HK1183435B - Frizzled-binding agents and uses thereof - Google Patents

Description

Frizzled protein binding agents and uses thereof

Technical Field

The field of the invention relates generally to antibodies and other agents that bind to human frizzled receptors, and to methods of using the antibodies or other agents for treating diseases such as cancer.

Background

Cancer is one of the leading causes of death in developed countries, with over one million people diagnosed with cancer and 500,000 deaths per year in the united states alone. It is estimated that in total more than 1 out of 3 people will develop some form of cancer in their lives. There are 200 or more different types of Cancer, 4 of which-breast, lung, colorectal and prostate-cancers-account for more than half of all new cases (Jemal et al, 2003, Cancer J. Clin.53: 5-26).

The Wnt signaling pathway has been identified as a potential target for cancer therapy. The Wnt signaling pathway is one of several key regulatory pathways for embryonic pattern formation, post-embryonic tissue maintenance, and stem cell biology. More specifically, Wnt signaling plays an important role in cell polarity generation and cell fate specification including self-renewal of stem cell populations. Unregulated activation of the Wnt pathway has been implicated in a variety of human cancers, where the activation can alter the developmental fate of tumor cells, thereby maintaining the tumor cells in an undifferentiated and proliferative state. Thus carcinogenesis can occur through the usurping of homeostatic mechanisms that control normal development and tissue repair by stem cells (reviewed in Reya and Clevers, 2005, Nature 434: 843; Beach et al, 2004, Nature 432: 324).

The Wnt signaling pathway was first elucidated in Drosophila developmental finless (wg) mutants, from the murine protooncogene int-1 (now known as Wnt1) (Nusse and Varmus, 1982, Cell 31: 99-109; Van Ooyen and Nusse, 1984, Cell 39: 233-40; Cabrera et al, 1987, Cell 50: 659-63; Rijsewijk et al, 1987, Cell 50: 649-57). The Wnt gene encodes a secreted lipid-modified glycoprotein, 19 of which have been identified in mammals. These secreted ligands activate a receptor complex consisting of a Frizzled (Fzd) receptor family member and Low Density Lipoprotein (LDL) receptor-associated protein 5 or 6(LPR 5/6). The Fzd receptor is a 7 transmembrane domain protein belonging to the G protein-coupled receptor (GPCR) superfamily, which contains a large extracellular N-terminal ligand binding domain with 10 conserved cysteines, namely a cysteine-rich domain (CRD) or Fri domain. There are 10 types of human FZD receptors: FZD 1-FZD 10. For particular Wnts, different Fzd CRDs have different affinities (Wu and Nusse, 2002, J.biol.chem.277: 41762-9), and the Fzd receptors have been classified as activating the classical β -catenin pathway described below and as activating non-classical pathways (Miller et al, 1999, Oncogene 18: 7860-72). To form receptor complexes that bind FZD ligands, FZD receptors interact with LRP5/6, LRP5/6 is a single transmembrane protein with four extracellular EGF-like domains separated by 6 YWTD amino acid repeats (Johnson et al, 2004, j. bone Mineral res.19: 1749).

The canonical Wnt signaling pathway is activated upon binding to the receptor and is mediated by a Dishevelled (Dsh), a cytoplasmic protein that interacts directly with the Fzd receptor, resulting in the stabilization and accumulation of β -catenin in the cytoplasm. In the absence of Wnt signaling, β -catenin localizes to the cytoplasmic degradation complex (destructionalomplex) which includes tumor suppressor proteins: adenomatous polyposis coli protein (APC) and Axin (Axin). These proteins perform the following functions: as a key backbone, Glycogen Synthase Kinase (GSK) -3 β is bound and phosphorylates β -catenin, labeling it for degradation via the ubiquitin/proteasome pathway. Activation of Dsh results in the dissociation of the phosphorylation and degradation complexes of GSK3 β. The accumulated cytoplasmic β -catenin is then transported to the nucleus where it interacts with the DNA binding proteins of the Tcf/Lef family, activating transcription.

In addition to the canonical signaling pathway, Wnt ligands activate the β -catenin-independent pathway (Veeman et al, 2003, Dev. cell 5: 367-77). Non-canonical Wnt signaling has been shown to be involved in a variety of processes, but the most compelling of these is the participation in gastrulation via a mechanism similar to the drosophila Planar Cell Polarity (PCP) pathway. Other potential mechanisms of non-canonical Wnt signaling include calcium flux, JNK, and small and heterotrimeric G proteins. Antagonism is often observed between the classical and non-classical pathways and some evidence suggests that non-classical signaling can inhibit cancer formation (Olson and Gibo, 1998, exp. cell Res.241: 134; Topol et al, 2003, J.CellBiol.162: 899-908). Thus, in certain contexts, Fzd receptors may act as negative regulators of the canonical Wnt signaling pathway. For example, FZD6 inhibits Wnt-3 a-induced canonical signaling through the TAK1-NLK pathway when co-expressed with FZD1 (Golan et al, 2004, JBC 279: 14879-88). Similarly, Fzd2 antagonizes canonical Wnt signaling through the TAK1-NLK MAPK cascade in the presence of Wnt-5a (Ishitani et al, 2003, mol.cell.biol.23: 131-9).

The canonical Wnt signaling pathway also plays a central role in the maintenance of small intestinal and colonic stem cell populations, and inappropriate activation of this pathway plays a dominant role in colorectal cancer (Reya and Clevers, 2005, Nature 434: 843). The intestinal absorptive epithelium is constructed of villi and crypts. Stem cells reside in the crypts and divide slowly to produce rapidly proliferating cells that produce all the differentiated cell populations that leave the crypts up and occupy the intestinal villi. The Wnt signaling cascade plays a dominant role in controlling cell fate along the crypt-villus axis and is essential for maintaining a stem cell population. Overexpression of either Tcf7/2 genetic loss (Koriek et al, 1998, nat. Genet.19: 379) or the strong secretory Wnt antagonist Dickkopf-1(Dkkl) (Pinto et al, 2003, Genes Dev.17: 1709-13; Kuhnert et al, 2004, Proc. nat' l.Acad. Sci.101: 266-71) disrupts Wnt signaling, leading to exhaustion of the intestinal stem cell population.

Colorectal cancer is most commonly caused by activating mutations in the Wnt signaling cascade. Approximately 5% to 10% of all colorectal cancers are inherited, with one of the major forms being Familial Adenomatous Polyposis (FAP), a disease that is autosomal dominant, with about 80% of affected individuals having germline mutations in the Adenomatous Polyposis Coli (APC) gene. Mutations have also been identified in other components of the Wnt pathway including Axin and β -catenin. Individual adenomas are clonal outgrowths of epithelial cells containing a second inactivated allele, while a large number of FAP adenomas inevitably lead to the development of adenocarcinomas by increasing mutations in oncogenes and/or tumor suppressor genes. In addition, activation of the Wnt signaling pathway, including gain-of-function mutations in APC and β -catenin, can trigger hyperplastic development and tumor growth in a mouse model (Oshima et al, 1997, Cancer Res.57: 1644-9; Harada et al, 1999, EMBO J.18: 5931-42).

With the identification of Wnt1 (protointl) as an oncogene in breast tumors transformed by the proximal insertion of murine virus, the role of Wnt signaling in cancer was first revealed (Nusse and Varmus, 1982, Cell 31: 99-109). Additional evidence for the role of Wnt signaling in breast cancer has been accumulated later. For example, transgenic overexpression of β -catenin in the mammary Gland results in hyperplasia and adenocarcinoma (Imbert et al, 2001, J.cell biol.153: 555-68; Michaelson and Leder, 2001, Oncogene 20: 5093-9), while loss of Wnt signaling disrupts normal mammary Gland development (Tepera et al, 2003, J.cell Sci.116: 1137-49; Hatsell et al, 2003, J.mammary Gland biol.Neopalasia 8: 145-58). It has recently been shown that Wnt signaling can activate mammary stem cells (Liu et al, 2004, Proc.Nat' l Acad.Sci.101: 4158). In human breast cancer, accumulation of β -catenin in more than 50% of the cancers implies activated Wnt signaling, and although no specific mutations were identified, upregulation of frizzled receptor expression has been observed (Brennan and Brown, 2004, J.M. Glan and Neoplasia 9: 119-31; Malovanovic et al, 2004, int.J.Oncol.25: 1337-42).

FZD10, FZD8, FZD7, FZD4, and FZD5 are 5 of 10 identified human Wnt receptors. Fzd10 was co-expressed with Wnt7b in the lung, and cell transfection studies have shown that the Fzd10/LRP5 co-receptor activates the canonical Wnt signaling pathway in response to Wnt7b (Wang et al, 2005, mol.cell biol.25: 5022-30). FZD10mRNA is upregulated in a variety of cancer cell lines, including cervical, gastric, and glioblastoma cell lines, as well as in primary cancers, including approximately 40% of primary gastric, colon, and synovial sarcomas (Saitoh et al, 2002, int.j.oncol.20: 117-20; Terasaki et al, 2002, int.j.mol.med.9: 107-12; Nagayama et al, 2005, Oncogene 1-12). FZD8 is upregulated in several human cancer cell lines, primary gastric and renal cancers (Saitoh et al, 2001, int.J. Oncol.18: 991-96; Kirikoshi et al, 2001, int.J. Oncol.19: 111-5; Janssens et al, 2004, Tumor biol.25: 161-71). FZD7 expression is throughout the gastrointestinal tract and is upregulated in one of 6 human primary gastric cancers (Kirikoshi et al, 2001, int.j. oncol.19: 111-5). Expression of the extracellular domain of FZD7 in colon cancer cell lines causes morphological changes and reduced tumor growth in xenograft models (Vincan et al, 2005, Differentiation 73: 142-53). FZD5 plays an essential role in yolk sac and placental angiogenesis (Ishikawa et al, 2001, Dev.128: 25-33), and is upregulated in kidney cancers associated with the activation of Wnt/β -catenin signaling (Janssens et al, 2004, Tumor biology 25: 161-71). FZD4 is highly expressed in intestinal crypt epithelial cells and is one of several factors that show differential expression in normal tissues relative to neoplastic tissues (Gregorieff et al, 2005, Gastroenterology 129: 626-38). Therefore, the identification of FZD receptors as markers for cancer stem cells (cancer stem cells) makes these proteins ideal targets for cancer therapy.

Disclosure of Invention

The present invention provides novel agents that bind to one or more human frizzled receptors (FZD), including but not limited to: antibodies or other agents that bind to two or more human frizzled receptors, and methods of using these agents. The invention also provides novel polypeptides, such as antibodies that bind to one or more frizzled receptors, fragments of such antibodies, and other polypeptides related to such antibodies. In certain embodiments, the agent, antibody, other polypeptide, or agent that binds to FZD binds to a region of FZD herein referred to as a Biological Binding Site (BBS), which the inventors have now identified for the first time as a target for inhibition of Wnt signaling and/or tumor growth. Antibodies and other polypeptides having an antigen binding site that binds to more than one FZD are also provided. Also provided are polynucleotides comprising nucleic acid sequences encoding the polypeptides, and vectors comprising the polynucleotides. Cells containing the polypeptides and/or polynucleotides of the invention are also provided. Also provided are compositions (e.g., pharmaceutical compositions) comprising the novel FZD-binding agents or antibodies. Also provided are methods of making and using the novel FZD-binding agents or antibodies, e.g., methods of using the novel FZD-binding agents or antibodies to inhibit tumor growth and/or treat cancer.

Accordingly, in one aspect, the invention provides an agent that specifically binds to a human frizzled receptor. In certain embodiments, the agent inhibits binding of a ligand (e.g., Wnt) to the Biological Binding Site (BBS) of a human frizzled receptor. In certain embodiments, the agent binds to at least a portion of a Biological Binding Site (BBS) in a human frizzled receptor. In certain embodiments, binding of the agent to the BBS results in inhibition of Wnt signaling and/or tumor growth. In certain embodiments, the human frizzled receptor is FZD8, and the agent binds to at least a portion of (a), (b), and/or (c) below: (a) a conformational epitope of FZD8 consisting of amino acids 72(F), 74-75 (PL), 78(I), 92(Y), 121-122 (LM) and 129-132 (WPDR (SEQ ID NO: 70)); (b) a region of FZD8 consisting of sequence QDEAGLEVHQFWPL (SEQ ID NO: 67); (c) a region of FZD8 consisting of the sequence QYGFA (SEQ ID NO: 66). In certain embodiments, the human frizzled receptor is selected from the group consisting of FZD1, FZD2, FZD5, FZD7, or FZD8, and the agent binds to at least a portion of sequence Q (DE/ED) aglevqf (Y/W) PL (SEQ ID NO: 24) in the human frizzled receptor. For example, in certain embodiments, the human frizzled receptor is FZD8 and the agent binds to at least a portion of sequence QDEAGLEVHQFWPL (SEQ ID NO: 67) in FZD 8. In certain embodiments, the agent binds to at least a portion of the sequence GLEVHQ (SEQ ID NO: 25). In certain embodiments, the human frizzled receptor is FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD9, or FZD10, and the agent binds to at least a portion of a region of the human frizzled receptor corresponding to a region of FZD8 consisting of QDEAGLEVHQFWPL (SEQ ID NO: 67). In certain embodiments, the agent binds to at least a portion of sequence (K/Q) (F/Y) GF (Q/A) (SEQ ID NO: 69) in FZD1, FZD2, FZD5, FZD7 and/or FZD 58. For example, in certain embodiments, the human frizzled receptor is FZD8, and the agent binds to at least a portion of the sequence QYGFA (SEQ ID NO: 66) in FZD 8. In certain embodiments, the human frizzled receptor is FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD9, or FZD10, and the agent binds to at least a portion of the human frizzled receptor region corresponding to FZD8 region consisting of QYGFA (SEQ ID NO: 66). In certain embodiments, the agent specifically binds to two or more, three or more, or four or more human frizzled receptors. In certain embodiments, the agent specifically binds to a human frizzled receptor comprising FZD5 and FZD 8.

In another aspect, the invention provides an agent that competes for specific binding to a human frizzled receptor with an antibody (e.g., in an in vitro competitive binding assay), wherein the antibody has a heavy chain variable region comprising SEQ ID NO: 10 and a light chain variable region comprising SEQ ID NO: 12 or SEQ ID NO: 14, light chain variable region. In certain embodiments, the antibody has an amino acid sequence comprising SEQ ID NO: 10 and a light chain variable region comprising SEQ ID NO: 14, light chain variable region. In certain embodiments, the agent competes for specific binding to two or more, three or more, or four or more human frizzled receptors. In certain embodiments, the agent competes for specific binding to FZD1, FZD2, FZD5, FZD7, or FZD 8.

In a further aspect, the present invention provides an agent that competes for specific binding to FZD5 and/or FZD8 with an antibody, wherein the antibody has a heavy chain variable region comprising SEQ ID NO: 85 and a light chain variable region comprising SEQ ID NO: 86 light chain variable region.

In yet another aspect, the invention provides agents that specifically bind to two or more human frizzled receptors. In certain embodiments, the two or more frizzled receptors comprise: (a) FZD1 and a second frizzled receptor selected from the group consisting of FZD2, FZD5, FZD7, and FZD 8; (b) FZD2 and a second frizzled receptor selected from the group consisting of FZD5, FZD7, and FZD 8; (c) FZD5 and FZD 7; or (d) FZD7 and FZD 8. In certain embodiments, the agent specifically binds three or more (i.e., 3, 4, or 5) human frizzled receptors, wherein the three or more human frizzled receptors comprise FZD1, FZD2, FZD5, FZD7, and/or FZD 8. In certain embodiments, the three or more human receptors comprise FZD5 and FZD 8. In certain embodiments, the three or more human frizzled receptors further comprise FZD3, FZD4, FZD6, FZD9, and/or FZD 10.

In another aspect, the present invention provides a polypeptide that specifically binds to a human frizzled receptor, wherein the polypeptide has a heavy chain variable region comprising the following (a), (b) and/or (c): (a) heavy chain CDR1 comprising GFTFSHYTLS (SEQ ID NO: 1) or a variant thereof having 1, 2, 3, or 4 amino acid substitutions; (b) heavy chain CDR2 comprising VISGDGSYTYYADSVKG (SEQ ID NO: 2) or a variant thereof having 1, 2, 3, or 4 amino acid substitutions; (c) comprises the heavy chain CDR3 of NFIKYVFAN (SEQ ID NO: 3) or a variant thereof having 1, 2, 3 or 4 amino acid substitutions. In certain embodiments, the polypeptide specifically binds FZD1, FZD2, FZD5, FZD7, and/or FZD 8. In certain embodiments, the polypeptide specifically binds to two or more human frizzled receptors including FZD5 and FZD 8. In certain embodiments, the amino acid substitution is a conservative substitution.

In another aspect, the present invention provides a polypeptide that specifically binds to a human frizzled receptor, wherein the polypeptide has a light chain variable region comprising the following (a), (b), and/or (c): (a) a light chain CDR1, the light chain CDR1 comprising SGDKLGKKYAS (SEQ ID NO: 4) or SGDNIGSFYVH (SEQ ID NO: 7), or SEQ ID NO: 4 or SEQ ID NO: 7; (b) a light chain CDR2, said light chain CDR2 comprising EKDNRPSG (SEQ ID NO: 5) or DKSNRRPSG (SEQ ID NO: 8), or SEQ ID NO: 5 or SEQ ID NO: 8, a variant thereof; (c) a light chain CDR3, the light chain CDR3 comprising SSFAGNSLE (SEQ ID NO: 6) or QSYANTLSL (SEQ ID NO: 9), or SEQ ID NO: 6 or SEQ ID NO: 9. In certain embodiments, the polypeptide specifically binds FZD1, FZD2, FZD5, FZD7, and/or FZD 8. In certain embodiments, the polypeptide specifically binds to two or more human frizzled receptors including FZD5 and FZD 8. In certain embodiments, the amino acid substitution is a conservative substitution.

In another aspect, the present invention provides a polypeptide having the following (a) and/or (b): (a) heavy chain CDR1 comprising GFTFSHYTLS (SEQ ID NO: 1), heavy chain CDR2 comprising VISGDGSYTYYADSVKG (SEQ ID NO: 2) and heavy chain CDR3 comprising NFIKYVFAN (SEQ ID NO: 3); (b) light chain CDR1 comprising SGDKLGKKYAS (SEQ ID NO: 4) or SGDNIGSFYVH (SEQ ID NO: 7), light chain CDR2 comprising EKDNRPSG (SEQ ID NO: 5) or DKSNRRPSG (SEQ ID NO: 8), and light chain CDR3 comprising SSFAGNSLE (SEQ ID NO: 6) or QSYANTLSL (SEQ ID NO: 9). In certain embodiments, the polypeptide specifically binds to a human frizzled receptor. In certain embodiments, the polypeptide specifically binds two or more (e.g., at least FZD5 and FZD8), three or more, or four or more human frizzled receptors.

In yet another aspect, the present invention provides an antibody that specifically binds to a human frizzled receptor, wherein the human frizzled receptor is selected from the group consisting of FZD1, FZD2, FZD5, FZD7, and FZD8, wherein the antibody has: a heavy chain variable region comprising (a) a heavy chain CDR1 comprising GFTFSHYTLS (SEQ ID NO: 1) or a variant thereof having 1, 2, 3, or 4 amino acid substitutions; heavy chain CDR2 comprising VISGDGSYTYYADSVKG (SEQ ID NO: 2) or a variant thereof having 1, 2, 3 or 4 amino acid substitutions; and a heavy chain CDR3 comprising NFIKYVFAN (SEQ ID NO: 3) or a variant thereof having 1, 2, 3, or 4 amino acid substitutions; and/or (b) a light chain CDR1, the light chain CDR1 comprising SGDKLGKKYAS (SEQ ID NO: 4), SGDNIGSFYVH (SEQ ID NO: 7), or SEQ ID NO: 4 or SEQ ID NO: 7; a light chain CDR2, said light chain CDR2 comprising EKDNRPSG (SEQ ID NO: 5), DKSNRPSG (SEQ ID NO: 8), or SEQ ID NO: 5 or SEQ ID NO: 8, a variant thereof; a light chain CDR3, the light chain CDR3 comprising SSFAGNSLE (SEQ ID NO: 6), QSYANTLSL (SEQ ID NO: 9), or SEQ ID NO: 6 or SEQ ID NO: 9. In certain embodiments, the antibody has (a) a heavy chain CDR1 comprising GFTFSHYTLS (SEQ ID NO: 1), a heavy chain CDR2 comprising VISGDGSYTYYADSVKG (SEQ ID NO: 2), and a heavy chain CDR3 comprising NFIKYVFAN (SEQ ID NO: 3); and/or (b) a light chain CDR1 comprising SGDKLGKKYAS (SEQ ID NO: 4) or SGDNIGSFYVH (SEQ ID NO: 7), a light chain CDR2 comprising EKDNRPSG (SEQ ID NO: 5) or DKSNRRPSG (SEQ ID NO: 8), and a light chain CDR3 comprising SSFAGNSLE (SEQ ID NO: 6) or QSYANTLSL (SEQ ID NO: 9). In certain embodiments, the antibody has (a) a heavy chain CDR1 comprising GFTFSHYTLS (SEQ ID NO: 1), a heavy chain CDR2 comprising VISGDGSYTYYADSVKG (SEQ ID NO: 2), and a heavy chain CDR3 comprising NFIKYVFAN (SEQ ID NO: 3); and/or (b) a light chain CDR1 comprising SGDNIGSFYVH (SEQ ID NO: 7), a light chain CDR2 comprising DKSNRPSG (SEQ ID NO: 8), and a light chain CDR3 comprising QSYANTLSL (SEQ ID NO: 9).

