of FZD10 surface expression of NC-1 siRNA
transfected samples was arbituary set to 100% for comparing the % of reduction of surface expression by FZD10-1 siRNA transfected sample. Likewise, The percent of FZD10 surface expression of NC-2 siRNA transfected samples was arbituary set to 100% for comparing the %
of reduction of surface expression by FZD10-2 siRNA transfected sample. Mock untransfected sample was relative to NC-1. The results showed that FZD10-1 siRNA reduced FZD10 surface expression more pronounce at 48 and 72 hour timepoints and FZD10-2 siRNA also showed the strongest knockdown of FZD10 surface expression at 48 and 72 hour timepoints, but not as potent of knockdown when compare FZD10-1 siRNA, since there are remaining at least 20% of surface expression of FZD10 on FZD10-2 siRNA transfected sample. Figure 5 also shows the histogram data for each sample at 72 hours timepoint to demonstrate the amount of signals detected in each transfected sample. Overall, both RT-PCR and GUAVA
Express analysis demonstrated reduction of FZD10 surface expression by both siRNAs and is more effective than FZD10-2.

[000348] Cell titer glow (ATP measurement; Promega) assays were used to measure anchorage-dependent cell growth. At 24 hours post siRNAs transfection, cells were plated on 96-well plates @ 1000-5000 cells/well depending on the cell type, for NCI-H460, 1600 cells/well were seeded. At various time-points, (24 hours, 48 hours, 72 hours, 96 hours) post-transfection; cells were lysed and assayed using cell titer glow, according to manufacturer's instructions. The output of the cell titer glow assay provides fluorescence that is proportional to relative cell number. As shown in Figure 6 and Figure 7, a decrease in anchorage-dependent cell growth was observed in NCI-H460 lung cancer cells and EKVX lung cancer cells, by both the FZD10-1 and FZD10-2 siRNAs, as comparable to control anti-proliferative siRNAs against Eg5 (Eg5-1 and Eg5-2). Proliferation was not inhibited by the two negative controls (NC-1 & NC-2) of two different siRNAs synthesis chemistries. These results demonstrated knockdown of FZD10 in NCI-H460 and EKVX lung cancer cell line inhibit proliferation.

[000349] Caspase 3/7 Glow (Promega # G8090) assays were used to measure apoptosis induced by siRNA transfection in cancer cell lines. At 24 hours post siRNA
transfection, cells were plated on 96-well plates @ 1000-5000 cells/well depending on the cell type, NCI-H460, 1600 cells/well were seeded. At various time-points, (48 hours & 72 hours) post-transfection, cells were lysed and assayed using Cell titre blue viability assay (Promega #G8080), according to manufacturer's instructions. The output of the cell titer blue assay provides fluorescence that is proportional to relative cell number. Upon recording the fluorescence signals by a plate reader, the same 96-well plate was used to measure caspase 3/7 activity by incubating with equal volume of Caspase 3/7 Glow reagents for 1 hour in the dark.
Luminescence signals were measured by plate reader according to manufacturer's instructions and data were analyzed by normalizing with viability signals recorded earlier using Cell titre Blue assay. The higher the normalized signal indicated increase of caspase 3/7 activities and is indicative of apoptosis. As shown in Figure 6 and Figure 7, an increase of caspase 3/7 activities was observed in NCI-H460 lung cancer cells and EKVX lung cancer cells, by both the FZD10-1 and FZD10-2 siRNAs, as comparable to positive control siRNAs against Eg5 (Eg5-1 and Eg5-2). Caspase 3/7 activity was not induced by the two negative controls (NC-1 & NC-2) of two different siRNAs synthesis chemistries. These results demonstrated knockdown of FZD10 in NCI-H460 and EKVX lung cancer cell line induced apoptosis via mechanism of inducing caspase 3/7 activities.

[000350] Soft-agar assays were used to measured anchorage-independent cell growth. Soft agar assays were performed by first coating a non-tissue culture treated plate with Poly-HEMA to prevent cells from attaching to the plate. Non-transfected cells were harvested using trypsin and washing twice in media. The cells were counted using a hemacytometer and resuspended to 104 cells per ml in media. Fifty l aliquots were placed in po1yHEMA coated 96-well plates and transfected.

[000351] The day after transfection, cells were trypsinized, resuspended and counted. Cells were diluted to about 1000 cells/ 100 1/well and transferred to a deep well block (max volume = lmUwell, in triplicate, following standard placement). Cells were plated in two plates:
Corning #7007 Ultra Low Adherent U-plate for the assay, and Corning # 3799 for the plating efficency check. Seaplaque GTG Agarose 3% was melted in a microwave oven by heating for about 1 minute. When fully melted, about 10 ml was poured into pre-warmed 50 ml polypropylene tubes (Falcon # 35-2070) and incubated in a 60 C heatblock for at least 10 minutes. About 18.6m1 complete media was added to a 50m1 polypropylene tube and incubated at about 37 C in a water bath. A MultimekTM pipettor was used to dispense agarose to cells in 96 well plates. About 18.6 ml warm media was poured into the IOml of agarose and mixed well by gentle inversion. Plates were incubated at about 4 C
for 20-30 min to let the agarose solidify quickly. After the agarose was solidified, 100 1 complete media was added over the cells. To measure the Day 0 plating efficiency, about 25 l/well Alamar Blue was added and incubated overnight at 37 C. Plates were read after 18-24 hours @
530ex/ 590em on a TECAN plate reader. Assay plates were incubated at 37 C for 7 days before developing with Alamar Blue (25 Uwell).