In another aspect, the present invention provides a polypeptide comprising the following (a) and/or (b): (a) and SEQ ID NO: 10, a polypeptide having at least about 80% sequence identity; (b) and SEQ ID NO: 12 or SEQ ID NO: 14 is at least about 80% polypeptide. In certain embodiments, the present invention provides a polypeptide comprising the following (a) and/or (b): (a) and SEQ ID NO: 10, a polypeptide having at least about 80% sequence identity; (b) and SEQ ID NO: 14 is at least about 80% polypeptide. In certain embodiments, the polypeptide specifically binds to a human frizzled receptor. In certain embodiments, the polypeptide specifically binds to two or more, three or more, or four or more human frizzled receptors. In certain embodiments, the human frizzled receptor is selected from the group consisting of FZD1, FZD2, FZD5, FZD7, and FZD 8.

In a further aspect, the present invention provides an agent such as an antibody that specifically binds to human FZD5 and/or FZD8, wherein the antibody has: (a) heavy chain CDR1 comprising GFTFSSYYIT (SEQ ID NO: 77) or a variant thereof having 1, 2, 3, or 4 conservative amino acid substitutions; heavy chain CDR2 comprising TISYSSSNTYYADSVKG (SEQ ID NO: 78) or a variant thereof having 1, 2, 3, or 4 conservative amino acid substitutions; and a heavy chain CDR3 comprising SIVFDY (SEQ ID NO: 79) or a variant thereof having 1, 2, 3, or 4 conservative amino acid substitutions; and/or (b) a light chain CDR1 comprising SGDALGNRYVY (SEQ ID NO: 80) or a variant thereof having 1, 2, 3, or 4 conservative amino acid substitutions; light chain CDR2 comprising SG (SEQ ID NO: 81) or a variant thereof having 1, 2, 3, or 4 conservative amino acid substitutions; light chain CDR3 comprising GSWDTRPYPKY (SEQ ID NO: 82) or variants thereof having 1, 2, 3, or 4 conservative amino acid substitutions. In certain embodiments, the antibody has: (a) heavy chain CDR1 comprising GFTFSSYYIT (SEQ ID NO: 77), heavy chain CDR2 comprising TISYSSSNTYYADSVKG (SEQ ID NO: 78) and heavy chain CDR3 comprising SIVFDY (SEQ ID NO: 79); and/or (b) a light chain CDR1 comprising SGDALGNRYVY (SEQ ID NO: 80), a light chain CDR2 comprising SG (SEQ ID NO: 81), and a light chain CDR3 comprising GSWDTRPYPKY (SEQ ID NO: 82).

In another aspect, the present invention provides a polypeptide that specifically binds FZD5 and/or FZD8, wherein the polypeptide comprises: (a) and SEQ ID NO: 85 is at least about 80% identical; and/or (b) a nucleotide sequence substantially identical to SEQ ID NO: 86 is at least about 80% polypeptide.

In a further aspect, the present invention provides an agent that competes for specific binding to human FZD1, FZD2, FZD5, FZD7, and/or FZD8 with any one of the following IgG antibodies: 18R8, 18R5, 18R4605 and 18R 4805.

In a further aspect, the present invention provides an agent that competes with an anti-FZD IgG antibody 44R24 for specific binding to human FZD5 and/or FZD 8.

In certain embodiments of each of the foregoing aspects and others described herein, the agent or polypeptide is an antibody. In certain alternative embodiments, the agent is not an antibody.

In certain embodiments of each of the foregoing aspects and others described herein, the agent or polypeptide or antibody specifically binds to the extracellular domain (ECD) of one or more human frizzled receptors to which it binds. In certain embodiments of each of the foregoing aspects and others described herein, the agent or polypeptide or antibody specifically binds to a Fri domain (Fri) of one or more human frizzled receptors to which it binds.

In certain embodiments of each of the foregoing aspects and others described elsewhere herein, a single antigen-binding site of the antibody or other polypeptide specifically binds (or is capable of binding) more than one human frizzled receptor.

In certain embodiments of each of the foregoing aspects and others described herein, the agent or polypeptide or antibody inhibits binding of a ligand to a human frizzled receptor. In certain embodiments, the ligand is Wnt.

In certain embodiments of each of the foregoing aspects and others described herein, the agent or polypeptide or antibody that binds to a FZD is an antagonist of that FZD.

In certain embodiments of each of the foregoing aspects and others described herein, the agent or polypeptide or antibody inhibits Wnt signaling. In certain embodiments, the Wnt signaling that is inhibited is canonical Wnt signaling. In some embodiments, the Wnt signaling inhibited by the FZD-binding agent is non-canonical Wnt signaling. In certain embodiments, Wnt signaling is non-canonical Wnt signaling.

In certain embodiments of each of the foregoing aspects as well as other aspects described herein, the FZD-binding agent or polypeptide or antibody inhibits tumor growth.

The invention also provides antibodies 18R8, 18R5, 18R4605, 44R24 and 18R4805 and fragments thereof.

The present invention also provides compositions (e.g., pharmaceutical compositions) comprising FZD-binding agents or antibodies.

The present invention provides methods of inhibiting Wnt signaling (e.g., canonical Wnt signaling) and/or inhibiting tumor growth in a subject comprising administering a therapeutically effective amount of a FZD-binding agent or polypeptide or antibody.

Also provided are methods of reducing the tumorigenicity of a tumor containing cancer stem cells. In certain embodiments, the method comprises administering to a subject having a tumor a therapeutically effective amount of an FZD-binding agent or polypeptide or antibody. In certain embodiments, the frequency of cancer stem cells in a tumor is reduced by administering the antibody. In certain embodiments, administration of the FZD-binding agent results in differentiation of tumorigenic cells in the tumor to a non-tumorigenic state.

Also provided are methods of inducing differentiation of cells in a tumor in a subject, the method comprising administering to the subject a therapeutically effective amount of a FZD-binding agent, polypeptide, or antibody.

Further provided are methods of treating cancer in a subject comprising administering to the subject a therapeutically effective amount of a FZD-binding agent, polypeptide, or antibody.

Further, a method of reducing myofibroblast activation in a solid tumor matrix is provided, the method comprising contacting the matrix with a therapeutically effective amount of a FZD-binding agent, polypeptide, or antibody.

In certain embodiments, a method comprising administering a FZD-binding agent, polypeptide, or antibody further comprises administering to the subject a second anti-cancer agent (e.g., a chemotherapeutic agent). In certain embodiments, the second agent is gemcitabine (gemcitabine), irinotecan (irinotecan), or paclitaxel. In certain embodiments, the second agent is an angiogenesis inhibitor and/or a Notch signaling inhibitor.

In another aspect, the invention provides a polypeptide comprising a sequence selected from the group consisting of SEQ ID NOs: 10-15 in a sequence of the group. Also provided is a polypeptide comprising a sequence selected from the group consisting of SEQ ID NOs: 85-86 in the sequence list. Also provided are polynucleotides comprising nucleic acid sequences encoding the polypeptides.

In yet another aspect, the present invention provides a polypeptide comprising a sequence selected from the group consisting of SEQ ID NOs: 17-12. Further provided is a polypeptide comprising a sequence selected from the group consisting of SEQ ID NOs: 87 to 90, 92 and 94 to 95.

In yet another aspect, the invention provides a polynucleotide comprising a sequence that hybridizes under high stringency conditions with a polynucleotide selected from the group consisting of SEQ ID NOs: 17. 19, 21, 87-90, 92 and 94-95 or a polynucleotide that hybridizes to or encodes a polynucleotide selected from the group consisting of SEQ ID NOs: 10. 12, 14 and 85 to 86 to the polypeptide of the group. In certain embodiments, the invention encompasses a nucleic acid sequence that hybridizes under high stringency conditions to a nucleic acid sequence consisting of SEQ ID NO: 17. 19 or 21 or to a polynucleotide encoding SEQ ID NO: 10. 12 or 14.

In certain embodiments of each of the foregoing aspects and others described herein, the agent or polypeptide or antibody or polynucleotide is isolated. In certain embodiments, the agent or polypeptide or antibody or polynucleotide is substantially pure.

The invention also provides Wnt gene signatures that can be used to identify tumors and/or patients that are likely to respond to treatment with FZD-binding agents (e.g., antagonists of human frizzled receptors and/or inhibitors of Wnt signaling) or other inhibitors of Wnt signaling. Also provided are methods of using Wnt gene signatures to select patients for treatment with FZD-binding agents or other inhibitors of Wnt signaling. In certain embodiments, the methods involve assessing the level of one or more genes in the Wnt gene signature. Also provided are methods of screening for drug candidates against tumors identified using the Wnt gene signature. Arrays, kits, and other compositions useful in the methods are also provided.

The invention also provides methods of screening for potential drug candidates or other agents. These methods include, but are not limited to: a method comprising comparing the level of one or more differentiation markers in a first solid tumor to the level of one or more differentiation markers in a second solid tumor, the first solid tumor having been exposed to the agent and the second solid tumor not having been exposed to the agent. In certain embodiments, these methods comprise: (a) exposing a first solid tumor to the agent while a second solid tumor is not exposed to the agent; (b) assessing the level of one or more differentiation markers in the first solid tumor and the second solid tumor; and (c) comparing the level of one or more differentiation markers in the first solid tumor and the second solid tumor.

Where aspects or embodiments of the invention are described in terms of markush groups or other groupings of optional elements, the invention encompasses not only the entire group listed as a whole, but also each member of the group individually as well as all possible subgroups of the major group, as well as the major group lacking one or more group members. The invention also includes the explicit exclusion of any one or more members of this group in the claimed invention.

Drawings

FIG. 1: 18R8 binds to a variety of human frizzled receptors. FACS analysis showed that 18R8 binds to FZD1, FZD2, FZD5, FZD7, and FZD8 sequences on transiently transfected HEK293 cells. FACS mapping of 18R8 binding to HEK293 cells transfected with expression vectors encoding the indicated FZD and GFP expression vectors is shown. Increased staining in the co-transfected (GFP positive) cell population indicated binding of 18R 8. The FACS plots for FZD1, FZD2, FZD5, FZD7, and FZD8 are boxed with thick lines to highlight the FZD that binds to 18R 8.

FIG. 2: anti-FZD antibody 18R5 binds to a variety of human frizzled receptors on cells. FACS analysis showed that 18R5 binds to FZD1, FZD2, FZD5, FZD7, and FZD8 sequences on cells as with 18R 8. Different concentrations of antibodies 18R8 and 18R5 were co-incubated with HEK293 cells overexpressing the indicated FZD and antibody binding was assessed by flow cytometry. Although 18R8 and 18R5 both bind FZD1, FZD2, FZD5, FZD7, and FZD8, the binding of 18R5 has higher affinity.

FIG. 3: luciferase reporter assays were performed in STF293 cells stably expressing the 8xTCF promoter element linked to luciferase. Cells were treated with conditioned media containing Wnt3A and varying concentrations of 18R8 and 18R5 and then assayed 18 hours later using the Dual-Glo luciferase detection reporter system (Promega). The results indicate that both 18R8 and 18R5 inhibit Wnt signaling, and that 18R5 has higher binding affinity than 18R 8.

FIG. 4: 18R8 blocks TCF signaling through multiple wnts. Luciferase reporter assays were performed in STF293 cells stably expressing the 8xTCF promoter element linked to luciferase. Various cells overexpressing Wnt were generated by transfecting HEK293 cells (ATCC) with Fugene 6(Roche) with an expression vector encoding the indicated Wnt proteins. STF293 cells were treated with 18R8 and Wnt-overexpressing HEK293 cells were added, followed by assay after 18 hours using Dual-Glo luciferase detection reporter system.

FIG. 5: 18R8 directly inhibits Wnt binding to FZD. Luciferase reporter assays were performed in STF293 cells stably expressing the 8xTCF promoter element. Mixtures containing Wnt3A conditioned medium, purified FZD8-Fc and/or 18R8 as shown were co-incubated for 2 hours at 4 ℃ with or without protein a sepharose microspheres. After incubation, protein a sepharose microspheres were removed and added to STF293 cells. The treated STF293 cells were assayed 18 hours later using the Dual-Glo luciferase assay reporter System. This experiment shows that Fzd8-Fc was able to inhibit the ability of Wnt3A to stimulate signaling in the absence of 18R8, but 18R8 was able to block the ability of Fzd8 to bind to Wnt3A, as evidenced by the restoration of signaling when Fzd8-Fc (and 18R8) was removed from the co-incubations using agarose a microspheres.

FIG. 6: FACS analysis of binding of 18R8 to mutant FZD8 (compared to wild-type FZD 8). To evaluate the 18R8 epitope on FZD, an epitope mapping study was performed. An expression vector encoding GFP was used in transient transfection studies with an expression construct capable of expressing the Fri domain of human FZD8 with an N-terminal FLAG tag and a C-terminal CD4 transmembrane and intracellular domain. Variants of this expression vector having selected amino acid substitutions within the sequence of FZD8 to encode amino acids at corresponding positions in the Fri domain of other specific FZD that do not bind to 18R8 were also prepared. The ability of 18R8 to bind to these FZD8 sequence variants was subsequently assessed by flow cytometry. As shown by the reduced staining in the co-transfected (GFP positive) cell population, it was found that the binding of 18R8 requires amino acids at certain positions of FZD8, including amino acids 66-71 and 126-127 of FZD 8. FACS mapping regions showing binding of 18R8 to co-transfected cell populations are highlighted in a box, and a box showing significantly reduced amino acid substitutions associated with 18R8 is shown in bold.

FIG. 7: 18R5 and 18R8 are shown to have similar binding epitopes on human FZD 8. The ability of 18R5 to bind an epitope similar to the epitope to which 18R8 binds was assessed by flow cytometry using a series of amino acid variants previously shown to disrupt 18R8 binding. As shown by the reduced staining in the co-transfected (GFP positive) cell population, it was found that positions including amino acids 66-71 and 126-127 of FZD8 were required for binding to both 18R8 and 18R 5. FACS mapping regions showing binding of 18R8 to co-transfected cell populations are highlighted in a box and the box corresponding to amino acid substitutions showing significantly reduced binding of 18R8 and 18R5 is bolded.

FIG. 8: comparison between partial amino acid sequences of the Fri domain sequences of the individual human frizzled receptors. Sites with conserved residues are shaded in black; sites with similar amino acid residues are shaded in grey. The FZD epitopes of 18R8 and 18R5 include regions marked by underlining and labeled "upper edge" and "lower edge". Based on a scrutiny of the crystal structure of the Fri domain, it can be recognized that these regions flank the cleft on the surface of the FZD protein, and the terms "upper edge" and "lower edge" reflect this recognition. The cleft comprises a plurality of highly conserved residues. The amino acids that make up the cleft are highlighted by the triangular symbols at each corresponding position in the alignment. The specific function of the area has not been previously recognized. The discovery of antibodies that bind to this region, the discovery that these antibodies inhibit Wnt binding and Wnt signaling, and the inventors' knowledge of the conservation of this cleft, have enabled the inventors to identify this region as a key functional part of the FZD protein.

FIG. 9: biological Binding Site (BBS) of FZD. Structural images of the Fzd Fri domain are shown. The image is based on an analysis of the previously reported crystal structure of mouse FZD8 (Dann CE et al, Nature 412(6842)86-90, 2001) and an analysis done using the software program Pymol. In the upper left panel is shown a surface view of the FZD Fri domain, where the FZD protein region contains the Biological Binding Site (BBS) that the inventors have found circled in white ovals. This region is the region to which antibodies 18R8 and 18R5 bind. This region contains structural elements referred to by the inventors as "upper edge", "lower edge" and "cleft". Each of the structural elements is highlighted with darker surface coloration in the separate images at the bottom of the figure. The upper right panel highlights residues conserved in 9 or 10 of the 10 human Fzd family members with darker surfaces and highlights the following recognition: a distinct cluster of these residues occurs within the center of the "cleft" region flanked by epitopes for binding to antibodies that inhibit Fzd function.

FIG. 10: wnt dependent tumor growth was prevented by mabs against FZD. NOD/SCID mice injected with 50,000 MMTV WNT1 tumor-derived cells and tumor growth were monitored weekly until growth was detected, followed by two measurements of tumor growth weekly. 10 mice with established tumors were treated with 18R8 or a control antibody as a control. Tumor growth was almost eliminated in animals treated with 18R8 compared to that observed in animals treated with control antibody.

FIG. 11: treatment with 18R5 in combination with irinotecan reduced the growth of OMP-C28 xenograft tumors. NOD/SCID mice were injected with 10,000 OMP-C28 colon tumor cells, and on day 24, the average tumor volume was 129mm³Mice were randomly grouped and treatment as indicated was initiated. Tumor growth was monitored weekly. In animals treated with 18R5, tumor growth was significantly reduced. Furthermore, the combination of 18R5 with irinotecan showed a significant reduction compared to treatment with either agent alone.

FIG. 12: treatment with 18R5 in combination with gemcitabine reduced the growth of OMP-PN4 xenograft tumors. NOD/SCID mice were injected with 50,000 OMP-PN4 pancreatic tumor cells, and on day 38, the average tumor volume was about 120mm ³Mice were randomly grouped and treatment as indicated was started after 2 days. Tumor growth was significantly reduced in animals treated with 18R5 in combination with gemcitabine compared to treatment with gemcitabine alone.

FIG. 13: 18R8 and 18R5, including VH and VL sequences.

FIG. 14: nucleotide sequences encoding the heavy and VH sequences of 18R8 and 18R 5.

FIG. 15: nucleotide sequences encoding the light chain and VL sequences of 18R8 and 18R 5.

FIG. 16: an amino acid sequence of FZD7ECD Fc protein and a nucleotide sequence for coding the protein.

FIG. 17: the amino acid sequences of human FZD1(SEQ ID NO: 26), the extracellular domain (ECD) of FZD1(SEQ ID NO: 27, shown as underlined amino acids 1-321 in SEQ ID NO: 26), and the Fri domain of FZD1(SEQ ID NO: 28).

FIG. 18: the amino acid sequences of human FZD2(SEQ ID NO: 30), the extracellular domain (ECD) of FZD2(SEQ ID NO: 31, shown as underlined amino acids 1-250 in SEQ ID NO: 30), and the Fri domain of FZD2(SEQ ID NO: 32).

FIG. 19: a nucleotide sequence encoding human FZD 1.

FIG. 20: a nucleotide sequence encoding human FZD 2.

FIG. 21: the amino acid sequences of human FZD3(SEQ ID NO: 34), the extracellular domain (ECD) of FZD3(SEQ ID NO: 35, shown as underlined amino acids 1-204 in SEQ ID NO: 34), and the Fri domain of FZD3(SEQ ID NO: 36).

FIG. 22: the amino acid sequences of human FZD4(SEQ ID NO: 38), the extracellular domain (ECD) of FZD4(SEQ ID NO: 39, shown as underlined amino acids 1-224 in SEQ ID NO: 38), and the Fri domain of FZD4(SEQ ID NO: 40).

FIG. 23: a nucleotide sequence encoding human FZD 3.

FIG. 24: a nucleotide sequence encoding human FZD 4.

FIG. 25: the amino acid sequences of human FZD5(SEQ ID NO: 42), the extracellular domain (ECD) of FZD5(SEQ ID NO: 43, shown as underlined amino acids 1-233 in SEQ ID NO: 42), and the Fri domain of FZD5(SEQ ID NO: 44).

FIG. 26: the amino acid sequences of human FZD6(SEQ ID NO: 46), the extracellular domain (ECD) of FZD6(SEQ ID NO: 47, shown as underlined amino acids 1-207 in SEQ ID NO: 46), and the Fri domain of FZD6(SEQ ID NO: 48).

FIG. 27 is a schematic view showing: a nucleotide sequence encoding human FZD 5.

FIG. 28: a nucleotide sequence encoding human FZD 6.

FIG. 29: the amino acid sequences of human FZD7(SEQ ID NO: 50), the extracellular domain (ECD) of FZD7(SEQ ID NO: 51, shown as underlined amino acids 1-255 in SEQ ID NO: 50), and the Fri domain of FZD7(SEQ ID NO: 52).