[000352] Results of representative proliferation, apoptosis and soft agar assays for NCI-H460 cells are displayed in Figure 6, and show that both FZD10-1 and FZD10-2 siRNAs were able to inhibit proliferation (both anchorage-dependent and independent) and induce apoptosis of this cell line.

[000353] The functional consequence of FZD10 knockdown was also tested by examining the effect of siRNAs on PARP cleavage in the lung cancer cell line, NCI-H460.
PARP
cleavage is indicative of caspase activation, which is a mediator of apoptosis. The cells transfected with the siRNAs with appropriate positive and negative controls were harvested and lyse in RIPA buffer at 48 hrs and 72 hrs. Then cell lysates were analyzed by Western blot for expression and processing of PARP and (3-tubulin as loading control. PARP
cleavage was increased in the positive controls (Eg5-1 and Eg5-2) with stronger signal at 72 hours timepoint. Transfection of siRNAs against FZD10 (FZD10-1 & FZD10-2) also resulted appearance of weak PARP cleavage products but not in negative control transfected (NC-1 &
NC-2) samples. As shown in Figure 6, FZD10-1 siRNA, which is more effective of inhibiting proliferation gave detectable PARP cleavage at both the 48hrs and 72 hours timepoint, whereas FZD10-2 siRNA, which is less effective of inhibiting proliferation gave detectable PARP cleavage only at the later 72 hours timepoint. Expression of (3-tubulin is uniform across all samples indicated of equal amount of input cell lysates. These data suggested growth inhibition induced by knockdown of FZD 10 is due in part by apoptosis mechanism.

[000354] Not all the cell lines that express FZD10 can be inhibited by FZD10 siRNAs. Cell lines that do not express FZD10, such as MCF7 and A549 were tested by transfection of the same siRNA and no functional effects (proliferation, soft agar growth and apoptosis) were observed. Cell lines such as SW620 (Figure 8) and Co1o320DM (Figure 9)which express FZD10 on the cell surface, and whose expression can be blocked by siRNAs did not have any functional effects post-siRNA transfection, despite good effects demonstrated by the Eg5 positive controls (Eg5-1 & Eg5-2). These colon can cell lines harbour APC
mutations which may bypass the extracellular requirement of FZD10 to mediate WNT signaling, thus not responded to siRNAs treatment. In summary, these siRNAs data suggested the requirement of FZD10 for cell growth and survival is context dependent. Those cell lines that harbour (3-catenin activating mutation or inactivating mutation on APC may be insensitive to treatment with FZD10 antagonists.

Effect seen on:

LKB1 R-catenin Proliferation A o tosis Soft A ar Cell lines Levels status status si-1 si-2 si-1 si-2 si-1 si-2 ; .:.:.: :::::::: . . . :::::: ;.;.;.; .: . ..:>:>:>:: :>:>:>:... :.: :::::: :
H460 Med truncation +/+ : ::>::: :~ >:>:: : ': :::>::::>:
EKVX Med unknown +/+
:::::::::::~I~:::;:::i:::::::i::::::::~`~::::<::::::::::::<:i::::~~:i::<:::::i:
::::::i<:::::~~ ::;:i:::::::i::::;:::i::~~ :i< :::::::
: ::~~:::
:..................... .... .... . . .. .... . ... ... ... .... .... .... ....
. . . .... .... .... ....
..................... ..................... .....................
H2126 Hi h loss unknown :::::::::::'#:~1 ::::::::::::::
:::::::::::::f::l'I::::::::::::::::::::1::::::::::::
................. .....................
H23 low truncation +/+ ND ND ND ND ND ND
MCF7 no unknown +/+
A549 no truncation +/+ ND ND
MRC9 no +/+ +/+ ND ND ND ND ND ND
DOV13 Med unknown unknown ND ND ND ND ND ND
SW620 High unknown APC mut CoIo320DM High unknown APC mut [000355] Example 5: Anti-FZD10 ECD antibody production [000356] FZD10 Antigen used to immunize the rabbits was prepared and purified as follows.

[000357] The growth media of baculovirus infected TN5 cells (10 L concentrated down to 1.6 L) which contained secreted FZD10-ECD protein was used as starting material for protein purification. Imidazole was added to the supernatant to 5mM final concentration. Working with endotoxin free buffers and equipment the supernatant was loaded onto a 25 ml Ni HP
(G.E.) column ( 6cm height x 2.6 cm length) after equilibration with Ni Buffer A (PBS pH
7.4/0.35M NaC1/5mM Imidazole). The column was washed to baseline with Buffer A
and the protein was eluted with a 30CV gradient to 100% Ni Buffer B (PBS pH 7.4/0.35M
NaC1/500mM Imidazole). Arginine was added to the fractions to 100mM final concentration.
Fractions containing FZD10 were analyzed using SDS-PAGE reducing gel and pooled. The pool was dialyzed vs. SP Buffer A (25mM MES pH 5.7/0.1M Arginine) to a final conductivity of 8 mS/cm. The dialyzed pool was loaded onto a 5m1 SP Fast Flow Hi Trap column (G.E.) equilibrated with SP Buffer A. The column was washed to baseline with SP
buffer A and eluted with 30CV gradient to 100% SP Buffer B (25mM MES pH
5.7/0.1M

Arginine/500mM NaC1). Fractions containing FZD10 were analyzed using SDS-PAGE
reducing gel and pooled. The SP pool was loaded onto a 5m1 Ni HP Hi Trap (G.E.) column equilibrated with Ni2 Buffer A ( PBS/0.35M NaC1/5mM Imidazole/100 mM
Arginine). The column was washed to baseline with Ni2 Buffer A and eluted with 20 CV gradient to 100%
Ni2 Buffer B (PBS/0.35M NaC1/500mM Imidazole/100 mM Arginine). Fractions containing FZD10 were analyzed using SDS-PAGE reducing gel and pooled. The second Ni pool was concentrated using Ultrafree 15m1 concentrators (Millipore) to a final volume of 10m1. The concentrate was loaded onto a Superdex 75 26/60 Gel Filtration column (G.E.) equilibrated with GFC Buffer (PBS pH 7.4/100mM Arginine). Fractions containing FZD10 were analyzed using SDS-PAGE reducing gel and pooled. Monomer and dimer were pooled separately and monomer material was used to generate antibodies. Monomer material was 95%
pure by Coomassie SDS-PAGE and endotoxin levels were less than 0.3 EU/mg protein.
Final concentration was 1.1 mg/ml stored -80 C.

[000358] FZD10 protein was conjugated to rabbit serum albumin (RSA) with m-maleimido benzoic acid N-hydroxysuccinimide ester (MBS) (Pierce, Rockford, Ill.).
Immunization protocols with the conjugated protein was performed according to standard methods. Initially, a pre-bleed of the rabbits was performed prior to immunization. The first immunization included Freund's complete adjuvant and 500 g conjugated protein. All subsequent immunizations, performed four weeks after the previous injection, included Freund's incomplete adjuvant with the same amount of protein. Bleeds were conducted seven to ten days after the immunizations. Day 77 post-immunization, bleeds were collected for analysis and purification.

[000359] Rabbit anti-FZD 10- specific antibody was prepared and purified as follows.
FZD10 antigen was coupled to HP resin using NHS activated HP (G.E. catalog #
10-0716-01) following manufacturers protocols. Day 77 Rabbit serum was loaded onto the FZD10 affinity column equilibrated with Buffer A (75mM Tris-HC1 pH 8.0). The column was washed to baseline with Buffer A. The antibody was step eluted with Buffer B(100mM
Glycine pH 2.7) into fractions containing Tris pH 8.0 for a final concentration of 100mM Tris pH 8Ø
Fractions were analyzed by SDS-PAGE and antibody containing fractions were pooled.
Western blot analysis showed that the antibody did not bind to a control antigen, EPHB3 or to FZD9.

[000360] Protein lysates were made from cell pellets of different cell lines with RIPA buffer (Teknova), and the lysates were separated by acrylamide gel electrophoresis and then subjected to Western blot analysis using an in-house purified anti-FZD10 antibodies (Rabbit A and Rabbit B representing two separate immunized animals). Cell lines included 293T
human embryonic kidney fibroblast which do not express FZD10 (Negative control); 293T
cells that was transiently transfected with FZD10 mammalian expression plasmid as positive control; a series human lung cancer cell lines that do not express FZD10 based on quantitative RT-PCR, NCI-H661, NCI-H1915, NCI-H446; a series of human lung cancer cell lines that express FZD10 based on quantitative RT-PCR, NCI-H2126, NCI-H23, EKVX and NCI-H460; an human ovarian cancer cell line PAI (Figure 10).

[000361] Three bands (-60 kD, -120 kD &-180kD) were observed in positive control (FZD10 transfected 293T) cell lysates. The faint band (180kDa) higher molecular weight product could be the trimeric form of the protein that cannot be completely denatured by SDS-PAGE, where as the 120kDa band could be the dimer form of FZD10. Rabbit A anti-antibody appeared to have a stronger preference towards the monomeric form of FZD10, but also picked up at least two background bands across all the human cancer lines with various degree of intensity. Rabbit A anti-FZD10 antibody also cross reacted with FZD9, which suggested these other bands may represent FZD9 or other potential FZD family members.
Rabbit B anti-FZD10 antibody appeared to have a equal preference towards the monomeric and dimeric form of FZD10, but also picked up two background bands at 50 and 36 kDa.
Since rabbit B anti-FZD10 antibody did not cross react with FZD9 (closest homologue), suggesting these other bands are likely non-specific proteins recognized by this antibody. In general, FZD10 protein expression is in agreement with quantitative RT-PCR Ct level, which were determined using methods described in Example 2 (Table 2). The Ct value indicates the threshold of detection following a certain number of cycles of PCR. A higher Ct value indicates that a higher number of cycles is required to detect the cDNA, and is therefore indicative of lower mRNA level. Table 2 indicates that FZD10 is expressed at significant levels in multiple human lung cancer cell lines.
Table 2 ATCC Cell Line ung cancer ZD10 qPCR Description Derived from CRL-5803 CI-H1299 SCLC Ct=35 Carcinoma -Lymph node mets CRL-5911 CI-H2009 SCLC Ct=37 Adenocarcinoma ymph node mets CRL-5930 CI-H2172 SCLC Ct=34 /A /A