FIG. 30: the amino acid sequences of human FZD8(SEQ ID NO: 54), the extracellular domain (ECD) of FZD8(SEQ ID NO: 55, shown as underlined amino acids 1-277 in SEQ ID NO: 54), and the Fri domain of FZD8(SEQ ID NO: 56).

FIG. 31: a nucleotide sequence encoding human FZD 7.

FIG. 32: a nucleotide sequence encoding human FZD 8.

FIG. 33: the amino acid sequences of human FZD9(SEQ ID NO: 58), the extracellular domain (ECD) of FZD9(SEQ ID NO: 59, shown as underlined amino acids 1-230 in SEQ ID NO: 58), and the Fri domain of FZD9(SEQ ID NO: 60).

FIG. 34: the amino acid sequences of human FZD10(SEQ ID NO: 62), the extracellular domain (ECD) of FZD10(SEQ ID NO: 63, shown as underlined amino acids 1-227 in SEQ ID NO: 62), and the Fri domain of FZD10(SEQ ID NO: 64).

FIG. 35: a nucleotide sequence encoding human FZD 9.

FIG. 36: a nucleotide sequence encoding human FZD 10.

FIG. 37: treatment with the 18R5 antibody in combination with paclitaxel reduced the growth of PE-13 breast tumors. NOD/SCID mice were injected with 10,000 PE-13 breast tumor cells, and on day 22, the average tumor volume was about 120mm³Mice were randomly grouped. The mice were subsequently treated with a control antibody ("control Ab"), an 18R5 antibody ("anti-FZD"), paclitaxel ("Taxol"), or a combination of an 18R5 antibody and paclitaxel ("anti-FZD + Taxol"). Treatment with the 18R5 antibody in combination with paclitaxel had anti-tumor activity.

FIG. 38: breast tumor growth in individual animals treated with the 18R5 antibody in combination with paclitaxel. Combination therapy with the 18R5 antibody and paclitaxel resulted in regression of established breast tumors.

FIG. 39: flow cytometric analysis of colorectal tumor cells following treatment with control antibody, 18R5 antibody, irinotecan, or a combination of 18R5 antibody and irinotecan.

FIG. 40: tumor growth in mice implanted with 30, 90, 270, or 810 tumor cells obtained from mice that have been treated with a control antibody ("control"), an 18R5 antibody ("anti-FZD"), gemcitabine ("gemcitabine"), or a combination of 18R5 and gemcitabine ("combination") for 41 days.

FIG. 41: cancer Stem Cell (CSC) frequency in PN-4 pancreatic tumors was determined by limiting dilution analysis after treatment with a control antibody ("control Ab"), with an 18R5 antibody alone ("anti-FZD"), with gemcitabine alone ("gemcitabine"), or a combination of an 18R5 antibody and gemcitabine ("combination").

FIG. 42: chromogranin (chromagram) gene expression in pancreatic tumor cells that have been treated with a control antibody ("LZ-1"), with 18R5 antibody ("18R 5") alone, with gemcitabine ("gemcitabine") alone, or with a combination of gemcitabine and 18R5 ("combination").

FIG. 43: treatment with anti-FZD antibody 18R5 promoted differentiation of tumor cells into non-proliferating mucinous cells. NOD/SCID mice were injected with 50,000 OMP-PN13 pancreatic tumor cells, and on day 23, the average tumor volume was about 107mm³Mice were randomly grouped and treatment with control antibody or 18R5 was started after 4 days. Tumors were collected and sectioned after 20 days. Tumor sections from 18R 5-treated mice or control antibody-treated mice were stained with alcian blue dye to show mucinous cells, and proliferating cells were shown by immunohistochemistry with ki67 antibody. Exemplary mucinous and proliferating cells are highlighted by arrows.

FIG. 44: anti-FZD antibody 18R5 used alone or in combination with(paclitaxel) the anti-FZD antibody 18R5 used in combination inhibited the growth of OMP-LU24 xenograft tumors. With a control antibody ("control Ab"), anti-FZD 18R5 ("18R 5")("Taxol") or 18R5 withThe combination of (3) (18R 5+ Taxol) was used to treat mice bearing OMP-LU24 human lung tumors.

FIG. 45: anti-FZD antibody 18R5 used alone or in combination with(bevacizumab) the anti-FZD antibody 18R5 used in combination inhibited the growth of OMP-LU33 xenograft tumors. With control antibody (Square), (upward pointing triangle), anti-FZD 18R5 (downward pointing triangle) or 18R5 andthe combination of (circles) was used to treat mice bearing OMP-LU33 human lung tumor.

FIG. 46: anti-FZD antibody 18R5 and(trastuzumab) inhibits the growth of T3 xenograft tumors. With control antibody (square), anti-FZD 18R5 (triangle),(filled circles) or 18R5 withThe combination of (open circle) on mice bearing T3 human breast tumor was treated.

FIG. 47: fzd binding patterns of 18R5 and 44R 24. Dose-response curves (Dose-response curves) representing the binding of each mAb to Fzd1, 2, 5, 7, and 8.

FIG. 48: inhibition of Wnt3 a-induced reporter activity in STF cells by 18R5 and 44R24 (dose response curves).

FIG. 49: inhibition of background levels of axin2 gene expression by 18R5 and 44R 24.

FIG. 50: IHC detection of Muc16 in OMP-PN13 tumors treated with 18R5 and 44R 24.

FIG. 51: inhibition of smooth muscle actin in pancreatic tumors treated with 18R 5. A. ACTA2 gene expression levels detected by microarray. B. Detection of SMA in OMP-PN4 tumors treated with control mAb (top panel) and 18R5 (bottom panel).

FIG. 52: 18R 5-induced Muc16+ OMP-PN13 cells were tested for tumorigenicity. A. FACS mapping of Muc16 stained lin-depleted (lin-depleted) OMP-PN13 tumor cells. The two sorted populations are circled. B. Representative images of tumors resulting from injection of Muc16- (top panel) and Muc16+ (bottom panel) cells. C. Tumor growth curve.

FIG. 53: inhibition of tumor recurrence by anti-FZD mAb 18R5 in PE13 breast tumor xenograft.

FIG. 54: mAb 18R5 against FZD decreased the frequency of cancer stem cells in breast.

FIG. 55: inhibition of tumor recurrence by anti-FZD mAb 18R5 in PN4 pancreatic tumor xenograft.

FIG. 56: inhibition of tumor growth by anti-FZD mAb 44R24 in combination with gemcitabine in PN4 pancreatic tumor xenograft.

FIG. 57: FACS analysis of binding of anti-FZD mAb 44R24 to mutant FZD8 (relative to wild-type FZD 8). The region of the FACS plot showing binding of 44R24 to the co-transfected cell population is highlighted by a line box. Arrows mark the wireframe of those amino acid substitutions that show a significant decrease in binding of 44R 24.

FIG. 58: anti-tumor activity of anti-FZD antibodies 44R24 and 18R5 in C25 colon tumor xenografts.

FIG. 59: induction of cytokeratin 7 expression in C28 colon tumor xenografts treated with anti-FZD antibodies 44R24 or 18R 5.

Detailed Description

The present invention provides novel agents, including but not limited to polypeptides (e.g., antibodies) that bind to one or more human frizzled receptors (FZD). Related polypeptides and polynucleotides, compositions containing FZD-binding agents, and methods of making the FZD-binding agents are also provided. Methods of using the novel FZD-binding agents, such as methods of inhibiting tumor growth and/or treating cancer, are also provided.

The present invention is based, in part, on the identification of a region in a human frizzled receptor that is a suitable target for an anti-cancer agent that binds FZD. Two anti-FZD antibodies, 18R8 and 18R5, were found to specifically bind FZD7 and also cross-react with FZD1, FZD2, FZD5, and FZD8 (examples 1 and 2 below). In vitro experiments with the 18R8 antibody showed that the antibody was able to inhibit Wnt signaling (example 3, below) and inhibit binding of Wnt ligands to FZD8 (example 4, below). In cell-based assays, 18R5 antibody has also been shown to be able to inhibit Wnt signaling as well (examples 3 and 20 below). In vivo experiments with the 18R5 antibody showed that the antibody was able to inhibit tumor growth or recurrence (examples 7, 17 and 23 below). The inventors also demonstrated that anti-FZD antibody 18R5 is capable of reducing the frequency of cancer stem cells in tumors (examples 8 and 23 below) and inducing differentiation and/or reducing tumorigenicity of tumor cells (examples 16, 21, 22 and 25 below). Epitope mapping experiments with these active 18R8 and 18R5 antibodies showed that both of these antibodies could bind to at least a portion of the sequence GLEVHD (SEQ ID NO: 25) and at least a portion of the sequence YGFA (SEQ ID NO: 74) in FZD8 (example 5, below). Based on the biological activities demonstrated for these two antibodies, the crystal structure of mouse frizzled 8 (Dann et al, Nature, 412: 86-90(2001)) was analyzed and the extracellular region of frizzled, which contains these sequences not previously thought to have any particular function, was first identified as playing an important functional role in FZD biology and Wnt signaling (example 6). This region of the human frizzled receptor, known as the Biological Binding Site (BBS), is a suitable target for anti-cancer therapy.

In addition, the third antibody 44R24 was found to specifically bind to human FZD5 and FZD8 (example 19 below). The antibodies have additionally been shown to be capable of inhibiting Wnt signaling in cell-based assays (example 20, below) and have anti-tumor utility in vivo (examples 23 and 25, below). Treatment of a tumor with 44R24 resulted in an increase in the level of differentiation markers in the tumor, as in treatment with anti-FZD antibodies 18R8 and 18R5 (example 25, below). Epitope mapping has also shown that the epitope of anti-FZD antibody 44R24 overlaps with the epitope of anti-FZD antibodies 18R8 and 18R 5. More specifically, 44R24 has been shown to bind to at least a portion of the region YGFA (SEQ ID NO: 74) in the BBS (example 24, below).

I. Definition of

To facilitate an understanding of the present invention, a number of terms and phrases are defined below.

The term "antibody" refers to an immunoglobulin molecule that recognizes and specifically binds a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combination thereof, through at least one antigen recognition site within the variable region of the immunoglobulin molecule. The term "antibody" as used herein encompasses intact polyclonal antibodies, intact monoclonal antibodies, antibody fragments (e.g., Fab ', F (ab') 2, and Fv fragments), single chain Fv (scfv) mutants, multispecific antibodies (e.g., bispecific antibodies) generated from at least two intact antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising an epitope of an antibody, and any other modified immunoglobulin molecule comprising an antigen recognition site, so long as the antibodies exhibit the desired biological activity. Depending on the heavy chain constant domains of the antibody (referred to as α, γ and μ, respectively), the antibody may be any of the 5 major classes of immunoglobulins (IgA, IgD, IgE, IgG and IgM) or subclasses (isotypes) thereof (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA 2). The different classes of immunoglobulins have different and well-known subunit structures and three-dimensional configurations. The antibody may be a naked antibody or conjugated to other molecules such as toxins, radioisotopes, and the like.

The term "antibody fragment" refers to a portion of an intact antibody, and to the epitope variable region of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab ', F (ab') 2, and Fv fragments, linear antibodies, single chain antibodies, and multispecific antibodies formed from antibody fragments.

"monoclonal antibody" refers to a homogeneous population of antibodies involved in recognizing and binding a single antigenic determinant or epitope with high specificity. In contrast, polyclonal antibodies typically include different antibodies directed against different antigenic determinants. The term "monoclonal antibody" encompasses intact full-length monoclonal antibodies and antibody fragments (e.g., Fab ', F (ab') 2, Fv), single chain (scFv) mutants, fusion proteins comprising an antibody portion, and any other modified immunoglobulin molecule comprising an antigen recognition site. In addition, "monoclonal antibodies" refer to such antibodies made in a variety of ways including, but not limited to, hybridomas, phage selection, recombinant expression, and transgenic animals.

The term "humanized antibody" refers to a form of non-human (e.g., murine) antibody having minimal non-human (e.g., murine) sequences, said form having a specific immunoglobulin chain, chimeric immunoglobulin, or fragment thereof. Typically, humanized antibodies are human immunoglobulins in which residues from the Complementarity Determining Regions (CDRs) are replaced by residues from CDRs from non-human species (e.g., mouse, rat, rabbit, hamster) having the desired specificity, affinity, and capacity (Jones et al, 1986, Nature, 321: 522-153525; Riechmann et al, 1988, Nature, 332: 323-327; Verhoeyen et al, 1988, Science, 239: 1534-1536). In some cases, residues from antibodies of the non-human species having the desired specificity, affinity, and capacity are substituted for the corresponding residues of the Fv Framework Region (FR) of the human immunoglobulin. Humanized antibodies can be further modified by substitution of additional residues in the Fv framework region and/or in the substituted non-human residues to improve and optimize antibody specificity, affinity, and/or capacity. In general, a humanized antibody will comprise substantially all of at least one, and typically two or three, variable regions comprising all or substantially all of the CDR regions corresponding to a non-human immunoglobulin, while all or substantially all of the FR regions belong to a human immunoglobulin consensus sequence. The humanized antibody may also comprise at least a portion of a constant region or domain (Fc) of an immunoglobulin, typically a human immunoglobulin. An example of a method for producing humanized antibodies is described in U.S. Pat. No. 5,225,539.

The term "human antibody" refers to an antibody produced by a human, or an antibody having an amino acid sequence corresponding to an antibody produced by a human, prepared by any technique known in the art. Such definitions of human antibodies include whole or full-length antibodies, fragments thereof, and/or antibodies comprising at least one human heavy and/or light chain polypeptide (e.g., antibodies comprising murine light chains and human heavy chain polypeptides).

The term "chimeric antibody" refers to an antibody in which the amino acid sequences of the immunoglobulin molecules are derived from two or more species. Typically, the variable regions of the light and heavy chains correspond to those of an antibody derived from one mammalian species (e.g., mouse, rat, rabbit, etc.) with the desired specificity, affinity, and capacity, while the constant regions are homologous to sequences in an antibody derived from another species (typically human), thereby avoiding the induction of an immune response in that species.

The terms "epitope" or "antigenic determinant" are used interchangeably herein to refer to a portion of an antigen that is capable of being recognized and specifically bound by a particular antibody. When the antigen is a polypeptide, the epitope can be composed of contiguous amino acids, and can also be composed of non-contiguous amino acids juxtaposed by tertiary structure folding of the protein. Epitopes consisting of contiguous amino acids are usually retained when proteins are denatured, whereas epitopes formed by tertiary structural folding are usually lost when proteins are denatured. Epitopes typically comprise at least 3, more typically at least 5 or 8-10 amino acids with a unique spatial conformation.

An antibody "specifically binds" to an epitope or protein means that the antibody reacts or associates with the epitope or protein more frequently, more rapidly, for a longer period of time, with greater affinity, or some combination thereof, than an alternative substance, including an unrelated protein. In certain embodiments, "specifically binds" refers to, for example, K to which an antibody binds to a protein_DValues are below about 0.1mM, but more often less than about 1. mu.M. In certain embodiments, "specifically binds" refers to K to which an antibody binds to a protein_DSometimes at least about 0.1. mu.M or less, and at other times at least about 0.01. mu.M or less. Due to sequence identity between homologous proteins in different species, specific binding may include antibodies that recognize specific proteins (e.g., frizzled receptors) in more than one species. Also, due to homology between different FZD receptors (e.g., FZD5 and FZD8) in certain regions of these receptor polypeptide sequences, specific binding may include antibodies (or other polypeptides or agents) that recognize more than one frizzled receptor. It will be appreciated that the antibody, or binding moiety, that specifically binds to the first target may or may not specifically bind to the second target. As such, "specific binding" does not necessarily require (although it may include) exclusive binding, i.e., binding to a single target. Thus, in certain embodiments, an antibody may specifically bind to more than one target (e.g., human FZD1, FZD2, FZD5, FZD7, and/or FZD 8). In certain embodiments, the same antigen binding site on an antibody may bind to multiple targets. For example, in some cases, an antibody may contain two The same antigen binding sites, wherein each site specifically binds two or more human frizzled receptors (e.g., human FZD1, FZD2, FZD5, FZD7, and/or FZD 8). In certain alternative embodiments, the antibody may be bispecific and contain at least two antigen binding sites that differ in specificity. By way of non-limiting example, a bispecific antibody may have one antigen-binding site that recognizes an epitope on one frizzled receptor (e.g., human FZD5), and also have another, different antigen-binding site that recognizes a different epitope on a second frizzled receptor (e.g., human FZD 8). Generally, but not necessarily, reference to binding refers to specific binding.

An "isolated" polypeptide, antibody, polynucleotide, vector, cell, or composition refers to a polypeptide, antibody, polynucleotide, vector, cell, or composition that is in a form not found in nature. An isolated polypeptide, antibody, polynucleotide, vector, cell or composition includes a polypeptide, antibody, polynucleotide, vector, cell or composition that has been purified to the extent that it is no longer in the form in which it is found in nature. In some embodiments, the isolated antibody, polynucleotide, vector, cell or composition is substantially pure.

As used herein, "substantially pure" refers to a material that is at least 50% pure (i.e., free of impurities), more preferably at least 90% pure, even more preferably at least 95% pure, even more preferably at least 98% pure, and even more preferably at least 99% pure.

The terms "cancer" and "cancerous" refer to or are used to describe a physiological condition in a mammal in which a population of cells is characterized by dysregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specific examples of such cancers include squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer (liver cancer), bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrioma or carcinoma of the uterus, salivary adenoma, kidney cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, liver cancer (hepatotic carcinoma), and various types of head and neck cancer.

"tumor" and "neoplasm" refer to any benign (noncancerous) or malignant (cancerous) tissue mass, including precancerous lesions, resulting from excessive cell growth or proliferation.

The terms "cancer stem cell", "tumor stem cell" or "solid tumor stem cell" are used interchangeably herein to refer to a population of cells from a solid tumor that have the following characteristics: (1) has stronger proliferation capacity; (2) capable of asymmetric cell division to produce one or more differentiated progeny having reduced proliferative or developmental potential; and (3) capable of symmetric splitting for self-renewal or self-maintenance. These properties of "cancer stem cells", "tumor stem cells" or "solid tumor stem cells" allow these cancer stem cells to form palpable tumors when serially transplanted into immunocompromised mice as compared to most tumor cells that are unable to form tumors. Cancer stem cells self-renew in a disordered manner relative to differentiation, thereby forming tumors with abnormal cell types that can change over time when mutated.

The terms "cancer cell," "tumor cell," and grammatically equivalent terms refer to the total cell population derived from a tumor or precancerous lesion, including non-tumorigenic cells (which includes a large population of tumor cells) and tumorigenic stem cells (cancer stem cells). The term "tumor cell" as used herein is modified by the term "non-tumorigenic" when referring to only those tumor cells lacking the ability to renew and differentiate, thereby distinguishing these tumor cells from cancer stem cells.

The term "tumorigenicity" refers to the functional characteristics of solid tumor stem cells, which include the self-renewal property (producing additional tumorigenic cancer stem cells) and the property of proliferating to produce all other tumor cells that cause solid tumor stem cells to form tumors (producing differentiated tumor cells that are thus not non-tumorigenic). Upon serial transplantation of immunocompromised mice, the nature of these self-renewing and proliferating to produce all other tumor cells enables cancer stem cells to form palpable tumors compared to non-tumorigenic tumor cells, which are unable to form tumors upon serial transplantation. It has been observed that non-tumorigenic tumor cells can form tumors upon initial transplantation of immunocompromised mice after tumor cells are obtained from a solid tumor, but that these non-tumorigenic tumor cells do not produce tumors upon serial transplantation.

The term "subject" refers to any animal (e.g., a mammal), including but not limited to humans, non-human primates, rodents, and the like, which will be the recipient of a particular treatment. Generally, the terms "subject" and "patient" are used interchangeably herein when referring to a human subject.

"pharmaceutically acceptable salt" refers to a salt of a compound that is pharmaceutically acceptable, which possesses the desired pharmacological activity of the parent compound.

By "pharmaceutically acceptable excipient, carrier or adjuvant" is meant an excipient, carrier or adjuvant that is capable of being administered to a subject with at least one antibody of the invention, does not destroy its pharmacological activity, and is non-toxic when administered at a dose sufficient to deliver a therapeutic amount of the compound.

By "pharmaceutically acceptable carrier" is meant a diluent, adjuvant, excipient, or vehicle with which at least one antibody of the invention is administered.

The term "therapeutically effective amount" refers to the amount of an antibody, polypeptide, polynucleotide, small organic molecule, or other drug that is effective to "treat" a disease or disorder in a subject or mammal. For cancer, a therapeutically effective amount of the drug is capable of reducing the number of cancer cells; reducing the size of the tumor; inhibiting or preventing cancer cell infiltration into peripheral organs, including, for example, cancer spread into soft tissue and bone; inhibit and prevent tumor metastasis; inhibiting or preventing tumor growth; alleviating to some extent one or more symptoms associated with cancer; reducing morbidity and mortality; the life quality is improved; reducing the tumorigenicity, tumorigenicity rate or tumorigenicity ability of the tumor; reducing the number or frequency of cancer stem cells in a tumor; differentiating the tumorigenic cells into a non-tumorigenic state; or a combination of these effects. In the case of drugs that prevent growth and/or kill existing cancer cells, this may be referred to as cytostatic and/or cytotoxic.