CRL-5935 CI-H2228 SCLC Ct=39 Adenocarcinoma /A

HTB-180 CI-H345 SCLC Ct=33 Carcinoma Bone marrow mets Papillary HTB-174 CI-H441 SCLC Ct=39 adenocarcinoma Pericardial fluid Adenosquamous HTB-178 CI-H596 SCLC Ct=39 carcinoma Tumor mass HTB-119 CI-H69 SCLC Ct=28 Carcinoma /A

Bronchoalveolar CRL-5894 CI-H1781 SCLC Ct=35 carcinoma Pleural effusion CRL-5844 CI-H838 SCLC Ct=32 Adenocarcinoma ymph node mets HTQ-183 \CI-H661 \SCLC' Ct=37 C'm-cinomn L~m h oode mets Poor1Ndifferentiated 'RL-5904 \CI-H 191 5 NSCLC Ct=38 carcinoma [3rain mets HT[3-17t \CI-H446 SCLC Ct=39 C'm-cinoma Pleural effusio^
'CL-256 \CI-H? 1?6 \SCLC' Ct=?3 Adenocarcinoma Pleural effusio^
RL-5800 NCI-H23 \SCLC Ct=31 Adenocarcinoma Tunior mass HTB-177 \CI-H460 \SCLC' Ct=?7 C'm-cinoinn Pleural effusio^
CCL-185 A549 SCLC Ct=39 Carcinoma Tumor mass NC160 EKVX \SCLC' 0=27 Adeuocarcinouia \/~ N, CRL-2062 DMS 53 SCLC Ct=29 Carcinoma ediastinal CRL-2066 DMS 114 SCLC Ct=31 Carcinoma ediastinal [000362] Example 6: Immunohistochemistry analyses of FZD10 on lung tumor microarray (TMA) and commercial multi-tumor TMA

[000363] Tissue sections were deparaffinized and antigen retrieval was performed on a Ventana Discovery instrument (Ventana Medical Systems, Inc., Tucson, AZ).
Standard cell conditioning was performed, and then cells were incubated for 60 minutes with primary antibodies. A rabbit anti-human FZD10 RbBd77 antibody (Chiron, Emeryville, CA) and rabbit IgG Prebleed control (Chiron, Emeryville, CA) were used at 10 g/ml.
Ventana Universal Secondary Reagent (Ventana Medical Systems, Inc.) followed by Ventana DAB
Map Kit (Ventana Medical Systems, Inc.) was used for detection. Ventana Hematoxylin and Bluing Reagents (Ventana Medical Systems, Inc.) were used for counterstain, and sections were dehydrated in graded alcohols, cleared in xylene and coverslipped using a synthetic mounting media.
FZD10 expression was observed as a nuclear punctate, BL-2+ cytoplasmic expression in the vasculature and tumor epithelium. The overall FZD10 expression was weak and observed in the tumor epithelium. Preimmune RbBdO showed some cytoplasmic diffuse blush in several of the tumor elements, as well as reactivity on inflammatory cells. Expression of FZD10 was observed in most of lung cancer elements, some colon cancer and ovarian cancer elements.
Bladder, esophagus, cervix, breast, thyroid, stomach, pancreas, skin, prostate and rectum derived cancers were also assessed for FZD10 expression as set forth below in Tables 3 and 4.
Table 3: IHC of Cybridi Multi-Tumor CA TMA

CC00-01-006 C brdi Multi-Tumor TMA
Preimmune No Organ Pathology Diagnosis Grade RbBdO FZD10 RbBd77 1 Brain Astrocytoma II 0 vasculature 0-1 2 Esophagus Squamous cell carcinoma III few reactive cells stromal 0-1 overall cytoplasmic 3 Stomach Adenocarcinoma I blush 0 overall cytoplasmic 4 Liver Hepatocellular carcinoma III blush few + cells Colon Adenocarcinoma III 0 few + cells cytoplasmic blush in overall cytoplasmic epithelium vasculature 6 Rectum Adenocarcinoma III blush 1-1+

7 Lung Squamous cell carcinoma III few reactive cells vasculature 1-1+
stromal 1-1+
8 Bladder Transitional cell carcinoma II 0 vasculature 1 -1 +
overall cytoplasmic overall cytoplasmic 9 Heart Myxoma - blush blush 0-1 vasculature Brain Astrocytoma II 0 0-1 vasculature 11 Eso ha us Squamous cell carcinoma III 0 0-1 vasculature overall cytoplasmic overall cytoplasmic 12 Stomach Adenocarcinoma III blush blush 1-i+vasculature overall cytoplasmic 13 Liver Hepatocellular carcinoma II blush rare 0-1 vasculature overall cytoplasmic overall cytoplasmic 14 Colon Adenocarcinoma II blush blush 1-1+stromal overall cytoplasmic 15 Rectum Adenocarcinoma III blush BI-1 c to lasmic tumor overall cytoplasmic 16 Lung Squamous cell carcinoma III 0 blush overall cytoplasmic BI-1 + tumor epithelium 17 Bladder Transitional cell carcinoma I blush 10% 1-1+ vasculature 18 Heart Myxoma - 0 1-2vasculature 19 Brain Astrocytoma 1 0 few + cells BL-1 + tumor epithelium 20 Esophagus Squamous cell carcinoma II 0 [10%]
overall cytoplasmic BI-1 tumor epithelium 21 Stomach Adenocarcinoma II blush 1%

22 Liver Cholangiocellular carcinoma II 0 few + cells overall cytoplasmic BL-1 + tumor epithelium 23 Colon Adenocarcinoma II blush [90%]
overall cytoplasmic BL-1 + tumor epithelium 24 Rectum Adenocarcinoma II blush [70%]

25 Lung A little squamous cell carcinoma III Element Missing Element Missing overall cytoplasmic over cytoplasmic blush 26 Bladder Transitional cell carcinoma II blush 0-1 + vasculature overall cytoplasmic 27 Heart Myxoma - blush few + cells 28 Kidney Clear cell carcinoma - 0 few + cells few + cells overall overall cytoplasmic cytoplasmic blush in 29 Thyroid Papillary carcinoma - blush tumor epithelium overall cytoplasmic BI-1 tumor epithelium 30 Pancreas Adenocarcinoma III blush [40%]
overall cytoplasmic blush in tumor, few 31 cervix Squamous cell carcinoma III few reactive cells reactive cells 32 Skin Squamous cell carcinoma III Element Missing Element Missing Nonspecific infiltrating duct 33 Breast carcinoma III few reactive cells few + cells overall cytoplasmic BI-1 tumor epithelium;
34 Ovary Clear cell carcinoma - blush 1-2 vasculature 35 Prostate Adenocarcinoma I 0 1-1+vasculature 36 Testis B cell l m homa - few reactive cells few reactive cells 37 Kidney Clear cell carcinoma pigment pigment overall cytoplasmic overall cytoplasmic 38 Thyroid Pa illar carcinoma - blush blush overall cytoplasmic overall cytoplasmic 39 Pancreas Adenocarcinoma III blush blush 1-1+ vasculature; few 40 cervix Squamous cell carcinoma III Ffew reactive cells reactive cells overall cytoplasmic 41 Skin Steatadenoma - blush BL-1 tumor epithelium Nonspecific infiltrating duct 42 Breast carcinoma II 0 1 -1 +vasculature overall cytoplasmic BL-1 tumor epithelium;
43 Ovary Endometrioid carcinoma - blush 0-1 vasculature overall cytoplasmic 44 Prostate Adenocarcinoma II blush 0 45 Testis Seminoma - 0 few + cells 46 Kidney Clear cell carcinoma - few reactive cells few + cells 47 Thyroid Thyroid gland tissue - few reactive cells 1-1+ vasculature overall cytoplasmic overall cytoplasmic 48 Pancreas Adenocarcinoma III blush blush 49 cervix Squamous cell carcinoma III few reactive cells BI-1 vasculature 50 Skin Squamous cell carcinoma I 0 reactive stromal cells Nonspecific infiltrating duct overall cytoplasmic 51 Breast carcinoma II blush few + cells overall cytoplasmic 52 Ovary Mucous cystocarcinoma III blush BI-1 tumor epithelium blush in tumor 53 Prostate Transitional cell carcinoma III 0 epithelium 54 Testis Seminoma - 0 1-1+ rare vasculature Table 4. Expresion of FZD10 in different tumor tissues - IHC

FZD10-RbBd77 (tumor epithelium score) Tissue Cancer Total Blush Blush-1 Blush-3 Novartis contructed TMAs Colon Adenocarcinoma 32 0 0 0 Lung Squamous cell carcinoma 31 6 15 6 Small cell carcinoma 7 7 0 0 Small cell carcinoma-metastatic 2 0 1 0 Pa illar adenocarcinoma 2 2 0 0 Solid mucinous adenocarcinoma 1 0 1 0 bronchioloalveolar adenocarinoma 2 2 0 0 Adenocarcinoma 19 16 2 0 Ovary Serous papillary cystadenocarcinoma 72 4 5 0 Commercial TMA
Bladder Transitional cell carcinoma 3 1 1 0 Esophagus Squamous cell carcinoma 3 0 1 0 Colon Adenocarcinoma 3 1 1 0 Stomach Adenocarcinoma 3 1 1 0 Rectum Adenocarcinoma 3 1 2 0 Pancreas Adenocarcinoma 3 0 1 0 Ovary Clear cell carcinoma 1 0 1 0 Mucous cystocarcinoma 1 0 1 0 Endometrioid carcinoma 1 0 1 0 Skin Squamous cell carcinoma 3 0 1 0 Prostate Transitional cell carcinoma 3 1 0 0 Number of Tumor Samples with >1% of Cells Positive for WNT1 [000364] Example 7:Overexpression of FZD10 induced phosphorylation of Dishievelled family members.