Terms such as "treating" or "treatment" or "treat" or "alleviate" or "alleviative" refer to: 1) therapeutic measures that cure, alleviate, reduce the symptoms of and/or halt the progression of a diagnosed pathological state or condition, and (2) prophylactic or preventative measures that prevent and/or slow the development of the targeted pathological state or condition. Thus, subjects in need of treatment include subjects already having the disorder, subjects susceptible to the disorder, and subjects in whom the disorder is to be prevented. In certain embodiments, a subject has been successfully "treated" for cancer according to the methods of the present invention if the patient exhibits one or more of the following phenomena: a reduction in the number of cancer cells or complete disappearance; reduction in tumor size; the inhibition or disappearance of cancer cell infiltration into peripheral organs including, for example, the spread of cancer into soft tissues and bone; the tumor metastasis is inhibited or disappeared; the growth of the tumor is inhibited or disappeared; alleviating one or more symptoms associated with a particular cancer; decreased morbidity and mortality; the life quality is improved; a reduction in the tumorigenicity, tumorigenicity rate or tumorigenic capacity of the tumor; a decrease in the number or frequency of cancer stem cells in the tumor; differentiation of tumorigenic cells to a non-tumorigenic state; or some combination of these effects.

"Polynucleotide" or "nucleic acid" are used interchangeably herein to refer to a polymer of nucleotides of any length, including DNA and RNA. The nucleotide may be a deoxyribonucleotide, a ribonucleotide, a modified nucleotide or base and/or the likeOr any substrate capable of being introduced into the polymer by a DNA polymerase or an RNA polymerase. Polynucleotides may include modified nucleotides, such as methylated nucleotides and analogs thereof. Modifications to the nucleotide structure, if present, may be made before or after assembly of the polymer. The non-nucleotide component may interrupt the nucleotide sequence. The polynucleotide may be further modified after polymerization, for example by coupling with a labeling component. Other types of modifications include, for example: "end-capping"; replacing one or more naturally occurring nucleotides with an analog; internucleotide modifications such as those having an uncharged bond (e.g., methylphosphonate, phosphotriester, phosphoamidate, carbamate, etc.) and a charged bond (e.g., phosphorothioate, phosphorodithioate, etc.), those having a pendant moiety (e.g., protein (e.g., nuclease, toxin, antibody, signal peptide, poly-L-lysine, etc.), those having an intercalator (e.g., acridine, psoralen, etc.), those having a chelator (e.g., metal, radioactive metal, boron, oxidative metal, etc.), those having an alkylator, those having a modified bond (e.g., alpha anomeric nucleic acid, etc.); and unmodified forms of the polynucleotides. In addition, any hydroxyl group typically present in a sugar may be replaced, for example, by a phosphonate group, a phosphate group, may be protected with standard protecting groups, or may be activated to make additional bonds to other nucleotides, or may be coupled to a solid support. The 5 'and 3' OH groups may be substituted or phosphorylated with amines or organic end-capping moieties containing 1 to 20 carbon atoms. Other hydroxyl groups can also be derivatized as standard protecting groups. Polynucleotides may also comprise analogous forms of ribose or deoxyribose known in the art, including, for example, 2 '-O-methyl ribose, 2' -O-allyl ribose, 2 '-fluoro ribose, 2' -azidoribose, carbocyclic sugar analogs, alpha anomeric sugars, epimeric sugars (e.g., arabinose, xylose, or lyxose), pyranoses, furanoses, sedoheptulose, acyclic analogs, and non-base nucleoside analogs (e.g., methyl riboside). More than one phosphodiester bond may be replaced with an alternative linking group. These substitutes Substituted linking groups include, but are not limited to, the following embodiments: wherein P (O) S ("thioester"), P (S) S ("dithioester"), "(O) NR" is used₂("amidates"), P (O) R, P (O) OR', CO OR CH₂("methylal") substituted phosphate wherein R or R' are each independently H or substituted or unsubstituted alkyl (1-20C) and optionally contain an ether (- -O- -) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or aralkyl (araldyl). It is not necessary that all linkages be identical in a single polynucleotide. The above description applies to all polynucleotides described herein, including RNA and DNA.

The "variable region" of an antibody may refer to the variable region of an antibody light chain or the variable region of an antibody heavy chain, alone or in combination. The variable regions of the heavy and light chains each consist of 4 Framework Regions (FRs) connected by three Complementarity Determining Regions (CDRs), also known as hypervariable regions. FRs bring together the CDRs in each chain in close proximity, which, together with the CDRs of the other chain, contribute to the formation of the antigen-binding site of the antibody. There are at least two techniques for determining CDRs: (1) methods based on sequence variability across species (i.e., Kabat et al, Sequences of proteins of Immunological Interest, (5 th edition, 1991, National Institutes of Health, Bethesda Md.); and (2) methods based on crystallographic studies of antigen-antibody complexes (Al-lazikani et Al, (1997) J.Molec.biol.273: 927-948). Furthermore, a combination of these two methods is sometimes used in the art to determine CDRs.

The term "vector" refers to a construct capable of delivering, preferably expressing, one or more genes or sequences of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids, or phage vectors, DNA or RNA expression vectors linked to cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells (e.g., producer cells).

The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acids of any length. The polymer may be chain-like or branched, may comprise modified amino acids, and may be interrupted by non-amino acids. The term also encompasses amino acid polymers modified by natural modification or intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification (e.g., coupling to a labeling component). Also included within the present definition are, for example, polypeptides containing one or more amino acid analogs (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art. It will be appreciated that, because the polypeptides of the invention are antibody-based, in certain embodiments, the polypeptides may occur as individual chains or linked chains.

The term "identical" or percent "identity" in reference to two or more nucleic acids or polypeptides refers to: when comparing and aligning (introducing gaps, if necessary) to obtain maximum correspondence, two or more sequences or subsequences are the same or have a specified ratio of nucleotides or amino acid residues that are the same, and no conservative amino acid substitutions are considered part of the sequence identity. Percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that can be used to obtain an alignment of amino acid or nucleotide sequences are known in the art. One such non-limiting example of a sequence alignment algorithm is described in Karlin et al, 1990, proc.natl.acad.sci, 87: 2264-2268 and described in Karlin et al, 1993, Proc. Natl. Acad. Sci, 90: 5873-5877, and integrated into the NBLAST and XBLAST programs (Altschul et al, 1991, Nucleic Acids Res., 25: 3389-3402). In certain embodiments, the compounds described in Altschul et al, 1997, Nucleic Acids Res., 25: 3389 gapped BLAST (gapped BLAST) as described in 3402-. BLAST-2, WU-BLAST-2(Altschul et al, 1996, Methods in Enzymology 266: 460-. In certain embodiments, the percent identity between two nucleotide sequences is determined using the GAP program in GCG software (e.g., using nwsgapdna. cmp matrix with GAP weights of 40, 50, 60, 70, or 90 and length weights of 1, 2, 3, 4, 5, or 6). In certain alternative embodiments, the GAP program in the GCG software package incorporating the algorithm of Needleman and Wnsch (J.mol.biol. (48): 444-453(1970)) can be used to determine percent identity between two amino acid sequences (e.g., using a Blossum 62 matrix or a PAM250 matrix with GAP weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5). Alternatively, in certain embodiments, the percentage identity between nucleotide or amino acid sequences is determined using the Myers and Miller algorithm (CABIOS, 4: 11-17 (1989)). For example, percent identity can be determined using the ALIGN program (version 2.0) using PAM120 with a table of residues with a gap length penalty of 12 and a gap penalty of 4. One skilled in the art can determine the appropriate parameters to obtain the maximum alignment for a particular alignment software. In certain embodiments, default parameters of the alignment software are used. In certain embodiments, the percent identity "X" of an amino acid of a first amino acid sequence relative to an amino acid of a second sequence is calculated as 100X (Y/Z), where Y is the number of amino acid residues scored as an identical match in an alignment of the first and second sequences (an alignment made by visual inspection or a particular sequence alignment program), and Z is the total number of residues in the second sequence. If the length of the first sequence is longer than the second sequence, the percent identity of the first sequence relative to the second sequence will be longer than the percent identity of the second sequence relative to the first sequence.

As a non-limiting example, in certain embodiments, whether any particular polynucleotide has a certain percentage of sequence identity (e.g., at least 80% identity, at least 85% identity, at least 90% identity, and in some embodiments at least 95%, 96%, 97%, 98%, or 99% identity) with respect to a reference sequence can be determined using the Bestfit program (Wisconsin sequence analysis Package, version 8 (for the Unix platform), Genetics Computer Group, university research Park, 575 Science Drive, Madison, WI 53711). Bestfit uses Smith and Waterman, Advances in Applied Mathematics 2: 482489 (1981) to find the best homologous fragment between the two sequences. When using Bestfit or any other sequence alignment program to determine whether a particular sequence is, for example, 95% identical to a reference sequence of the invention, the parameters are set such that: the percentage of identity is calculated based on the full length of the reference nucleotide sequence and allows gaps in homology of up to 5% of the total number of nucleotides in the reference sequence.

In some embodiments, two nucleic acids or polypeptides of the invention are substantially identical, i.e., at least 70%, at least 75%, preferably at least 80%, more preferably at least 85%, further preferably at least 90%, and in some embodiments at least 95%, 96%, 97%, 98%, 99% nucleotide or amino acid residue identity thereof, as measured by sequence comparison algorithms or visual inspection, when compared and aligned for maximum correspondence. Preferably, identity exists over a region of the sequence that is at least about 10 residues, preferably about 20 residues, more preferably about 40-60 residues or any integer value therebetween in length, preferably over a region that is longer than 60-80 residues, more preferably over a region that is at least about 90-100 residues in length, and most preferably over a sequence that is substantially identical over the entire length of the sequence being compared (e.g., the coding region for a nucleotide sequence).

A "conservative amino acid substitution" is a substitution in which an amino acid residue is replaced with another amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine tryptophan, histidine). For example, substitution of tyrosine with phenylalanine is a conservative substitution. Preferably, conservative substitutions in the polypeptide and antibody sequences of the present invention do not abolish binding of the polypeptide or antibody comprising the amino acid sequence to an antigen (i.e., the human frizzled receptor or receptors to which the polypeptide or antibody binds). Methods for identifying conservative substitutions of nucleotides and amino acids that do not eliminate antigen binding are well known in the art (see, e.g., Brummell et al, biochem.32: 1180-1187 (1993); Kobayashi et al, Protein Eng.12 (10): 879-884 (1999); and Burks et al, Proc. Natl. Acad. Sci USA 94: 412-417 (1997)).

As used in this disclosure and the claims, the singular forms "a", "an" and "the" include the plural forms unless the context clearly dictates otherwise.

It should be understood that in any event where the term "comprising" is used herein to describe an embodiment, other similar embodiments described by "consisting of …" and/or "consisting essentially of …" are also provided.

The term "and/or" as used herein in phrases such as "a and/or B" is intended to include "a and B", "a or B", "a" and "B". Likewise, the term "and/or" as used in phrases such as "A, B and/or C" is intended to encompass each of the following embodiments: A. b and C; A. b or C; a or C; a or B; b or C; a and C; a and B; b and C; only A; only B; and only C.

"high stringency conditions" can be determined by: (1) washing with low ionic strength and high temperature, e.g., 0.015M sodium chloride/0.0015M sodium citrate/0.1% sodium lauryl sulfate, 50 ℃; (2) a denaturing agent (e.g., formamide) such as 50% (v/v) formamide containing 0.1% bovine serum albumin/0.1% Ficoll (Ficoll)/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer (ph6.5) containing 750mM sodium chloride, 75mM sodium citrate is used during hybridization, 42 ℃; or (3) at 42 ℃ with 50% formamide, 5 XSSC (0.75MNaCl, 0.075M sodium citrate), 50mM sodium phosphate (pH6.8), 0.1% sodium pyrophosphate, 5 XDenhardt's solution, sonicated salmon sperm DNA (50. mu.g/ml), 0.1% SDS and 10% dextran sulfate, and at 42 ℃ washing, at 55 ℃ in 0.2 XSSC (sodium chloride/sodium citrate) and 50% formamide, followed by 55 ℃ high stringency washing consisting of 0.1 XSSC with EDTA.

FZD binding agents

The present invention provides agents that specifically bind to one or more human frizzled receptors (FZD). These agents are referred to herein as "FZD-binding agents". In certain embodiments, the agent specifically binds two, three, four, five, six, seven, eight, nine, or ten frizzled receptors. The human frizzled receptor to which the agent binds may be selected from the group consisting of FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, and FZD 10. In certain embodiments, the one or more human frizzled receptors comprise FZD1, FZD2, FZD5, FZD7, and/or FZD 8. In certain embodiments, the one or more human frizzled receptors comprise FZD 7. In certain embodiments, the one or more human frizzled receptors comprise FZD5 and/or FZD 8. In certain embodiments, the agent specifically binds FZD1, FZD2, FZD5, FZD7, and FZD 8. The full-length amino acid (aa) and nucleotide (nt) sequences of FZD 1-FZD 10 are known in the art and are also provided herein as SEQ ID NO: 26(FZD1 aa), SEQ ID NO: 30(FZD2 aa), SEQ ID NO: 34(FZD3 aa), SEQ ID NO: 38(FZD4 aa), SEQ ID NO: 42(FZD5 aa), SEQ ID NO: 46(FZD6aa), SEQ ID NO: 50(FZD7 aa), SEQ ID NO: 54(FZD8 aa), SEQ ID NO: 58(FZD9 aa), SEQ ID NO: 62(FZD10 aa), SEQ ID NO: 29(FZD1 nt), SEQ ID NO: 33(FZD2 nt), seq id NO: 37(FZD3 nt), SEQ ID NO: 41(FZD4 nt), SEQ ID NO: 45(FZD5 nt), SEQ ID NO: 49(FZD6 nt), SEQ ID NO: 53(FZD7 nt), SEQ ID NO: 57(FZD8 nt), SEQ ID NO: 61(FZD9 nt) and SEQ ID NO: 65(FZD10 nt).

In certain embodiments, an antibody or other polypeptide or agent described herein specifically binds FZD 7. In certain embodiments, the antibody, polypeptide, or agent may also specifically bind to or cross-react with one or more other human frizzled receptors.

In certain embodiments, an antibody or other polypeptide or agent described herein specifically binds FZD 5. In certain embodiments, the antibody, polypeptide, or agent may also specifically bind to or cross-react with one or more other human frizzled receptors.

In certain embodiments, the agent specifically binds two or more human frizzled receptors. In certain embodiments, the two or more human frizzled receptors are selected from the group consisting of FZD2, FZD5, FZD7, and FZD 8. In certain embodiments, the two or more human frizzled receptors comprise FZD1 and a second human frizzled receptor selected from the group consisting of FZD2, FZD5, FZD7, and FZD 8. In certain embodiments, the two or more human frizzled receptors comprise FZD2 and a second frizzled receptor selected from the group consisting of FZD1, FZD5, FZD7, and FZD 8. In certain embodiments, the two or more human frizzled receptors comprise FZD5 and a second frizzled receptor selected from the group consisting of FZD1, FZD2, FZD7, and FZD 8. In certain embodiments, the two or more human frizzled receptors comprise FZD5 and FZD 8. In certain embodiments, the two or more human frizzled receptors comprise FZD7 and a second human frizzled receptor selected from the group consisting of FZD1, FZD2, FZD5, and FZD 8.

In certain embodiments, the agent specifically binds three or more human frizzled receptors. In certain embodiments, the three or more human frizzled receptors comprise three or more frizzled receptors selected from the group consisting of FZD1, FZD2, FZD5, FZD7, and FZD 8. In certain embodiments, the agent also specifically binds to one or more other human frizzled receptors.

In certain embodiments, the agent or antibody specifically binds to the extracellular domain (ECD) in the one or more human frizzled receptors to which it binds. The sequence of the extracellular domain of each human frizzled receptor is known in the art and is also provided as SEQ ID NO: 27(FZD1 ECD), SEQ ID NO: 31(FZD2 ECD), SEQ ID NO: 35(FZD3 ECD), SEQ ID NO: 39(FZD4 ECD), SEQ ID NO: 43(FZD5ECD), SEQ ID NO: 47(FZD6 ECD), SEQ ID NO: 51(FZD7 ECD), SEQ ID NO: 55(FZD8ECD), SEQ ID NO: 59(FZD9 ECD) and SEQ ID NO: 63(FZD10 ECD).

In certain embodiments, the agent or antibody specifically binds to a Fri domain (Fri), also known as a cysteine-rich domain (CRD), in the human frizzled receptor to which it binds. The sequence of the Fri domain of each human frizzled receptor is known in the art and is also provided as SEQ ID NO: 28(FZD1 FRI), SEQ id no: 32(FZD2 FRI), SEQ ID NO: 36(FZD3 FRI), SEQ ID NO: 40(FZD4 FRI), SEQ ID NO: 44(FZD5 FRI), SEQ ID NO: 48(FZD6 FRI), SEQ ID NO: 52(FZD7 FRI), SEQ ID NO: 56(FZD8 FRI), SEQ ID NO: 60(FZD9 FRI) and SEQ ID NO: 64(FZD10 FRI).

In certain embodiments, a single antigen-binding site of an FZD-binding antibody or polypeptide described herein binds or is capable of binding one, two, three, four, or five (or more) human frizzled receptors. In certain embodiments, the single antigen-binding site of the FZD-binding antibody or polypeptide is capable of specifically binding one, two, three, four, or five human frizzled receptors selected from the group consisting of FZD1, FZD2, FZD5, FZD7, and FZD 8. In certain embodiments, a single binding site of the antibody or polypeptide specifically binds at least FZD5 and FZD 8.

In certain embodiments, the FZD-binding agent or antibody binds to one or more (e.g., two or more, three or more, or four or more) human frizzled receptors and its dissociation constant (K)_D) About 1 μ M or less, about 100nM or less, about 40nM or less, about 20nM or less, or about 10nM or less. For example, in certain embodiments, the compounds described herein arefZD-binding agents or antibodies that bind to one or more fZDs, and Ks that bind to these fZDs_DAbout 100nM or less, about 20nM or less, or about 10nM or less. In certain embodiments, the dissociation constant for binding of a FZD-binding agent or antibody to each of one or more (e.g., 1, 2, 3, 4, or 5) FZD1, FZD2, FZD5, FZD7, and FZD8 is about 40nM or less. In certain embodiments, the FZD-binding agent or antibody binds each of FZD1, FZD2, FZD5, FZD7, and FZD8 with a dissociation constant of about 10nM or less. In certain embodiments, the FZD-binding agent or antibody binds to each of FZD1, FZD2, FZD5, FZD7, and FZD8 with a dissociation constant of about 10nM or less. In certain embodiments, the dissociation constant for binding of the agent or antibody to a particular FZD is determined by using FZD-Fc fusion proteins immobilized on a Biacore chip, said fusion proteins containing FZD extracellular domains or Fri domains.

In certain embodiments, the FZD-binding agent or antibody binds to one or more (e.g., two or more, three or more, or four or more) human frizzled receptors and the EC thereof₅₀About 1 μ M or less, about 100nM or less, about 40nM or less, about 20nM or less, about 10nM or less, or about 1nM or less. For example, in certain embodiments, FZD-binding agents or antibodies described herein that bind to more than one FZD are relative to the EC of the FZD₅₀About 40nM or less, about 20nM or less, or about 10nM or less. In certain embodiments, the FZD-binding agent or antibody has an EC relative to one or more (e.g., 1, 2, 3, 4, or 5) of FZD1, FZD2, FZD5, FZD7, and FZD8₅₀About 20nM or less. In certain embodiments, the FZD-binding agent or antibody has an EC50 of about 10nM or less relative to one or more (e.g., 1, 2, 3, 4, or 5) of FZD1, FZD2, FZD5, FZD7, and FZD 8. In certain embodiments, the FZD-binding agent or antibody is directed to EC that binds FZD5 and/or FZD8₅₀About 40nM or less or 20nM or less.

In certain embodiments, the FZD-binding agent (e.g., antibody) binds to an epitope selected from the group consisting of: the epitope is similar to a peptide having a sequence comprising SEQ ID NO: 10 and a light chain variable region comprising SEQ ID NO: 12 or SEQ ID NO: 14 (e.g., 18R5 or 18R8IgG antibody) are identical or overlapping in epitope. In certain embodiments, the FZD-binding agent or antibody binds to one of the following epitopes: the epitope is similar to a peptide having a sequence comprising SEQ ID NO: 11 and a light chain comprising SEQ ID NO: 13 or SEQ ID NO: 15, the epitopes of the antibodies of the light chains are identical or overlapping. In certain embodiments, the FZD-binding agent binds to one of the following epitopes: the epitope is similar to a peptide having a sequence comprising SEQ ID NO: 85 and a light chain variable region comprising SEQ ID NO: 86 (e.g., the 44R24 IgG antibody) are identical or overlapping in epitope.