[000365] 293T cells were transfected with mammalian expression vector expressing either FZD10, FZD9, LRP5, WNT7a and WNT7b. Cells were harvested 24 hours post-transfection and total cell lysates were generated by lysing the cells in RIPA buffer (Teknova). Western blot analysis was performed to detect expression of the various transgenes.
FZD10 expression was detected by anti-FZD10 RbBD77 rabbit polyclonal antibody. FZD9 expression was detected by anti-FZD9 goat polyclonal antibody from R&D systems. LRP5 was detected by anti-LRP5 antibody from Santa Cruz. Wnt7a-HA and Wnt7b-HA expression were detected by anti-HA tag monoclonal antibody from Covance. The phosphorylation state of Dv12 and Dv13 in these transfected 293T cell lysates were determined by mobility shifts of Dv12 and Dv13 by rabbit polyclonal antibodies from Cell Signaling, which recognized all post-translational modified and unmodified Dv12 and Dv13 proteins. Figure 11 demonstrated over expression of FZD9 or FZD10 resulted in hyper-phosphorylation of both Dv12 and Dv13. This observation is consistent with reported effect of Frizzled overexpression in cell lines suggesting Dv12 and Dv13 phosphorylation state can be used as a direct readout of FZD10 overexpression in cancer cell lines.

[000366] Example 8: siRNA knock down of FZD10 reduced phosphorylation of Dishievelled family members in human lung cancer cell line NCI-H460.

[000367] The effect of FZD10 knockdown by FZD10-1 and FZD10-2 siRNAs on phosphorylation state of Dv12 and Dv13 were tested in NCI-H4601ung cancer cell line (Figure 11). The cells were incubated with the siRNAs and corresponding negative controls for 24 hrs, and then cell lysates were analyzed by Western blot analysis. The anti-FZD10 antibody RbBd77 (Chiron, Emeryville, CA) was used to detect reduction of FZD10 protein.
Phosphorylation states of Dv12 and Dv13 were detected by anti-Dv12 and anti-Dv13 rabbit polyclonal antibodies (Cell Signaling) which recognized all post-translational modified and unmodified Dv12 and Dv13 proteins. The slower mobility species of Dv12 and Dv13 were reduced only by FZD10-1 siRNA at 24 hours, but not any of the negative controls. The less of an effect by FDZ10-2 siRNA may be attributed to the slow knockdown kinetics of this siRNA compare to FZD10-1 siRNA (Example 4). This data suggested phosphorylation state of Dv12 and Dv13 in cancer cell lines that overexpress FZD10 such as NCI-H460 can be used as target modulation biomarker for functional knockout of FZD 10 in cancer cell lines.

[000368] Example 9: Sequences SEQ ID NO:1 FZD10 nt sequence atgcagcgcccgggcccccgcctgtggctggtcctgcaggtgatgggctcgtgcgccgcc atcagctccatggacatggagcgcccgggcgacggcaaatgccagcccatcgagatcccg atgtgcaaggacatcggctacaacatgactcgtatgcccaacctgatgggccacgagaac cagcgcgaggcagccatccagttgcacgagttcgcgccgctggtggagtacggctgccac ggccacctccgcttcttcctgtgctcgctgtacgcgccgatgtgcaccgagcaggtctct acccccatccccgcctgccgggtcatgtgcgagcaggcccggctcaagtgctccccgatt atggagcagttcaacttcaagtggcccgactccctggactgccggaaactccccaacaag aacgaccccaactacctgtgcatggaggcgcccaacaacggctcggacgagcccacccgg ggctcgggcctgttcccgccgctgttccggccgcagcggccccacagcgcgcaggagcac ccgctgaaggacgggggccccgggcgcggcggctgcgacaacccgggcaagttccaccac gtggagaagagcgcgtcgtgcgcgccgctctgcacgcccggcgtggacgtgtactggagc cgcgaggacaagcgcttcgcagtggtctggctggccatctgggcggtgctgtgcttcttc tccagcgccttcaccgtgctcaccttcctcatcgacccggcccgcttccgctaccccgag cgccccatcatcttcctctccatgtgctactgcgtctactccgtgggctacctcatccgc ctcttcgccggcgccgagagcatcgcctgcgaccgggacagcggccagctctatgtcatc caggagggactggagagcaccggctgcacgctggtcttcctggtcctctactacttcggc atggccagctcgctgtggtgggtggtcctcacgctcacctggttcctggccgccggcaag aagtggggccacgaggccatcgaagccaacagcagctacttccacctggcagcctgggcc atcccggcggtgaagaccatcctgatcctggtcatgcgcagggtggcgggggacgagctc accggggtctgctacgtgggcagcatggacgtcaacgcgctcaccggcttcgtgctcatt cccctggcctgctacctggtcatcggcacgtccttcatcctctcgggcttcgtggccctg ttccacatccggagggtgatgaagacgggcggcgagaacacggacaagctggagaagctc atggtgcgtatcgggctcttctctgtgctgtacaccgtgccggccacctgtgtgatcgcc tgctacttttacgaacgcctcaacatggattactggaagatcctggcggcgcagcacaag tgcaaaatgaacaaccagactaaaacgctggactgcctgatggccgcctccatccccgcc gtggagatcttcatggtgaagatctttatgctgctggtggtggggatcaccagcgggatg tggatttggacctccaagactctgcagtcctggcagcaggtgtgcagccgtaggttaaag aagaagagccggagaaaaccggccagcgtgatcaccagcggtgggatttacaaaaaagcc cagcatccccagaaaactcaccacgggaaatatgagatccctgcccagtcgcccacctgc gtgtga SEQ ID NO:2 FZD10 aa sequence mqrpgprlwlvlqvmgscaaissmdmerpgdgkcqpieipmckdigynmtrmpnlmghen qreaaiqlhefaplveygchghlrfflcslyapmcteqvstpipacrvmceqarlkcspi meqfnfkwpdsldcrklpnkndpnylcmeapnngsdeptrgsglfpplfrpqrphsaqeh plkdggpgrggcdnpgkfhhveksascaplctpgvdvywsredkrfavvwlaiwavlcff ssaftvltflidparfryperpiiflsmcycvysvgylirlfagaesiacdrdsgqlyvi qeglestgctlvflvlyyfgmasslwwvvltltwflaagkkwgheaieanssyfhlaawa ipavktililvmrrvagdeltgvcyvgsmdvnaltgfvliplacylvigtsfilsgf fhirrvmktggentdkleklmvriglfsvlytvpatcviacyfyerlnmdywkilaaqhk ckmnnqtktldclmaasipaveifmvkifmllvvgitsgmwiwtsktlqswqqvcsrrlk kksrrkpasvitsggiykkaqhpqkthhgkyeipaqsptcv [000369] While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the present invention.