In certain embodiments, the FZD-binding agent competes for specific binding to the human frizzled receptor with an antibody in a competitive binding assay, wherein the antibody has an amino acid sequence comprising SEQ ID NO: 10 and a light chain variable region comprising seq id NO: 12 or SEQ ID NO: 14, light chain variable region. In certain embodiments, the FZD-binding agent binds to a polypeptide having an amino acid sequence comprising SEQ ID NO: 11 and a light chain comprising SEQ ID NO: 13 or SEQ ID NO: 15 to obtain specific binding to the human frizzled receptor. In certain embodiments, the antibody that competes with the agent for specific binding to the human frizzled receptor is an 18R5IgG antibody. In certain alternative embodiments, the antibody is an 18R8 IgG antibody.

In certain embodiments, the FZD-binding agent competes for specific binding to the human frizzled receptor with an antibody in a competitive binding assay, wherein the antibody has an amino acid sequence comprising SEQ ID NO: 85 and a light chain variable region comprising seq id NO: 86 light chain variable region.

In certain embodiments, the FZD-binding agent or antibody binds to at least a portion of the region of a human frizzled receptor designated by the present inventors as the Biological Binding Site (BBS) (fig. 9, example 6). In human FZD8(SEQ ID NO: 54), the BBS consists of the following (a), (b) and (c): (a) conformational epitopes consisting of amino acids 72(F), 74-75 (PL), 78(I), 92(Y), 121-122 (LM) and 129-132 (WPDR (SEQ ID NO: 70)) (the "cleft" of the BBS shown in FIGS. 8 and 9); (b) the region of FZD8 (the "upper edge" of the BBS shown in FIGS. 8 and 9) consisting of the sequence GLEVHQ (SEQ ID NO: 25); and (c) a region of FZD8 consisting of the sequence YGFA (SEQ ID NO: 74) ("lower border" of BBS shown in FIGS. 8 and 9). The corresponding residues of the BBS located on FZD 1-FZD 7, FZD9 and FZD10 are shown in table 1 below and fig. 8. In certain embodiments, an agent that blocks the binding of a ligand (e.g., Wnt) to FZD inhibits the binding of the ligand to the BBS. It will be appreciated that, in certain embodiments, an agent that binds to at least a portion of the BBS also binds to one or more other regions on the human frizzled receptor (i.e., outside the BBS). In other words, in certain embodiments, the epitope to which the FZD-binding agent or antibody binds is a region in the FZD receptor extracellular domain that overlaps, but is not completely contained in, the BBS. In certain alternative embodiments, the epitope to which the FZD-binding agent or antibody binds is completely contained within the BBS (i.e., the BBS contains the entire epitope to which the FZD-binding antibody or other agent binds).

Table 1: biological binding site of FZD receptor (BBS).

Without being bound by theory, it is believed that the BBS contains a potential ligand binding site, e.g., a site that binds Wnt. On FZD8, the potential ligand binding sites comprise conformational epitopes (the "cleft" of the BBS shown in FIGS. 8 and 9) consisting of amino acids 72(F), 74-75 (PL), 78(I), 92(Y), 121-122 (LM) and 129-132 (WPDR (SEQ ID NO: 70)). The corresponding residues at FZD 1-FZD 7, FZD9 and FZD10 of the potential ligand binding sites are shown in the sequence alignment of figure 8. In certain embodiments, an agent that blocks binding of a ligand (e.g., Wnt) to the BBS inhibits binding of the ligand to the conformational epitope. It will be appreciated that, in certain embodiments, an agent that binds to at least a portion of the ligand binding site also binds to other regions on the human frizzled receptor (e.g., regions outside the BBS). In certain alternative embodiments, the agent does not bind to any portion of the FZD other than the conformational epitope.

In certain embodiments, if the human frizzled receptor is FZD8, the agent binds to at least a portion of sequence QDEAGLEVHQFWPL (SEQ ID NO: 67) in the human frizzled receptor, or if the human frizzled receptor is FZD 1-FZD 7, FZD9, or FZD10, the agent binds to at least a portion of the corresponding sequence. This region located on the FZD contains the "upper edge" of the BBS shown in fig. 8 and 9. The sequences corresponding to epitope QDEAGLEVHQFWPL (SEQ ID NO: 67) of FZD8 in the various frizzled receptors are shown in Table 2 below and are also evident from the alignment of FIG. 8. In certain embodiments, the agent specifically binds to a human frizzled receptor selected from the group consisting of FZD1, FZD2, FZD5, FZD7, and FZD8, and the agent binds to at least a portion of sequence Q (DE/ED) aglevqf (Y/W) PL (SEQ ID NO: 24) in the human frizzled receptor. In certain embodiments, the agent specifically binds to at least a portion of the sequence aglevqf (SEQ ID NO: 68) in the human frizzled receptors FZD1, FZD2, FZD5, FZD7, and/or FZD 8. In certain embodiments, the agent binds to at least a portion of the sequence of FZD3, FZD4, FZD6, FZD9, and/or FZD10 that corresponds to the sequence aglevqf of FZD8 (SEQ ID NO: 68). In certain embodiments, the agent specifically binds to at least a portion of the sequence GLEVHQ (SEQ ID NO: 25) in the human frizzled receptors FZD1, FZD2, FZD5, FZD7 and/or FZD 8. This sequence is the "upper edge" of the BBS shown in fig. 9. In certain embodiments, the agent binds to at least a portion of the sequence of FZD3, FZD4, FZD6, FZD9, and/or FZD10 that corresponds to the sequence GLEVHQ in FZD8 (SEQ ID NO: 25). The sequence corresponding to the sequence GLEVHQ of FZD8 (SEQ ID NO: 25) is underlined in the second and third columns of Table 2 below and is evident from the alignment of the sequences in FIG. 8. In certain embodiments, the amino acid sequence of FZD8 that is identical to SEQ ID NO: 67 or SEQ ID NO: 68 or an agent that binds to a corresponding epitope in another FZD inhibits binding of a ligand (e.g., Wnt) to the FZD (e.g., to the BBS of the FZD). In certain embodiments, an agent that binds to a region indicated above may also bind to additional other (i.e., outside of the regions indicated above) regions on the human frizzled receptor.

Table 2: corresponding regions on the human frizzled receptor.

(a) The method comprises the following steps Underlined are sequences corresponding to aa 66-71 GLEVHQ (SEQ ID NO: 25) of FZD8(SEQ ID NO: 54).

(b) The method comprises the following steps Underlined are sequences corresponding to aa 125 to 128YGFA (SEQ ID NO: 74) of FZD8(SEQ ID NO: 54).

In certain embodiments, the FZD-binding agent (when the human frizzled receptor is FZD 8) binds to at least a portion of a region comprising the "lower border" of the BBS consisting of the sequence QYGFA (SEQ ID NO: 66) or (when the human frizzled receptor is FZD 1-FZD 7, FZD9, or FZD 10) to at least a portion of the corresponding sequence. The sequences corresponding to the region QYGFA (SEQ ID NO: 66) in the various frizzled receptors are shown in FIG. 8 and Table 2 above. In certain embodiments, the FZD-binding agent (when the human frizzled receptor is FZD 8) binds to at least a portion of a BBS "lower border" region consisting of sequence YGFA (SEQ ID NO: 74) or (when the human frizzled receptor is FZD 1-FZD 7, FZD9, or FZD 10) of the corresponding sequence. The sequences corresponding to region YGFA (SEQ ID NO: 74) in the various frizzled receptors are shown in FIG. 8 and underlined in the fourth column of Table 2 above. In certain embodiments, the amino acid sequence of FZD8 that is identical to SEQ ID NO: 66 or SEQ ID NO: 74 or its corresponding sequence in another FZD inhibits binding of a ligand (e.g., Wnt) to a FZD (e.g., to the BBS of a FZD). In certain embodiments, an agent that binds to this region can also bind to one or more amino acid residues at other positions on the human frizzled receptor (i.e., outside of the region).

In certain embodiments, the FZD-binding agent binds to at least a portion of a region that forms the "upper margin" of the BBS and at least a portion of a region that forms the "lower margin" of the BBS. In certain embodiments, an FZD-binding agent that binds to at least a portion of Q (DE/ED) aglevqf (Y/W) PL (SEQ ID NO: 24) in FZD1, FZD2, FZD5, FZD7, and/or FZD8, QDEAGLEVHQFWPL (SEQ ID NO: 67) in FZD8, aglevqff (SEQ ID NO: 68) in FZD8, and/or GLEVHQ (SEQ ID NO: 25) in FZD8 and/or a sequence in a different human frizzled receptor corresponding to any of these sequences (as defined in table 2 above) also binds to at least a portion of QYGFA (SEQ ID NO: 66) in FZD8 or YGFA (SEQ ID NO: 74) in f 8 and/or FZD 1-FZD 7, FZD9, or FZD10 corresponding to one of these sequences (as defined in table 2 above). In certain embodiments, the FZD-binding agent binds to at least a portion of the sequence GLEVHQ in FZD8 (SEQ ID NO: 25) and at least a portion of the sequence YGFA in FZD8 (SEQ ID NO: 74). In certain embodiments, the FZD-binding agent binds to at least a portion of the following regions: the regions are regions of FZD 1-FZD 7, FZD9 and FZD10 corresponding to the sequence GLEVHQ in FZD8 (SEQ ID NO: 25), and regions of FZD 1-FZD 7, FZD9 and FZD10 corresponding to the sequence YGFA in FZD8 (SEQ ID NO: 74). In certain embodiments, an FZD-binding agent that binds to an indicated sequence also binds to one or more sequences elsewhere in the human frizzled receptor to which it binds. In other words, in certain embodiments, the epitope to which the FZD-binding agent or antibody binds is a region of the FZD extracellular domain that only partially overlaps with the sequences indicated above. In certain alternative embodiments, the entire epitope bound by the FZD-binding agent is completely contained within the sequences indicated above.

In certain embodiments, the agent is a polypeptide. In certain embodiments, the agent or polypeptide is an antibody. In certain embodiments, the antibody is an IgG1 antibody or an IgG2 antibody. In certain embodiments, the antibody is a monoclonal antibody. In certain embodiments, the antibody is a human or humanized antibody. In certain embodiments, the antibody is an antibody fragment.

Specific binding of an antibody or other agent of the invention can be determined by any method known in the art. Immunoassays that can be used include, but are not limited to: competitive or non-competitive assay systems using techniques such as BIAcore analysis, FACS analysis, immunofluorescence, immunocytochemistry, western blotting, radioimmunoassay, ELISA, "sandwich" immunoassays, immunoprecipitation assays, precipitation reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement fixation assays, immunoradiometric assays, fluorescent immunoassays, and protein a immunoassays. Such assays are well known and conventional in the art (see, e.g., Ausubel et al, eds., 1994, Current Protocols in Molecular Biology, Vol.1, John Wiley & Sons, Inc., New York, which is incorporated herein by reference in its entirety).

For example, ELISA can be used to determine the specific binding of an antibody to a human frizzled receptor. The ELISA assay included: preparing an antigen, coating the wells of a 96-well microtiter plate with the antigen, adding FZD-binding antibodies or other FZD-binding agents conjugated to a detectable compound such as an enzyme substrate (e.g., horseradish peroxidase or alkaline phosphatase) to the wells, incubating for a period of time, and detecting the presence of the antigen. In some embodiments, the FZD-binding antibody or agent is not conjugated to a detectable compound, but a second conjugated antibody that recognizes the FZD-binding antibody or agent is added to the well. In some embodiments, instead of coating the wells with an antigen, the wells are coated with a FZD-binding antibody or agent, and a second antibody conjugated to a detectable compound is added after the antigen is added to the coated wells. Those skilled in the art know that: parameters that can be modified to increase the signal detected, as well as other ELISA variations known in the art (see, e.g., Ausubel et al eds., 1994, Current Protocols in molecular biology, Vol.1, John Wiley & Sons, Inc., New York, 11.2.1).

The affinity of the antibody or other agent for binding to human frizzled receptors and the off-rate of antibody-antigen interaction can be determined by competitive binding assays. An example of a competitive binding assay is a radioimmunoassay, which comprises: the labeled antigen (e.g., a labeled antigen) is added in the presence of a gradual addition of unlabeled antigen ³H or¹²⁵I label) or a fragment or variant thereof is incubated with the antibody of interest, followed by detection of the antibody bound to the labeled antigen. The affinity of the antibody to the frizzled receptor and the rate of detachment of the binding can be determined from the data by scatchard plot (scatchard plot) analysis. In some embodiments, BIAcore kinetic analysis is used to determine the rate of binding (on-rate) and the rate of detachment of an antibody or agent that binds to one or more human frizzled receptors. BIAcore kinetic analysis involves analysis of antibody binding and dissociation from a chip with FZD antibody immobilized on the surface.

In certain embodiments, the agent (e.g., antibody) is an antagonist of at least one human frizzled receptor (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 FZD) to which the agent binds. In certain embodiments, the agent is capable of forming at least about 10%, at least about 20%, at least about 30%, at least about 50%, at least about 75%, at least about 90%, or about 100% inhibition of the activity of one or more of the human frizzled antibodies to which the agent binds.

In certain embodiments, the FZD-binding agent inhibits binding of a ligand to at least one human frizzled receptor. In certain embodiments, the FZD-binding agent inhibits binding of a ligand to the biological binding site of a human frizzled receptor (BBS). In certain embodiments, the ligand is a human Wnt protein. 19 human Wnt proteins have been identified: WNT1, WNT2, WNT2B/13, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A (formerly WNT14), WNT9B (formerly WNT15), WNT10A, WNT10B, WNT11, and WNT 16. In certain embodiments, the agent inhibits the binding of WNT3A to FZD 8. In certain embodiments, the FZD-binding agent inhibits the binding of a particular ligand to a particular human protein frizzled receptor by at least about 10%, at least about 25%, at least about 50%, at least about 75%, at least about 90%, or at least about 95%. In certain embodiments, an agent that inhibits binding of a ligand (e.g., Wnt) to a FZD also inhibits Wnt signaling (e.g., inhibits canonical Wnt signaling).

In certain embodiments, the FZD-binding agent inhibits Wnt signaling. It will be appreciated that FZD-binding agents that inhibit Wnt signaling may, in certain embodiments, inhibit signaling by one or more, but not necessarily all, of the Wnt(s). In certain alternative embodiments, signaling by all human Wnt may be inhibited. In certain embodiments, signaling by one or more WNTs selected from the group consisting of Wnt1, Wnt2, Wnt2B/13, Wnt3, Wnt3A, Wnt4, Wnt5A, Wnt5B, Wnt6, Wnt7A, Wnt7B, Wnt8A, Wnt8B, Wnt9A (formerly Wnt14), Wnt9B (formerly Wnt15), Wnt10A, Wnt10B, Wnt11, and Wnt16 is inhibited. In certain embodiments, the Wnt signaling that is inhibited is signaling by Wnt1, Wnt2, Wnt3, Wnt3A, Wnt7a, Wnt7b, and/or Wnt 10B. In certain embodiments, the agent inhibits signaling by WNT1, WNT3A, WNT7b, and WNT10B (at least). In particular embodiments, the agent inhibits signaling by WNT3A (at least). In certain embodiments, the inhibitory effect of the FZD-binding agent on signaling by a Wnt decreases the level of signaling by the Wnt by at least about 10%, at least about 25%, at least about 50%, at least about 75%, at least about 90%, or at least about 95%. In certain embodiments, the Wnt signaling that is inhibited is canonical Wnt signaling.

In vivo and in vitro assays for determining whether a FZD-binding agent (or a candidate FZD-binding agent) inhibits Wnt signaling are known in the art. For example, canonical Wnt signaling levels can be measured in vitro using a cell-based luciferase reporter assay that utilizes a TCF/Luc reporter vector containing multiple copies of the TCF binding domain upstream of the firefly luciferase reporter (Gazit et al, 1999, Oncogene 18; 5959-66). For Wnt signaling in the presence of one or more Wnt (e.g., Wnt expressed by transfected cells or Wnt provided by Wnt conditioned media), its level in the presence of a FZD-binding agent is compared to its level in the absence of a FZD-binding agent. Non-limiting specific examples of the use of such luciferase reporter assays to assess inhibition of canonical Wnt signaling are provided in examples 3 and 11 below. In addition to TCF/Luc reporter gene assays, the effect of an FZD-binding agent (or candidate agent) on canonical Wnt signaling can be measured by measuring the effect of the agent on the expression level of a gene regulated by β -catenin (β -connexin), e.g., c-myc (He et al, Science, 281: 1509-12(1998)), cyclin D1(Tetsu et al, Nature, 398: 422-6(1999)) and/or fibronectin (Gradl et al, mol.cell biol., 19: 5576-87(1999)), in vitro or in vivo. In certain embodiments, the effect of an agent on Wnt signaling can also be assessed by measuring the effect of the agent on the phosphorylation status of disheveled protein-1, disheveled protein-2, disheveled protein-3, LRP5, LRP6, and/or β -catenin. In additional embodiments, the effect of the FZD-binding agent on Wnt signaling is determined by assessing the effect of the FZD-binding agent on the expression level of one or more genes in the Wnt signature.

In certain embodiments, the FZD-binding agent has one or more of the following effects: inhibiting tumor cell proliferation, reducing tumor tumorigenicity by reducing the frequency of cancer stem cells in a tumor, inhibiting tumor growth, increasing survival, triggering cell death of tumor cells, differentiating tumorigenic cells into a non-tumorigenic state, or preventing metastasis of tumor cells.

In certain embodiments, an antibody or other agent that specifically binds to one or more human frizzled receptors triggers cell death by a conjugated toxin, chemotherapeutic agent, radioisotope, or other such agent. For example, in certain embodiments, an antibody directed to a human frizzled antibody is conjugated to a toxin and the toxin is activated in FZD-expressing tumor cells by protein internalization. In certain alternative embodiments, the agent or antibody is not conjugated to a toxin, chemotherapeutic agent, or radioisotope.

In certain embodiments, the FZD-binding agent is capable of inhibiting tumor growth. In certain embodiments, the FZD-binding agent is capable of inhibiting tumor growth in vivo (e.g., in a xenograft mouse model and/or in a human suffering from cancer).

In certain embodiments, the FZD-binding agent is capable of reducing the tumorigenicity of a tumor. In certain embodiments, the agent or antibody is capable of reducing the tumorigenicity of a tumor containing cancer stem cells in an animal model (e.g., a mouse xenograft model). In certain embodiments, the number or frequency of cancer stem cells in a tumor is reduced by at least about 2-fold, about 3-fold, about 5-fold, about 10-fold, about 50-fold, about 100-fold, or about 1000-fold. In certain embodiments, the reduction in the number or frequency of cancer stem cells is determined by limiting dilution assay using an animal model. An example of a limiting dilution assay used to test the utility of anti-FZD antibodies is provided in example 8 below. Additional examples and guidance regarding the use of limiting dilution assays to determine a reduction in the number or frequency of cancer stem cells in a tumor can be found, for example, in international publication No. WO2008/042236, U.S. patent application publication No. 2008/0064049, and U.S. patent application publication No. 2008/0178305, each of which is incorporated by reference herein in its entirety.

In certain embodiments, the antibody to the human frizzled receptor mediates cell death of cells expressing the FZD protein by antibody-dependent cellular cytotoxicity (ADCC). ADCC involves the lysis of effector cells that recognize the Fc portion of antibodies. For example, many lymphocytes, monocytes, tissue macrophages, granulocytes and eosinophils have Fc receptors and are capable of mediating cytolysis (Dillman, 1994, j.clin.oncol.12: 1497).

In certain embodiments, an antibody to one or more FZD triggers cell death of a cell expressing the FZD protein by activating Complement Dependent Cytotoxicity (CDC). CDC involves the binding of serum complement to the Fc portion of antibodies and the subsequent activation of the complement protein cascade leading to cell membrane damage and ultimately cell death. The biological activity of antibodies is known to be determined in large part by the constant or Fc region of the antibody molecule (Uananue and Benacerraf, Textbook of Immunology, second edition, Williams & Wilkins, p.218 (1984)). Antibodies of different classes and subclasses differ in this respect, as do antibodies from the same subclass of different species. Among the human antibodies, IgM is the most efficient class of antibodies in binding complement, followed by IgG1, IgG3 and IgG2, whereas IgG4 appears to be very inefficient at activating the complement cascade (Dillman, 1994, J.Clin.Oncol.12: 1497; Jefferis et al, 1998, Immunol.Rev.163: 59-76). According to the present invention, antibodies of those classes that possess the desired biological activity are prepared.