Claims (60)

1. A composition comprising a FZD10 modulator and one or more pharmaceutically acceptable carriers, wherein said FZD10 modulator has one or more of the activities selected from the group consisting of Dishevelled phosphorylation, c-Jun phosphorylation, and .beta.-catenin phosphorylation.

2. The composition of claim 1 wherein the composition is a sterile injectable.

3. The composition of claim 1 wherein the FZD10 modulator is an inhibitor that decreases phosphorylation of Dishevelled, decreases phosphorylation of c-Jun, and increases phosphorylation of .beta.-catenin.

4. The composition of claim 1 wherein said FZD10 modulator decreases phosphorylation of Dishevelled.

5. The composition of claim 1 wherein said FZD10 modulator inhibits FZD10 binding to Dishevelled.

6. The composition of claim 1 wherein the FZD10 modulator is an oligonucleotide, a small molecule, a mimetic, a soluble receptor, a decoy receptor, or an antibody.

7. The composition of claim 1 wherein the FZD10 modulator is a monoclonal antibody which binds to FZD10 with an affinity of at least 1x10 8Ka.

8. The composition of claim 1 wherein the FZD10 modulator is a monoclonal antibody which selectively binds FZD10 and modulates one or more FZD10-related biological activities.

9. The composition of claim 7 wherein the monoclonal antibody is a human antibody, a humanized antibody or chimeric antibody.

10. The composition of claim 7 wherein the monoclonal antibody binds to an epitope of FZD10, said epitope selected from the group consisting of the first extracellular domain (ECD), the second ECD, the third ECD, the fourth ECD and the ligand-binding domain.

11. The composition of claim 7 wherein the monoclonal antibody binds to an epitope of the ECD of FZD10.

12. The composition of claim 11 wherein the monoclonal antibody binds to an epitope of the first ECD.

13. The composition of claim 7 wherein the monoclonal antibody does not bind to the first ECD.

14. The composition of claim 7 wherein the monoclonal antibody does not cross-react with FZD9.

15. The composition of claim 7 wherein the monoclonal antibody is conjugated to another diagnostic or therapeutic agent.

16. A method of treating cancer or a cancer symptom in a patient in need thereof comprising administering to the patient a therapeutically effective amount of the FZD10 modulator of claim 1.

17. The method of claim 16 wherein the FZD10 modulator decreases phosphorylation of Dishevelled.

18. The method of claim 17 wherein the FZD10 modulator inhibits FZD10 expression by at least 50% as compared to a control.

19. The method of claim 16 wherein the FZD10 modulator is an oligonucleotide, a small molecule, a mimetic, a soluble receptor, a decoy receptor, or an antibody.

20. The method of claim 16 wherein the FZD10 modulator is a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a human antibody, a humanized antibody, a single-chain antibody, or a Fab fragment.

21. The method of claim 20 wherein the antibody is labeled.

22. The method of claim 21 wherein the label is an enzyme, radioisotope, toxin or fluorophore.

23. The method of 20 wherein the antibody has a binding affinity less than about 1x10 5K a for a polypeptide other than FZD10.

24. The method of claim 16 wherein the FZD10 modulator is a monoclonal antibody.

25. The method of claim 24 wherein the monoclonal antibody binds to FZD10 with an affinity of at least 1x10 8Ka.

26. The method of claim 24 wherein the monoclonal antibody binds to an epitope of FZD10, said epitope selected from the group consisting of the first ECD, the second ECD, the third ECD, the fourth ECD and ligand-binding domain.

27. The method of claim 24 wherein the monoclonal antibody binds to an epitope of the first ECD of FZD10.

28. The method of claim 24 wherein the monoclonal antibody does not bind to the first ECD of FZD10.

29. The method of claim 24 wherein the monoclonal antibody does not bind to FZD9.

30. The method of claim 24 wherein the monoclonal antibody decreases phosphorylation of Dishevelled, decreases phosphorylation of c-Jun, and increases phosphorylation of .beta.-catenin.

31. The method of claim 16 wherein the cancer is ovarian, colon, lung, stomach or uterine cancer.

32. The method of claim 16 further comprising the administration of a traditional cancer therapeutic to the patient.

33. The method of claim 16 further comprising the treatment of the patient with one or more of chemotherapy, radiation therapy or surgery.

34. The method of claim 16 wherein the cancer symptom is selected from the group consisting of pain, death, weight loss, weakness, difficulty eating, blood in stool, nausea, vomiting, liver metastases, lung metastases, bone metastases, abdominal fullness, bloating, fluid in peritoneal cavity, vaginal bleeding, constipation, abdominal distension, perforation of colon, acute peritonitis (infection, fever, pain), vomiting blood, and difficulty swallowing.

35. A method of modulating a FZD10-related biological activity in a patient, the method comprising administering to the patient an amount of the FZD10 modulator of claim 1 effective to modulate the FZD10 biological activity.

36. The method of claim 35 wherein the FZD10 modulator is a monoclonal antibody which selectively binds FZD10.

37. The method of claim 35 wherein the patient has or is predisposed to one or more of ovarian, colon, lung, or uterine cancer.

38. The method of claim 16 wherein the FZD10 modulator is an antibody and is administered to the subject via in vivo therapeutic antibody gene transfer.

39. A method of identifying a patient susceptible to FZD10 therapy comprising:
(a) detecting the presence or absence of evidence of FZD10 expression in said sample, wherein the presence of evidence of FZD10 expression in said sample is indicative of a patient who is a candidate for FZD10 therapy and the absence of evidence of FZD10 expression in said sample is indicative of a patient who is not a candidate for FZD10 therapy;
(b) administering a therapeutically effective amount of the composition of claim 1 to the patient if the patient is a candidate for FZD10 therapy; and c) administering a traditional cancer therapeutic to the patient if the patient is not a candidate for FZD10 therapy.

40. The method of claim 39 wherein the expression of FZD10 is increased at least 30%
compared to a control.

41. The method of claim 39 wherein the expression of FZD10 is increased at least 50%
compared to a control.

41. The method of claim 39 wherein the expression of FZD10 is increased at least 100%
compared to a control.

42. The method of claim 39 wherein evidence of FZD10 expression is detected by measuring FZD10 RNA.

43. The method of claim 39 wherein evidence of FZD10 expression is detected by measuring FZD10 expression products.

44. The method of claim 39 wherein the patient has or is predisposed to one or more of ovarian, colon, lung, or uterine cancer.

45. The method of claim 39 further comprising the administration of a traditional cancer therapeutic to the patient.

46. A method of inhibiting cancer cell growth in a patient in need thereof comprising administering a therapeutically effective amount of the FZD10 modulator of claim 1 to the patient.

47. The method of claim 46 wherein the FZD10 modulator is a monoclonal antibody which specifically binds FZD10 and decreases phosphorylation of Dishevelled.

48. A method of inhibiting a cancer cell phenotype in a patient in need thereof, said method comprising administering to said patient a therapeutically effective amount of the FZD10 modulator of claim 1.

49. The method of claim 48 wherein the cancer cell phenotype is one or more of colony formation in soft agar and tubular network formation in a three dimensional basement membrane or extracellular membrane preparation.

50. The method of claim 48 further comprising the administration of a traditional cancer therapeutic to the patient.

51. The method of claim 48 further comprising the treatment of the patient with one or more of chemotherapy, radiation therapy or surgery.

52. The method of claim 48 wherein the cancer cells are selected from the group consisting of ovarian, colon, lung, or uterine cancer cells.

53. A method for detecting a tumor in a patient comprising administering to the patient a composition comprising the FZD10 modulator of claim 1 linked to an imaging agent and detecting the localization of the imaging agent in the patient.

54. The method of claim 53 wherein the FZD10 modulator is selected from the group consisting of a small molecule, an oligonucleotide, a mimetic, a soluble receptor, a decoy receptor, or an antibody.

55. The method of claim 53 wherein the composition comprises an anti-FZD10 antibody conjugated to an imaging agent.

56. The method of claim 55 wherein the imaging agent is 18F, 43K, 52Fe, 57Co, 67Cu, 67Ga, 77Br, 87MSr, 86Y, 90Y, 99MTc, 111In, 123I, 125I, 127Cs, 129Cs, 131I, 132I, 197Hg, 203Pb, or 206Bi.

57. The method of claim 53 wherein the FZD10 modulator is selected from the group consisting of a small molecule, an oligonucleotide, a mimetic, a soluble receptor, a decoy receptor, or an antibody.

58. The method of claim 53 wherein the FZD10 modulator is a monoclonal antibody which selectively binds FZD10 and decreases phosphorylation of Dishevelled.

59. A method of expressing an anti-FZD10 antibody in a CHO or myeloma cell wherein the anti-FZD10 antibody inhibits one or more FZD10-related biological activities, the method comprising expressing a nucleic acid encoding the anti-FZD10 antibody in said CHO or myeloma cell.

60. A method of identifying a cancer inhibitor, the cancer characterized by overexpression of FZD10 compared to a control, said method comprising contacting a cell expressing FZD10 with a candidate compound and determining whether an FZD10-related biological activity is modulated, wherein the induced FZD10-related biological activity is selected from the group consisting of phosphorylation of Dishevelled, c-Jun, and .beta.-catenin, wherein modulation of the FZD10-related biological activity is indicative of a cancer inhibitor.