The ability of any particular antibody against one or more FZD to mediate lysis of target cells by complement activation and/or ADCC can be determined. Growing and labeling cells of interest in vitro; adding to the cell culture a combination of antibodies and serum complement or immune cells capable of being activated by the antigen-antibody complex. For example, based on the release of the label from the lysed cells, cytolysis of the target cells may be detected. In fact, the patient's own serum can be used as a source of complement and/or immune cells to screen for antibodies. Antibodies capable of activating complement or mediating ADCC in an in vitro test may be used therapeutically in this particular patient.

The present invention provides polypeptides, including but not limited to antibodies that specifically bind to one or more human frizzled receptors, which antibodies comprise 1, 2, 3, 4, 5, and/or 6 of the CDRs of 18R5 and/or 18R8 (see table 4 of example 1 below), and each CDR has up to 4 (i.e., 0, 1, 2, 3, or 4) conservative amino acid substitutions. The invention thus provides polypeptides, including but not limited to antibodies that specifically bind to one or more human frizzled receptors, said antibodies comprising 1, 2, 3, 4, 5 and/or 6 of the CDRs of 18R5 and/or 18R 8. In certain embodiments, the polypeptide comprises a heavy chain CDR3 of 18R8 and/or a light chain CDR3 of 18R5 or 18R 8. In certain embodiments, the heavy chain CDRs are comprised within a heavy chain variable region and/or the light chain CDRs are comprised within a light chain variable region.

For example, the present invention provides a polypeptide (e.g., an antibody) that specifically binds to a human frizzled receptor, wherein the polypeptide has a heavy chain variable region comprising the following (a), (b), and/or (c): (a) a heavy chain CDR1 comprising GFTFSHYTLS (SEQ ID NO: 1) or a variant thereof having 1, 2, 3 or 4 amino acid substitutions; (b) heavy chain CDR2 comprising VISGDGSYTYYADSVKG (SEQ ID NO: 2) or a variant thereof having 1, 2, 3, or 4 amino acid substitutions; (c) comprises the heavy chain CDR3 of NFIKYVFAN (SEQ ID NO: 3) or a variant thereof having 1, 2, 3 or 4 amino acid substitutions. In certain embodiments, the polypeptide further has a light chain variable region comprising the following (a), (b), and/or (c): (a) a light chain CDR1, the light chain CDR1 comprising SGDKLGKKYAS (SEQ ID NO: 4) or SGDNIGSFYVH (SEQ ID NO: 7), or SEQ ID NO: 4 or SEQ ID NO: 7; (b) a light chain CDR2, said light chain CDR2 comprising EKDNRPSG (SEQ ID NO: 5) or DKSNRRPSG (SEQ ID NO: 8), or SEQ ID NO: 5 or SEQ ID NO: 8, a variant thereof; (c) a light chain CDR3, the light chain CDR3 comprising SSFAGNSLE (SEQ ID NO: 6) or QSYANTLSL (SEQ ID NO: 9), or SEQ ID NO: 6 or SEQ ID NO: 9. In certain embodiments, the amino acid substitution is a conservative substitution.

Thus, the invention provides a polypeptide or antibody, wherein the polypeptide or antibody has a heavy chain CDR1 comprising GFTFSHYTLS (SEQ ID NO: 1), a heavy chain CDR2 comprising VISGDGSYTYYADSVKG (SEQ ID NO: 2), and/or a heavy chain CDR3 comprising NFIKYVFAN (SEQ ID NO: 3). In certain embodiments, the light chain CDRs are contained within the variable region of an antibody heavy chain. In certain embodiments, the polypeptide or antibody has one or more heavy chain CDRs that specifically bind to one or more human frizzled receptors. In certain embodiments, the CDR is modified with 1, 2, 3, or 4 conservative amino acid substitutions. In certain embodiments, each heavy chain CDR is modified with no more than 1-2 conservative amino acid substitutions.

The present invention also provides an antibody that specifically binds to a human frizzled receptor, wherein the antibody has a heavy chain variable region comprising the following (a), (b), and (c): (a) heavy chain CDR1 comprising GFTFSHYTLS (SEQ ID NO: 1) or a variant thereof having 1, 2, 3, or 4 amino acid substitutions; (b) a heavy chain CDR2 comprising VISGDGSYTYYADSVKG (SEQ ID NO: 2) or a variant thereof having 1, 2, 3, or 4 amino acid substitutions; (c) comprises the heavy chain CDR3 of NFIKYVFAN (SEQ ID NO: 3) or a variant thereof having 1, 2, 3 or 4 amino acid substitutions. In certain embodiments, the antibody further has a light chain variable region comprising the following (a), (b), and (c): (a) a light chain CDR1, the light chain CDR1 comprising SGDKLGKKYAS (SEQ ID NO: 4) or SGDNIGSFYVH (SEQ ID NO: 7), or SEQ ID NO: 4 or SEQ ID NO: 7; (b) a light chain CDR2, said light chain CDR2 comprising EKDNRPSG (SEQ ID NO: 5) or DKSNRRPSG (SEQ ID NO: 8), or SEQ ID NO: 5 or SEQ ID NO: 8, a variant thereof; (c) a light chain CDR3, the light chain CDR3 comprising SSFAGNSLE (SEQ ID NO: 6) or QSYANTLSL (SEQ ID NO: 9), or SEQ ID NO: 6 or SEQ ID NO: 9. In some alternative embodiments, the antibody instead has a light chain variable region comprising the following (a), (b), and (c): (a) a light chain CDR1 comprising SGDKLGKKYAS (SEQ ID NO: 4) or SGDNIGSFYVH (SEQ ID NO: 7); (b) a light chain CDR2 comprising EKDNRPSG (SEQ ID NO: 5) or DKSNRRPSG (SEQ ID NO: 8); (c) comprises the light chain CDR3 of SSFAGNSLE (SEQ ID NO: 6) or QSYANTLSL (SEQ ID NO: 9). In certain embodiments, the antibody specifically binds FZD1, FZD2, FZD5, FZD7, and/or FZD 8. In certain embodiments, the antibody specifically binds to two or more human frizzled receptors including FZD5 and FZD 8. In certain embodiments, the amino acid substitution is a conservative substitution.

The invention also provides a polypeptide (e.g., an antibody) that specifically binds to a human frizzled receptor, wherein the polypeptide has a light chain variable region comprising the following (a), (b), and/or (c): (a) a light chain CDR1, the light chain CDR1 comprising SGDKLGKKYAS (SEQ ID NO: 4) or SGDNIGSFYVH (SEQ ID NO: 7), or SEQ ID NO: 4 or SEQ ID NO: 7; (b) a light chain CDR2, said light chain CDR2 comprising EKDNRPSG (SEQ ID NO: 5) or DKSNRRPSG (SEQ ID NO: 8), or SEQ ID NO: 5 or SEQ ID NO: 8, a variant thereof; (c) a light chain CDR3, the light chain CDR3 comprising SSFAGNSLE (SEQ ID NO: 6) or QSYANTLSL (SEQ ID NO: 9), or SEQ ID NO: 6 or SEQ ID NO: 9. In certain embodiments, the amino acid substitution is a conservative substitution.

Also provided are polypeptides or antibodies having the following (a), (b) and/or (c): (a) SEQ ID NO comprising the sequence SGD (K/N) (L/I) G (K/S) (K/F) Y (A/V) (S/H) (SEQ ID NO: 71) or up to 4 (i.e. 0, 1, 2, 3 or 4) conservative amino acid substitutions: 71, the light chain CDR 1; (b) comprises the sequence (E/D) K (D/S) NRPSG (SEQ ID NO: 72) or SEQ ID NO: 72, the light chain CDR 2; (c) comprises the sequence (S/Q) S (F/Y) A (G/N) (N/T) (without aa/L) SL (E/without aa) (wherein "without aa/L" denotes L or NO amino acid, "E/without aa" denotes E or NO amino acid; SEQ NO: 73) or SEQ ID NO: 73, or a light chain CDR 3.

The invention also provides polypeptides or antibodies having a light chain CDR1 comprising SGDKLGKKYAS (SEQ ID NO: 4) or SGDNIGSFYVH (SEQ ID NO: 7), a light chain CDR2 comprising EKDNRPSG (SEQ ID NO: 5) or DKSNRRPSG (SEQ ID NO: 8), and/or a light chain CDR3 comprising SSFAGNSLE (SEQ ID NO: 6) or QSYANTLSL (SEQ ID NO: 9). In certain embodiments, the polypeptide or antibody has a light chain CDR1 comprising SGDNIGSFYVH (SEQ ID NO: 7), a light chain CDR2 comprising DKSNRPSG (SEQ ID NO: 8), and a light chain CDR3 comprising QSYANTLSL (SEQ ID NO: 9). In certain alternative embodiments, the polypeptide or antibody has a light chain CDR1 comprising SGDKLGKKYAS (SEQ ID NO: 4), a light chain CDR2 comprising EKDNRPSG (SEQ ID NO: 5), and a light chain CDR3 comprising SSFAGNSLE (SEQ ID NO: 6). In certain embodiments, the light chain CDRs are contained within the variable region of an antibody light chain. In certain embodiments, the polypeptide or antibody specifically binds to one or more human frizzled receptors. In certain embodiments, a polypeptide or antibody having one or more light chain CDRs specifically binds to one or more human frizzled receptors. In certain embodiments, the CDRs have been modified by 1, 2, 3 or 4 conservative modifications. In certain embodiments, each light chain CDR has been modified by no more than 1-2 conservative amino acid substitutions.

In certain embodiments, the antibody has (a) a heavy chain CDR1 comprising GFTFSHYTLS (SEQ ID NO: 1), a heavy chain CDR2 comprising VISGDGSYTYYADSVKG (SEQ ID NO: 2), and a heavy chain CDR3 comprising NFIKYVFAN (SEQ ID NO: 3); and/or (b) a light chain CDR1 comprising SGDKLGKKYAS (SEQ ID NO: 4) or SGDNIGSFYVH (SEQ ID NO: 7), a light chain CDR2 comprising EKDNRPSG (SEQ ID NO: 5) or DKSNRRPSG (SEQ ID NO: 8), and a light chain CDR3 comprising SSFAGNSLE (SEQ ID NO: 6) or QSYANTLSL (SEQ ID NO: 9). In certain embodiments, the antibody has (a) a heavy chain CDR1 comprising GFTFSHYTLS (SEQ ID NO: 1), a heavy chain CDR2 comprising VISGDGSYTYYADSVKG (SEQ ID NO: 2), and a heavy chain CDR3 comprising NFIKYVFAN (SEQ ID NO: 3); and (b) a light chain CDR1 comprising SGDKLGKKYAS (SEQ ID NO: 4), a light chain CDR2 comprising EKDNRPSG (SEQ ID NO: 5), and a light chain CDR3 comprising SSFAGNSLE (SEQ ID NO: 6). In certain embodiments, the antibody has (a) a heavy chain CDR1 comprising GFTFSHYTLS (SEQ ID NO: 1), a heavy chain CDR2 comprising VISGDGSYTYYADSVKG (SEQ ID NO: 2), and a heavy chain CDR3 comprising NFIKYVFAN (SEQ ID NO: 3); and (b) a light chain CDR1 comprising SGDNIGSFYVH (SEQ ID NO: 7), a light chain CDR2 comprising DKSNRPSG (SEQ ID NO: 8), and a light chain CDR3 comprising QSYANTLSL (SEQ ID NO: 9). In certain embodiments, the CDRs have been modified by 1, 2, 3 or 4 conservative amino acid substitutions. In certain embodiments, each CDR has been modified by no more than 1-2 conservative amino acid substitutions.

The present invention also provides polypeptides, including, but not limited to, antibodies that specifically bind to one or more human frizzled receptors, which antibodies comprise 1, 2, 3, 4, 5, and/or 6 of the CDRs of anti-FZD antibody 44R24 (see table 7 of example 18, below), and each CDR has up to 4 (i.e., 0, 1, 2, 3, or 4) conservative amino acid substitutions. The invention thus provides polypeptides, including but not limited to antibodies that specifically bind to one or more human frizzled receptors, which antibodies comprise 1, 2, 3, 4, 5, and/or 6 of the CDRs of 44R 24. In certain embodiments, the polypeptide comprises the heavy chain CDR3 of 44R24 and/or the light chain CDR3 of 44R 24. In certain embodiments, the heavy chain CDRs are comprised within a heavy chain variable region and/or the light chain CDRs are comprised within a light chain variable region.

The present invention also provides a polypeptide (e.g., an antibody) that specifically binds to human FZD5 and/or FZD8, wherein the antibody has: (a) heavy chain CDR1 comprising GFTFSSYYIT (SEQ ID NO: 77) or a variant thereof having 1, 2, 3, or 4 conservative amino acid substitutions; heavy chain CDR2 comprising TISYSSSNTYYADSVKG (SEQ ID NO: 78) or a variant thereof having 1, 2, 3, or 4 conservative amino acid substitutions; and a heavy chain CDR3 comprising SIVFDY (SEQ ID NO: 79) or a variant thereof having 1, 2, 3, or 4 conservative amino acid substitutions; and/or (b) a light chain CDR1 comprising SGDALGNRYVY (SEQ ID NO: 80) or a variant thereof having 1, 2, 3, or 4 conservative amino acid substitutions; light chain CDR2 comprising SG (SEQ ID NO: 81) or a variant thereof having 1, 2, 3, or 4 conservative amino acid substitutions; and a light chain CDR3 comprising GSWDTRPYPKY (SEQ ID NO: 82) or a variant thereof having 1, 2, 3, or 4 conservative amino acid substitutions. In certain embodiments, the antibody (or other FZD-binding polypeptide) has: (a) heavy chain CDR1 comprising GFTFSSYYIT (SEQ ID NO: 77), heavy chain CDR2 comprising TISYSSSNTYYADSVKG (SEQ ID NO: 78) and heavy chain CDR3 comprising SIVFDY (SEQ ID NO: 79); and/or (b) a light chain CDR1 comprising SGDALGNRYVY (SEQ ID NO: 80), a light chain CDR2 comprising SG (SEQ ID NO: 81), and a light chain CDR3 comprising GSWDTRPYPKY (SEQ ID NO: 82).

Also provided are polypeptides comprising a single light chain or one of the heavy chains described herein, as well as polypeptides (e.g., antibodies) comprising both a light chain and a heavy chain.

Also provided are polypeptides comprising the following (a) and/or (b): (a) and SEQ ID NO: 10 is at least about 80% polypeptide sequence identity; (b) and SEQ ID NO: 12 or SEQ ID NO: 14 is at least about 80% polypeptide. In certain embodiments, the polypeptide comprises a sequence identical to SEQ ID NO: 10. 12 or 14 is at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% of the polypeptide. Thus, in certain embodiments, the polypeptide comprises (a) a sequence that is identical to SEQ ID NO: 10 is at least about 95% polypeptide sequence identity; and/or (b) a sequence that is identical to SEQ ID NO: 12 or SEQ ID NO: 14 is at least about 95% polypeptide. In certain embodiments, the polypeptide comprises (a) a polypeptide having the amino acid sequence of SEQ ID NO: 10; and/or (b) a polypeptide having the amino acid sequence of SEQ ID NO: 12 or SEQ ID NO: 14. In certain embodiments, the polypeptide comprises (a) a polypeptide having the amino acid sequence of SEQ ID NO: 11; and/or (b) a polypeptide having the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 15. In certain embodiments, the polypeptide is an antibody and/or a polypeptide that specifically binds to one or more human frizzled receptors (e.g., FZD1, FZD2, FZD5, FZD7, and/or FZD 8). For example, the invention provides an antibody that specifically binds to a human frizzled receptor, the antibody comprising (a) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 10; and (b) a peptide having the amino acid sequence of SEQ id no: 14. In certain embodiments, the polypeptide having the amino acid sequence of SEQ ID NO: 10 is a heavy chain variable region. In certain embodiments, the polypeptide having the amino acid sequence of SEQ ID NO: 12 or 14 is a light chain variable region. In certain embodiments, the polypeptide of SEQ ID NO: 10. 12 or 14 has a certain percentage of sequence identity to SEQ ID NO: 10. 12 or 14 differ only by conservative amino acid substitutions.

In certain embodiments, the polypeptide or antibody comprises: (a) SEQ ID NO: 10 and SEQ ID NO: 12; (b) SEQ ID NO: 10 and SEQ ID NO: 14; (c) SEQ ID NO: 11 and SEQ ID NO: 13; or (d) SEQ ID NO: 11 and SEQ ID NO: 15.

the present invention also provides an antibody or other polypeptide that specifically binds FZD5 and/or FZD8, comprising: (a) and SEQ ID NO: 85, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99%; and/or (b) a sequence that is identical to SEQ ID NO: 86 is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% polypeptide. In certain alternative embodiments, the polypeptide or antibody comprises SEQ ID NO: 85 and/or SEQ ID NO: 86.

in certain embodiments, the FZD-binding agent comprises, consists essentially of, or consists of an anti-FZD antibody selected from the group consisting of 18R8, 18R5, 18R4605, 18R4805, and 44R24 IgG antibodies.

In certain embodiments, the FZD-binding agent comprises the heavy and light chains of the 18R8 IgG2 antibody (with or without leader sequences). In certain embodiments, the FZD-binding agent is an 18R8 IgG2 antibody. DNA encoding the heavy and light chains of the 18R8 IgG2 antibody has been deposited at the american type culture collection (ATCC, 10801 University Boulevard, Manassas, VA, USA) on 29.9.2008 under the budapest treaty with the designation ATCC accession No. PTA-9540. In certain embodiments, the FZD-binding agent comprises the heavy and light chains of the 18R5 IgG2 antibody (with or without leader sequences). In certain embodiments, the FZD-binding agent is an 18R5 IgG2 antibody. DNA encoding the heavy and light chains of the 18R5 IgG2 antibody has been deposited with the ATCC as required by the budapest treaty on 29.9.2008, and designated ATCC accession No. PTA-9541.

In certain embodiments, the FZD-binding agent is an IgG antibody encoded by a plasmid deposited with the ATCC on 8/26 of 2009 and designated deposit number PTA-10307, PTA10309, or PTA-10311.

In certain embodiments, the FZD-binding agent is an agent that competes (e.g., in a competitive binding assay) for specific binding to FZD1, FZD2, FZD5, FZD7, and/or FZD8 with an antibody encoded by a plasmid having ATCC accession No. PTA-9540, PTA-9541, PTA-10307, or PTA-10309. In certain alternative embodiments, the FZD-binding agent is an agent that competes for specific binding to FZD5 and/or FZD8 with the antibody encoded by the plasmid deposited with ATCC as PTA 10311.

In certain embodiments, the FZD-binding agent has a circulatory half-life in a mouse, cynomolgus monkey (cynomologous monkey) or human of at least about 10 hours, at least about 24 hours, at least about 3 days, at least about 1 week, or at least about 2 weeks. In certain embodiments, the FZD-binding agent is an IgG (e.g., IgG1 or IgG2) antibody that has a circulating half-life in a mouse, cynomolgus monkey, or human that is at least about 10 hours, at least about 24 hours, at least about 3 days, at least about 1 week, or at least about 2 weeks. Methods of increasing the half-life of agents such as polypeptides and antibodies are known in the art. For example, known methods of increasing the circulating half-life of IgG antibodies include introducing mutations in the FC region that increase the pH-dependent binding of the antibody to neonatal FC receptor (FcRn) at pH 6.0 (see, e.g., U.S. patent publication nos. 2005/0276799, 2007/0148164, and 2007/0122403). Known methods to increase the circulating half-life of antibody fragments lacking an Fc region include techniques such as pegylation.

Polyclonal antibodies can be prepared by any known method. Animals (e.g., rabbits, rats, mice, donkeys, etc.) are immunized by multiple subcutaneous or intraperitoneal injections of the relevant antigen (purified peptide fragment, full-length recombinant protein, fusion protein, etc.) to generate polyclonal antibodies, optionally coupled to Keyhole Limpet Hemocyanin (KLH), serum albumin, etc., diluted in sterile saline and combined with an adjuvant (e.g., freund's complete adjuvant or freund's incomplete adjuvant) to form a stable emulsion. The polyclonal antibody is then recovered from the blood, ascites, etc. of the animal thus immunized. The collected blood was coagulated, followed by decanting the serum, centrifugation to clarify, and antibody titer determination. Polyclonal antibodies can be purified from serum or ascites fluid according to standard methods in the art, including affinity chromatography, ion exchange chromatography, gel electrophoresis, dialysis, and the like.

For example, Kohler and Milstein (1975) Nature 256: 495 to produce monoclonal antibodies. By using the hybridoma method, a mouse, hamster, or other suitable host animal, is immunized as described above, thereby eliciting the lymphocytes to produce antibodies that specifically bind to the immunizing antigen. Lymphocytes can also be immunized in vitro. Following immunization, lymphocytes are isolated and fused with a suitable myeloma cell line using, for example, polyethylene glycol, to form hybridoma cells, which can then be selected from unfused lymphocytes and myeloma cells. Hybridomas producing monoclonal antibodies specific for a selected antigen are determined by immunoprecipitation, immunoblotting, or by in vitro binding assays (e.g., Radioimmunoassay (RIA); enzyme-linked immunosorbent assay (ELISA)), which can then be propagated in vitro in culture using standard methods (Goding, monoclonal antibodies: Principles and Practice, Academic Press, 1986), or in vivo as ascites tumors in animals. The monoclonal antibodies can then be purified from the culture medium or ascites fluid as described above for the polyclonal antibodies.

Alternatively, monoclonal antibodies can also be prepared using recombinant DNA methods described in U.S. patent No. 4,816,567. Polynucleotides encoding monoclonal antibodies are isolated from mature B cells or hybridoma cells, for example, by RT-PCR using oligonucleotide primers that specifically amplify the genes encoding the heavy and light chains of the antibody, and their sequences are determined using conventional procedures. The isolated polynucleotides encoding the heavy and light chains are then cloned into suitable expression vectors, which are then transfected into host cells that do not otherwise produce immunoglobulin (e.g., e.coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells), and monoclonal antibodies are produced by the host cells. In addition, recombinant monoclonal antibodies and fragments thereof of a desired species can be isolated from phage display libraries expressing the CDRs of the desired species (McCafferty et al, 1990, Nature, 348: 552-554; Clackson et al, 1991, Nature, 352: 624-628; and Marks et al, 1991, J.mol.biol., 222: 581-597).

Polynucleotides encoding monoclonal antibodies can be further modified in a number of different ways using recombinant DNA techniques to produce alternative antibodies. In some embodiments, the constant regions of 1) such as human antibodies can be replaced with, for example, the light and heavy chains of a mouse monoclonal antibody to produce a chimeric antibody or with 2) a non-immunoglobulin polypeptide to produce a fusion antibody. In some embodiments, the constant region is truncated or removed to produce a desired antibody fragment of the monoclonal antibody. Site-directed or high-density mutations in the variable regions can be used to optimize the specificity, affinity, etc. of monoclonal antibodies.

In some embodiments, the monoclonal antibody against human frizzled receptor is a humanized antibody. In certain embodiments, such antibodies are used therapeutically to reduce antigenicity and HAMA (human anti-mouse antibody) responses when administered to a human subject. Humanized antibodies can be generated using various techniques known in the art. In certain alternative embodiments, the antibody to a human frizzled receptor is a human antibody.

Human antibodies can be made directly using various techniques known in the art. Immortalized human B lymphocytes isolated from immunized individuals or immunized in vitro can be prepared that produce Antibodies to the target antigen (see, e.g., Cole et al, Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, p.77 (1985); Boemer et al, 1991, J.Immunol., 147 (1): 86-95; and U.S. Pat. No. 5,750,373). Furthermore, the human antibodies can be selected from phage libraries expressing human antibodies, such as described in: vaughan et al, 1996, nat. biotech, 14: 309 and 314; sheets et al, 1998, proc.nat' l.acad.sci., 95: 6157-6162; hoogenboom and Winter, 1991, j.mol.biol., 227: 381; and Marks et al, 1991, j.mol.biol, 222: 581. in U.S. patent nos. 5,969,108, 6,172,197, 5,885,793, 6,521,404; 6,544,731 No; 6,555,313 No; 6,582,915 No; 6,593,081 No; U.S. Pat. No. 6,300,064; 6,653,068 No; 6,706,484 th and 7,264,963 th; and Rothe et al, 2007, j.mol.bio, doi: 10.1016/j.jmb.2007.12.018 (each incorporated herein by reference in its entirety) also describes techniques for generating and using antibody phage libraries. Affinity maturation strategies and chain shuffling strategies (Marks et al, 1992, Bio/Technology 10: 779- & 783, incorporated by reference in its entirety) are known in the art and can be used to generate high affinity human antibodies.

Humanized antibodies can also be prepared in transgenic mice containing human immunoglobulin loci capable of producing a full repertoire of human antibodies when immunized without the production of endogenous immunoglobulins. This method is described in U.S. Pat. nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425 and 5,661,016.

Bispecific antibodies that specifically recognize human frizzled receptors are also encompassed by the invention. Bispecific antibodies are antibodies that are capable of specifically recognizing and binding at least two different epitopes. The different epitopes may be within the same molecule (e.g., the same human frizzled receptor) or on different molecules, such that, for example, both antibodies are capable of specifically recognizing and binding to the human frizzled receptor and, for example, 1) an effector molecule on a leukocyte such as a T cell receptor (e.g., CD3) or Fc receptor (e.g., CD64, CD32, or CD16) or 2) a cytotoxic agent as described in detail below. In certain embodiments, the bispecific antibody specifically binds at least one human frizzled receptor and VEGF, a Notch ligand such as a like ligand (e.g., DLL4) or a Jagged ligand, or at least one Notch receptor selected from the group consisting of Notch1, Notch2, Notch3, and Notch 4. Bispecific antibodies can be whole antibodies or antibody fragments.

Exemplary bispecific antibodies are capable of binding two different epitopes, at least one of which is derived from a polypeptide of the invention. Alternatively, the anti-antigen arm of an immunoglobulin molecule may be combined with an arm that binds to a triggering molecule on a leukocyte, such as a T-cell receptor molecule (e.g., CD2, CD3, CD28, or B7) or the Fc receptor of IgG, thereby focusing cellular defense mechanisms on cells expressing a particular antigen. Bispecific antibodies can also be used to direct cytotoxic agents to cells expressing a particular antigen. These antibodies have an antigen-binding arm and an arm that binds a cytotoxic agent or radionuclide chelator (e.g., EOTUBE, DPTA, DOTA, or TETA). Techniques for the preparation of bispecific antibodies are common in the art (Millstein et al, 1983, Nature 305: 537-. Antibodies that are more than bivalent are also provided. For example, trispecific antibodies can be prepared (Tutt et al, J.Immunol.147: 60 (1991)). Thus, in certain embodiments, the antibody to the human frizzled receptor is multispecific.

Alternatively, in certain alternative embodiments, the FZD-binding agents of the present invention are not bispecific antibodies.

In certain embodiments, an antibody (or other polypeptide) described herein can be monospecific. For example, in certain embodiments, an antibody comprises one or more antigen binding sites that each bind or are capable of binding to the same one or more human FZD receptors (e.g., FZD1, FZD2, FZD5, FZD7, and/or FZD8, or homologous epitopes on FZD in some combination). In certain embodiments, the antigen binding site of a monospecific antibody described herein binds or is capable of binding one, two, three, four or five (or more) human frizzled receptors.

In certain embodiments, antibody fragments are provided, for example, to increase tumor penetration. Various techniques for producing antibody fragments are known. These fragments have traditionally been obtained by proteolytic cleavage of intact antibodies (e.g., Morimoto et al, 1993, Journal of Biochemical and Biophysical Methods 24: 107-. In certain embodiments, the antibody fragment is produced by recombinant means. Fab, Fv and scFv antibody fragments can be expressed and secreted in e.coli or other host cells, allowing the production of large quantities of these fragments. Such antibody fragments may also be isolated from the antibody phage libraries discussed above. Antibody fragments can also be linear antibodies, such as described in U.S. Pat. No. 5,641,870, and can be monospecific or bispecific. Other techniques for producing antibody fragments will be apparent to those skilled in the art.

According to the present invention, the techniques can be adapted to produce single chain antibodies specific for one or more human frizzled receptors (see U.S. Pat. No. 4,946,778). Furthermore, the method may be adapted to construct Fab expression libraries (Huse et al, Science 246: 1275-1281(1989)) so that monoclonal Fab fragments or derivatives, fragments, analogs or homologues thereof having the desired specificity for the FZD receptor may be identified rapidly and efficiently. Antibody fragments can be prepared by techniques in the art, including but not limited to: (a) f (ab') 2 fragments produced by pepsin digestion of the antibody molecule; (b) a Fab fragment produced by reducing the disulfide bond of the F (ab') 2 fragment, (c) a Fab fragment produced by treating an antibody molecule with papain and a reducing agent, and (d) an Fv fragment.

More desirably, the antibody is modified (particularly for antibody fragments) to increase its serum half-life. This can be achieved by: for example, a salvage receptor (sample receptor) binding epitope is introduced into an antibody fragment by mutation of an appropriate region in the antibody fragment, or an epitope is introduced into a peptide tag and the peptide tag is subsequently fused to either end or the middle of the antibody fragment (e.g., by DNA or peptide synthesis).

Heteroconjugate antibodies are also within the scope of the invention. Heteroconjugate antibodies consist of two covalently bound antibodies. For example, such antibodies have been proposed to target immune cells to unwanted cells (U.S. Pat. No. 4,676,980). The invention also includes: antibodies are prepared in vitro using known methods in synthetic protein chemistry, including those involving cross-linking agents. For example, immunotoxins may be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolane and methyl-4-mercaptobutyrimidate.

For the purposes of the present invention, it will be appreciated that the modified antibody can comprise any type of variable region used to associate the antibody with a polypeptide of a human FZD receptor. In this regard, the variable region may comprise or be derived from any type of variable region of a mammal that has been induced to produce a humoral response and produce immunoglobulins against the desired tumor-associated antigen. Thus, the variable region of the modified antibody can be of, for example, human, murine, non-human primate (e.g., cynomolgus monkey, etc.) or wolf origin. In some embodiments, the variable and constant regions of the modified immunoglobulin are both human. In other embodiments, the variable region of a compatible antibody (compatible antibody), typically derived from a non-human source, may be engineered or specially tailored to improve the binding properties of the molecule or reduce its immunogenicity. In view of this, the variable regions useful in the present invention may be humanized or altered by inclusion of an introduced amino acid sequence.

In certain embodiments, the variable domains in the heavy and light chains are altered by at least partial substitution of one or more CDRs, and if necessary by partial framework region substitution and sequence alteration. Although the CDRs may be derived from the same antibody class or even subclass as the antibody from which the framework regions are derived, it is contemplated that the CDRs are derived from a different class of antibody and preferably from a different species of antibody. In order to transfer the antigen binding ability of one variable domain to another, it may not be necessary to replace all CDRs with the complete CDRs from the donor variable region. Instead, only those residues necessary for maintaining the activity of the antigen binding site may have to be transferred. In view of the description made in U.S. Pat. nos. 5,585,089, 5,693,761 and 5,693,762, it is within the ability of those skilled in the art to obtain functional antibodies with reduced immunogenicity by performing routine experimentation or by trial and error.

Although alterations are made to the variable regions, those skilled in the art will appreciate that the modified antibodies of the invention will include the following antibodies (e.g., full length antibodies or immunoreactive fragments thereof): at least a portion of one or more constant region domains in the antibody have been deleted or altered to provide a desired biochemical property, such as increased tumor localization or reduced serum half-life, as compared to an antibody containing a native or unaltered constant region and having about the same immunogenicity. In some embodiments, the constant region of the modified antibody will comprise a human constant region. Constant region modifications compatible with the present invention include additions, deletions or substitutions of one or more amino acids in one or more domains. In other words, the modified antibodies disclosed herein may comprise alterations or modifications to more than one of the three heavy chain constant domains (CH1, CH2, or CH3) and/or the light chain constant domain (CL). In some embodiments, modified constant regions are provided in which one or more domains are partially or completely deleted. In some embodiments, the modified antibody will comprise a domain deletion construct or variant (Δ CH2 construct) in which the entire CH2 domain has been removed. In some embodiments, the deleted constant region domains are replaced with short amino acid spacers (e.g., 10 residues) that provide some molecular flexibility, which is typically provided by a missing constant region.

In addition to their construction, constant regions are known in the art to mediate several effector functions. For example, binding of complement C1 component to an antibody activates the complement system. Complement activation plays an important role in opsonization and cytolysis of cellular pathogens. Activation of complement also stimulates inflammatory responses and may also be involved in autoimmune hypersensitivity reactions. In addition, antibodies bind to cells via the Fc region, binding Fc receptors (fcrs) on cells with Fc receptor sites on the Fc region of the antibody. There are many Fc receptors that are specific for different classes of antibodies, including IgG (gamma receptor), IgE (eta receptor), IgA (alpha receptor), and IgM (mu receptor). Binding of antibodies to Fc receptors on cell surfaces triggers a number of important and diverse biological responses, including phagocytosis and destruction of antibody-coated particles, clearance of immune complexes, lysis of antibody-coated target cells by killer cells (known as antibody-dependent cell-mediated cytotoxicity, or ADCC), release of inflammatory mediators, placental transfer, and control of immunoglobulin production.

In certain embodiments, the FZD-binding antibody provides an altered effector function that in turn affects the biological characteristics of the administered antibody. For example, deletion or inactivation of the constant region domain (by point mutation or other means) can reduce binding of the modified antibody to Fc receptors in the circulation, thereby increasing tumor localization. In other cases, it is possible that constant region modifications consistent with the present invention will modulate complement binding and thus reduce serum half-life and reduce non-specific binding to the coupled cytotoxin. Other modifications to the constant region may also be used to eliminate disulfide bonds or oligosaccharide moieties, thereby enhancing localization due to increased antigen specificity or antibody flexibility. Similarly, modifications made to the constant region of the invention can be readily accomplished using biochemical or molecular engineering techniques well known to those skilled in the art.

In certain embodiments, the FZD-binding agent is an antibody that does not have one or more effector functions. For example, in some embodiments, the antibody does not have antibody-dependent cellular cytotoxicity (ADCC) activity and/or does not have complement-dependent cytotoxicity (CDC) activity. In certain embodiments, the antibody does not bind to an Fc receptor and/or complement factors. In certain embodiments, the antibody has no effector function.

It should be noted that in certain embodiments, the modified antibody may be engineered such that the CH3 domain is fused directly to the hinge region of the corresponding modified antibody. In other constructs it may be desirable to provide a peptide spacer sequence between the hinge region and the modified CH2 and/or CH3 domains. For example, a compatible construct may be expressed in which the CH2 domain has been deleted and the remaining CH3 domain (modified or unmodified) is linked to the hinge region with a 5-20 amino acid spacer sequence. For example, this spacer sequence may be added to ensure that regulatory elements of the constant domain remain free and accessible, or to ensure that the hinge region remains flexible. It should be noted, however, that in some cases it can be demonstrated that the amino acid spacer sequence is immunogenic and elicits an adverse immune response to the construct. Thus, in certain embodiments, any spacer sequence added to the construct will be relatively non-immunogenic or even omitted entirely, thereby preserving the desired biochemical quality of the modified antibody.

It will be appreciated that in addition to deletion of the entire constant region domain, the antibodies of the invention may also be provided by partial deletion or substitution of several or even a single amino acid. For example, a single amino acid mutation in a selected region of the CH2 domain is sufficient to substantially attenuate Fc binding, thereby enhancing tumor localization. Similarly, it may be desirable to make simple deletions to the above-described portion of one or more constant region domains that control the effector function to be modulated (e.g., complement CLQ binding). This partial deletion of the constant region can improve selected properties of the antibody (serum half-life) while retaining other desired functions associated with the treated constant region domain intact. In addition, as alluded to above, the constant regions of the antibodies disclosed herein may be modified by mutations or substitutions of one or more amino acids that enhance the properties of the resulting constructs. In this regard, it is possible to disrupt the activity provided by the conserved binding site (e.g., Fc binding) while substantially retaining the structural and immunogenic properties of the modified antibody. Certain embodiments can include the addition of one or more amino acids to the constant region, thereby enhancing a desired property, such as reducing or enhancing effector function, or providing attachment for more cytotoxins or carbohydrates. In such embodiments, it may be desirable to insert or replace specific sequences derived from selected constant region domains.

The invention also includes variants and equivalents substantially homologous to the chimeric, humanized, human antibodies or antibody fragments thereof set forth herein. Such variants and equivalents may comprise, for example, conservative substitution mutations, i.e., substitution of one or more amino acids with a similar amino acid. For example, a conservative substitution is one that replaces one amino acid with another amino acid of the same general class, such as replacing one acidic amino acid with another acidic amino acid, replacing one basic amino acid with another basic amino acid, or replacing one neutral amino acid with another neutral amino acid. The purpose of conservative amino acid substitutions is well known in the art.

The invention also relates to immunoconjugates comprising an antibody conjugated to a cytotoxic agent. Cytotoxic agents include chemotherapeutic agents, growth inhibitory agents, toxins (e.g., enzymatically active toxins derived from bacteria, fungi, plants, or animals, or fragments thereof), radioisotopes (i.e., radioactive conjugates), and the like. Chemotherapeutic agents that may be used to generate such immunoconjugates include, for example, methotrexate, adriamycin (adriamycin), doxorubicin (doxorubicin), melphalan, mitomycin C, chlorambucil, nordstamycin, or other intercalating agents. Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin (alpha-sarcin), Aleurites fordii protein (Aleurites fordiprotein), dianthin protein, pokeweed protein (PAPI, PAPII and PAP-S), Momordica charantia inhibitor (momordica charantia inhibitor), lepra curcin (curcin), crotin, Saponaria officinalis inhibitor (sapaonaria officinalis inhibitor), gelonin (gelonin), mitogen (ogellin), restrictocin (restrictocin), phenomycin (phenomycin), enomycin (enomycin) and trichothecin (tricothecene). A variety of radionuclides can be used to generate radioconjugated antibodies, including 212Bi, 131I, 131In, 90Y, and 186 Re. The manufacture of conjugates of antibodies and cytotoxic agents uses a variety of bifunctional protein-coupling agents, such as N-succinimidyl-3- (2-pyridyldithiol) propionate (SPDP), Iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate HCl, dimethyl adipimidate HCL), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azido compounds (e.g., bis (p-azidobenzoyl) hexamethylenediamine), bis (diazo) derivatives (e.g., bis (p-diazobenzoyl) ethylenediamine), diisocyanates (e.g., benzylidene 2, 6-diisocyanate), and bis-reactive fluorine compounds (e.g., 1, 5-difluoro-2, 4-dinitrobenzene). Conjugates of an antibody and one or more small molecule toxins, such as calicheamicin (calicheamicin), maytansinol (maytansinoid), fusarium trispora and CC1065, and toxin-active derivatives of these toxins, can also be used.

The conjugated antibody consists of two covalently bound antibodies. For example, such antibodies have been proposed to target immune cells to unwanted cells (U.S. Pat. No. 4,676,980). It is also contemplated that antibodies can be prepared in vitro using known methods in synthetic protein chemistry, including those involving cross-linking agents. For example, immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolane and methyl-4-mercaptobutyrimidate.

Regardless of how useful amounts are obtained, the antibodies of the invention can be used in any of a variety of conjugated (i.e., immunoconjugate) or unconjugated forms. Alternatively, the antibodies of the invention can be used in unconjugated or "naked" form. In certain embodiments, the antibody is used in an unconjugated formThereby destroying malignant cells using the subject's natural defense mechanisms, including complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC). In some embodiments, the antibody may be conjugated to the radioisotope using any of a variety of well-known chelators or direct labels, e.g. ⁹⁰Y、¹²⁵I、¹³¹I、¹²³I、¹¹¹In、¹⁰⁵Rh、¹⁵³Sm、⁶⁷Cu、⁶⁷Ga、¹⁶⁶Ho、¹⁷⁷Lu、¹⁸⁶Re and¹⁸⁸re. In other embodiments, the disclosed compositions may comprise an antibody conjugated to a drug, prodrug, or biological response modifier, such as methotrexate, adriamycin, and lymphokines (e.g., interferon). Additional embodiments of the invention include the use of antibodies conjugated to specific biological toxins, such as ricin or diphtheria toxin. In yet further embodiments, the modified antibody can be complexed with other immunologically active ligands (e.g., antibodies or fragments thereof), wherein the resulting molecule binds to both neoplastic cells and effector cells (e.g., T cells). The choice of which conjugated or unconjugated modified antibody to use will depend on the type and stage of the cancer, the use of adjunctive therapies (e.g., chemotherapy or external radiation), and the patient's condition. It will be appreciated that given the teachings herein, one skilled in the art can readily make such a selection.

The polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide, which comprises an antibody against a human FZD receptor or a fragment thereof. It will be appreciated in the art that some amino acid sequences of the invention can be altered without significantly affecting the structure or function of the protein. Thus, the present invention also includes variants of the polypeptides which exhibit substantial activity or comprise antibody regions or fragments thereof against the human FZD receptor protein. Such mutants include deletions, insertions, inversions, repeats and type substitutions.

Polypeptides and analogs thereof may be further modified to include additional chemical moieties not normally part of a protein. These derived moieties can improve the solubility, biological half-life or adsorption of the protein. These moieties can also reduce or eliminate any desirable side effects of the protein, etc. A review of these sections can be found in REMINGTON' S PHARMACEUTICAL SCIENCES, 20 th edition, Mack Publishing Co., Easton, Pa (2000).

The isolated polypeptides described herein can be produced by any suitable method known in the art. These methods include direct protein synthesis methods as well as the construction of DNA sequences encoding the sequences of the isolated polypeptides and the expression of these sequences in a suitable transformed host. In some embodiments, the DNA sequence is constructed using recombinant techniques by isolating or synthesizing a DNA sequence encoding the wild-type protein of interest. Alternatively, the sequence can be mutated by site-specific mutagenesis to provide a functional analog thereof. See, e.g., Zoeller et al, proc.nat' l.acad.sci.usa 81: 5662 5066(1984) and U.S. Pat. No. 4,588,585.

In some embodiments, the DNA sequence encoding the polypeptide of interest can be constructed by chemical synthesis using an oligonucleotide synthesizer. Such oligonucleotides can be designed based on the amino acid sequence of the desired polypeptide and selecting codons that are favored in the host cell that will produce the recombinant polypeptide of interest. An isolated polynucleotide sequence encoding an isolated polypeptide of interest can be synthesized using standard methods. For example, the complete amino acid sequence can be used to construct a reverse translated gene. In addition, DNA oligomers can be synthesized that comprise nucleotide sequences encoding specific isolated polypeptides. For example, several small oligonucleotides encoding portions of the desired polypeptide can be synthesized and subsequently ligated. Individual oligonucleotides typically contain 5 'or 3' overhangs for complementary assembly.

Once assembled (by synthesis, site-directed mutagenesis, or other means), the polynucleotide sequence encoding a particular isolated polypeptide of interest is inserted into an expression vector and operably linked to expression control sequences suitable for expression of the protein in a desired host. Correct assembly can be confirmed by nucleotide sequencing, restriction mapping and expression of the biologically active polypeptide in a suitable host. As is well known in the art, in order to obtain high expression levels of a transfected gene in a host, it is necessary to operatively link the gene to transcriptional and translational expression control sequences that are functional in the chosen expression host.

In certain embodiments, recombinant expression vectors are used to amplify and express DNA encoding an antibody or fragment thereof against human frizzled receptors. A recombinant expression vector is a replicable DNA construct having a synthetic or cDNA-derived DNA fragment encoding a polypeptide chain of an anti-FZD antibody or a fragment thereof operatively linked to suitable transcriptional or translational regulatory elements of a gene derived from a mammal, microorganism, virus or insect. The transcription unit generally comprises an aggregate of the following (1), (2) and (3): (1) one or more genetic elements that play a regulatory role in gene expression, such as a transcriptional promoter or enhancer, (2) structural or coding sequences that are transcribed into mRNA and translated into protein, (3) suitable transcriptional and translational initiation and termination sequences as described in detail below. Such regulatory elements may include operator sequences that control transcription. The ability to replicate in the host, which is usually conferred by an origin of replication, and a selection gene to facilitate recognition of the transformant may be additionally added. D operably links the other DNA regions when they are functionally related to each other. For example, if expressed as a precursor involved in secretion of a polypeptide, DNA for a signal peptide (secretion leader) is operably linked to DNA for that polypeptide; a promoter is operably linked to a coding sequence if transcription of the coding sequence is controlled; or operably linked to a coding sequence if the ribosome binding site is positioned so as to permit translation. Structural elements designed for use in yeast expression systems include leader sequences that enable the host cell to secrete the translated protein extracellularly. Alternatively, when the recombinant protein is expressed without a leader or transporter sequence, the protein may comprise an N-terminal methionine residue. This residue can then optionally be cleaved from the expressed recombinant protein to yield the final product.

The choice of expression control sequences and expression vectors will depend on the choice of host. A variety of expression host/vector combinations can be employed. Expression vectors useful in eukaryotic hosts include, for example, vectors containing expression control sequences from SV40, bovine papilloma virus, adenovirus (adenovims), and cytomegalovirus. Expression vectors useful in bacterial hosts include known bacterial plasmids such as those from E.coli (including pCR1, pBR332, pMB9, and derivatives thereof), as well as broad-host plasmids such as M13 and filamentous single-stranded DNA phages.

Suitable host cells for expressing FZD-binding polypeptides or antibodies (or FZD proteins used as antigens) include prokaryotes, yeast, insect or higher eukaryote cells controlled by suitable promoters. Prokaryotes include gram-negative or gram-positive organisms, such as e. Higher eukaryote cells include established mammalian derived cell lines as described below. Cell-free translation systems may also be employed. Pouwels et al describe suitable Cloning and expression Vectors for bacterial, fungal, yeast and mammalian cell hosts (Cloning Vectors: organism Manual, Elsevier, N.Y., 1985), and the relevant disclosures therein are hereby incorporated by reference. Additional information regarding methods of producing proteins, including antibodies, can be found, for example, in U.S. patent publication No. 2008/0187954, U.S. patent nos. 6,413,746 and 6,660,501, and international patent No. WO04009823, each of which is hereby incorporated by reference in its entirety.

It may also be advantageous to employ various mammalian or insect cell culture systems for expression of recombinant proteins. The reason that recombinant proteins can be expressed in mammalian cells is that such proteins are usually correctly folded, appropriately modified and fully functional. Examples of suitable mammalian host Cell lines include the COS-7 monkey kidney Cell line described by Gluzman (Cell 23: 175, 1981) and other Cell lines capable of expressing suitable vectors, including, for example, L-Cell, C127, 3T3, Chinese Hamster Ovary (CHO), HeLa and BHK Cell lines. Mammalian expression vectors may contain non-transcriptional elements such as origins of replication, suitable promoters and enhancers for linkage to the gene to be expressed, and other 5 'or 3' side non-transcribed sequences, as well as 5 'or 3' non-translated sequences such as necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, and transcription termination sequences. Baculovirus system for the production of heterologous proteins in insect cells the virus system was described in Luckow and Summers, Bio/Technology 6: 47 (1988).

Any suitable method can be used to purify the protein produced by the transformed host. Such standard methods include chromatography (e.g., ion exchange chromatography, affinity chromatography, and size exclusion column chromatography), centrifugation, differential solubility (differential solubility), or any other standard method of protein purification. Affinity tags such as hexahistidine, maltose binding domain, influenza coat protein sequence (inflenza coat sequence) and glutathione-S-transferase can be attached to the protein, allowing the protein to be easily purified when passed through a suitable affinity column. The isolated protein may also be physically characterized using techniques such as proteolysis, nuclear magnetic resonance, and X-ray crystallography.

For example, for a supernatant of a system that secretes a recombinant protein into a culture medium, concentration can first be performed using a commercially available protein concentration filter (e.g., Amicon or Millipore Pellicon ultrafiltration unit). After the concentration step, the concentrate may be added to a suitable purification matrix. Alternatively, an anion exchange resin may be employed, such as a matrix or substrate having pendant Diethylaminoethyl (DEAE) groups. The matrix may be acrylamide, agarose, dextran, cellulose or other types of matrices commonly used for protein purification. Alternatively, a cation exchange step may be used. Suitable cation exchangers include various insoluble matrices containing sulfopropyl or carboxymethyl groups. Finally, the FZD-binding agents can be further purified using one or more reverse phase high performance liquid chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media, such as silica gel with overhanging methyl or other aliphatic groups. Various combinations of some or all of the above purification steps may also be employed to provide a homogeneous recombinant protein.

Recombinant proteins produced in bacterial culture can be isolated by, for example, the following steps: initial extraction from the cell pellet is followed by one or more concentration, salting out, aqueous phase ion exchange or size exclusion chromatography steps. High Performance Liquid Chromatography (HPLC) can be used for the final purification step. Microbial cells used to express the recombinant protein may be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or the use of a lytic agent.

Methods known in the art for purifying antibodies and other proteins also include, for example, those described in U.S. patent nos. 2008/0312425, 2008/0177048, and 2009/0187005, each of which is hereby incorporated by reference in its entirety.

In certain embodiments, the FZD-binding agent is a non-antibody polypeptide. Various methods are known in the art for identifying and producing non-antibody polypeptides that bind with high affinity to a protein target. See, e.g., Skerra, curr, opin, biotechnol, 18: 295-304 (2007); hosse et al, Protein Science, 15: 14-27 (2006); gill et al, curr, opin, biotechnol, 17: 653-; nygren, FEBS j., 275: 2668-76 (2008); and Skerra, FEBS j., 275: 2677-83(2008), each of which is incorporated herein by reference in its entirety. In certain embodiments, phage display technology has been used to identify/prepare FZD-binding polypeptides. In certain embodiments, the polypeptide comprises a type of protein framework selected from the group consisting of protein a, lipocalin (lipocalin), fibronectin (fribryonectin) domain, ankyrin consensus repeat domain, and thioredoxin.

In some embodiments, the agent is a non-protein molecule. In certain embodiments, the agent is a small molecule. Combinatorial chemistry libraries and techniques useful in identifying non-protein FZD-binding agents are known to those of skill in the art. See, e.g., Kennedy et al, j.comb. chem, 10: 345-; belle et al, j. 855-902 (2007); and Bhattacharyya, curr. med. chem., 8: 1383-404(2001), each of which is incorporated herein by reference in its entirety. In certain other embodiments, the agent is a saccharide, glycosaminoglycan, glycoprotein, or proteoglycan.

In certain embodiments, the agent is a nucleic acid aptamer (aptamer). Aptamers are polynucleotide molecules that are selected for their ability to bind another molecule (e.g., from random pools or mutant pools). In some embodiments, the aptamer comprises a DNA polynucleotide. In certain alternative embodiments, the aptamer comprises an RNA polynucleotide. In certain embodiments, the aptamer comprises one or more modified nucleic acid residues. Methods of generating and screening for aptamers that bind to proteins are well known in the art. See, for example, U.S. patent No. 5,270,163, U.S. patent No. 5,683,867, U.S. patent No. 5,763,595, U.S. patent No. 6,344,321, U.S. patent No. 7,368,236, U.S. patent No. 5,582,981, U.S. patent No. 5,756,291, U.S. patent No. 5,840,867, U.S. patent No. 7,312,325, U.S. patent No. 7,329,742, international patent No. WO 02/077262 publication, international patent No. WO 03/070984 publication, U.S. patent application No. 2005/0239134 publication, U.S. patent application No. 2005/0124565 publication, and U.S. patent application No. 2008/0227735 publication, each of which is incorporated herein by reference in its entirety.

The invention also provides methods of screening for agents having Wnt signaling inhibitory, anti-tumor, and/or anti-cancer stem cell utility. These methods include, but are not limited to: a method comprising comparing the level of one or more differentiation markers in a first solid tumor to the level of the one or more differentiation markers in a second solid tumor, the first solid tumor having been exposed to the agent and the second solid tumor not having been exposed to the agent. In certain embodiments, these methods comprise: (a) exposing a first solid tumor but not a second solid tumor to the agent; (b) assessing the level of one or more differentiation markers in the first solid tumor and the second solid tumor; and (c) comparing the level of the one or more differentiation markers in the first solid tumor to the second solid tumor. In certain embodiments, the agent is an inhibitor of the canonical Wnt signaling pathway and/or inhibits binding of one or more human Wnt proteins to one or more human frizzled receptors. In certain embodiments, the agent is an antibody that specifically binds to one or more human frizzled receptors. In certain embodiments, the level of one or more differentiation markers is increased in the first solid tumor as compared to the second solid tumor, indicating utility against solid tumor stem cells. In certain alternative embodiments, the level of one or more differentiation markers (i.e., negative markers of differentiation) is decreased in the first solid tumor as compared to the second solid tumor, indicating the utility of the anti-solid tumor stem cells. In certain embodiments, the solid tumor is a pancreatic tumor. In certain embodiments, the solid tumor is a pancreatic tumor and the one or more differentiation markers may comprise one or more mucins (e.g., Muc16) and/or chromogranin a (chga). In certain alternative embodiments, the solid tumor is a colon tumor. In some embodiments, the solid tumor is a colon tumor and the one or more differentiation markers comprise cytokeratin 7. Other potential differentiation markers for pancreas and colon and other tumor types are known to those skilled in the art. One skilled in the art can readily assess the usefulness of a potential differentiation marker in a screening method by treating a desired tumor type with one or more of the anti-FZD antibodies disclosed herein (e.g., 18R5 and/or 44R24) and then assessing changes in marker expression in the treated tumor relative to a control. Non-limiting examples of these methods can be found, for example, in the specific examples below.

Polynucleotides of

In certain embodiments, the present invention encompasses polynucleotides comprising a polynucleotide encoding a polypeptide that specifically binds to a human FZD receptor or a fragment of such a polypeptide. For example, the invention provides a polynucleotide comprising a nucleic acid sequence encoding an antibody against a human frizzled receptor or encoding a fragment of such an antibody. The polynucleotide of the present invention may be in the form of RNA or DNA. DNA includes cDNA, genomic DNA and synthetic DNA; and may be double-stranded or single-stranded, and if single-stranded, may be the coding strand or the non-coding (anti-sense) strand.

In certain embodiments, the polynucleotide is isolated. In certain embodiments, the polynucleotide is substantially pure.

The present invention provides a polynucleotide comprising a polynucleotide encoding a polypeptide comprising: the polypeptide comprises a sequence selected from the group consisting of SEQ ID NO: 10. 12, 14. The present invention also provides a polynucleotide comprising a polynucleotide encoding a polypeptide comprising: the polypeptide comprises a sequence selected from the group consisting of SEQ ID NO: 85. SEQ ID NO: 86, or a sequence of the group consisting of seq id no. Also provided is a polynucleotide comprising a polynucleotide encoding a polypeptide comprising: the polypeptide comprises the amino acid sequence of SEQ ID NO: 11. 13 or 15.

The present invention further provides a polypeptide comprising a sequence selected from the group consisting of SEQ ID NOs: 17. 19 and 21. Alternatively, in certain embodiments, the polynucleotide may comprise a sequence selected from the group consisting of SEQ id nos: 87-90, 92 and 94-95. Also provided is a polypeptide comprising SEQ ID NO: 18. 20 or 22.

Also provided is a polynucleotide comprising a nucleotide sequence identical to a nucleotide sequence having the sequence of SEQ ID NO: 17. 19 or 21 and/or a polynucleotide encoding a polypeptide having the sequence of SEQ ID NO: 10. 12 or 14. Also provided is a polynucleotide comprising a nucleotide sequence identical to a nucleotide sequence having a sequence selected from the group consisting of SEQ id nos: 87-90, 92 and 94-95 and/or to a polynucleotide encoding a polypeptide having the sequence of SEQ ID NO: 85 or 86, or a pharmaceutically acceptable salt thereof. In certain embodiments, the hybridization is performed under high stringency conditions.

In certain embodiments, the polynucleotide comprises a coding sequence for a mature polypeptide fused in the same reading frame to a polynucleotide that assists in expressing and secreting the polypeptide, e.g., from a host cell (e.g., a leader sequence that functions as a secretory sequence for controlling the transport of the polypeptide from the cell). The polypeptide having a leader sequence is a proprotein and the leader sequence can be cleaved off by the host cell to form the mature form of the polypeptide. The polynucleotide can also encode a proprotein consisting of the mature protein with the addition of an additional 5' amino acid residue. The mature protein with the prosequence is a proprotein and is the inactive form of the protein. Once the prosequence is cleaved, the active mature protein is left.

In certain embodiments, the polynucleotide comprises a coding sequence for the mature polypeptide fused in frame with a marker sequence, e.g., for purification of the encoded polypeptide. For example, the tag sequence may be a hexahistidine tag provided by the pQE-9 vector for purification of the mature polypeptide fused to the tag when the host is bacterial, or a Hemagglutinin (HA) tag derived from an influenza hemagglutinin protein when a mammalian host (e.g., COS-7 cells) is used.

The invention also relates to variants of the above polynucleotides, which variants encode, for example, fragments, analogs and derivatives.

In certain embodiments, the present invention provides an isolated polynucleotide comprising a polynucleotide having at least 80%, at least 85%, at least 90%, at least 95%, and in some embodiments at least 96%, 97%, 98%, or 99% nucleotide sequence identity to a polynucleotide encoding a polypeptide comprising an antibody or fragment thereof against a human FZD receptor described herein.

A polynucleotide has at least, e.g., 95% nucleotide sequence "identity" to a reference nucleotide sequence, which means that the nucleotide sequence of the polynucleotide is identical to the reference sequence, except that the sequence of the polynucleotide may contain up to 5 point mutations in every 100 nucleotides of the reference nucleotide sequence. In other words, in order to obtain a polynucleotide having at least 95% nucleotide sequence identity to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with other nucleotides, or nucleotides up to 5% in number of the total nucleotides of the reference sequence may be inserted into the reference sequence. The mutations made to a reference sequence can occur at the amino-terminal or carboxy-terminal positions of the reference nucleotide sequence, or at any position between these terminal positions, and are interspersed either individually among the nucleotides of the reference sequence or in more than one contiguous group within the reference sequence.

Variants of a polynucleotide may comprise alterations of coding regions and/or non-coding regions. In some embodiments, a variant of a polynucleotide comprises an alteration that results in a silent substitution, addition, or deletion, but does not alter the properties or activity of the encoded polypeptide. In some embodiments, the nucleotide variant is produced by silent substitutions due to the degeneracy of the genetic code. Polynucleotide variants can be produced for a variety of reasons, for example, to optimize codon expression in a particular host (to change codons in human mRNA to codons preferred by a bacterial host (e.g., e.

Vectors and cells containing the polynucleotides described herein are also provided.

Methods of use and pharmaceutical compositions

FZD-binding agents (including polypeptides and antibodies) of the present invention are useful in a variety of applications, including but not limited to therapeutic methods (e.g., treatment of cancer). In certain embodiments, the agent can be used to inhibit Wnt signaling (e.g., canonical Wnt signaling), inhibit tumor growth, induce differentiation, reduce tumor volume, and/or reduce tumor tumorigenicity. These methods of use may be in vivo, ex vivo (ex vivo) or in vitro. In certain embodiments, the FZD-binding agent or polypeptide or antibody is an antagonist of one or more human frizzled receptors to which it binds.

In certain embodiments, the FZD-binding agent or antagonist is used to treat a disease associated with the activation of Wnt signaling. In a particular embodiment, the disease is a Wnt signaling dependent disease. In a particular embodiment, the Wnt signaling is canonical Wnt signaling. In certain embodiments, the FZD-binding agent or antagonist is used to treat a disorder characterized by elevated stem cell and/or progenitor cell levels.

In certain embodiments, the disease treated with a FZD-binding agent or antagonist (e.g., an anti-FZD antibody) is cancer. In certain embodiments, the cancer is characterized by a Wnt-dependent tumor. In certain embodiments, the cancer is characterized by a tumor that expresses one or more frizzled receptors that bind to the FZD-binding agent (e.g., antibody). In certain embodiments, the cancer is characterized by a tumor that expresses one or more genes in a Wnt gene signature.

In certain embodiments, the disease treated with the FZD-binding agent or antagonist is not cancer. For example, the disease may be a metabolic disorder, such as obesity or diabetes (e.g., type II diabetes) (Jin T., Diabetologia, 2008 Oct; 51 (10): 1771-80). Alternatively, the disease may be a Bone disease, such as osteoporosis, osteoarthritis or rheumatoid arthritis (Corr M, Nat Clin practice Rheumatotol, 2008 Oct; 4 (10): 550-6; Day et al, Bone Joint Surg Am, 2008 Feb; 90 Suppl 1: 19-24). The disease may also be a renal disease, such as polycystic Kidney disease (Harris et al, Annu Rev Med, 2008 Oct.23; Schmidt-Ott et al, Kidney Int, 2008 Oct; 74 (8): 1004-8; Benzing et al, J Am Soc N ephrol, 2007 May; 18(5): 1389-98). Alternatively, ocular diseases may be treated, including but not limited to macular degeneration and familial exudative vitreoretinopathy (Lad et al, Stem Cells Dev, 2008 aug.8). Cardiovascular diseases, including myocardial infarction, atherosclerosis, and valvular disorders May also be treated (Al-Aly Z., Transl Res, 2008 May; 151 (5): 233-9; Kobayashi et Al, Nat Cell Biol, 2009 Jan; 11 (1): 46-55; van Gijn et Al, Cardiovasc Res, 2002 Jul; 55 (1): 16-24; Christman et Al, Am J Physiol Heart Circuit Physiol, 2008 Jun; 294 (6): H2864-70). In some embodiments, the disease is a pulmonary disease, such as idiopathic pulmonary hypertension (idiopathic pulmonary arterial hypertension) or pulmonary fibrosis (laumans et al, Am JRespir Cell Mol Biol, 2008 Nov 21;etc., PLoS ONE, 2008May 14; 3(5): e2142) In that respect In some embodiments, the disease treated with the FZD-binding agent is a Liver disease, such as cirrhosis or fibrosis of the Liver (Cheng et al, Am J Physiol Gastrointest Liver Physiol, 2008 Jan; 294 (1): G39-49).

The present invention provides methods of treating cancer comprising administering to a subject (e.g., a subject in need of treatment) a therapeutically effective amount of an FZD-binding agent. In certain embodiments, the cancer is a cancer selected from the group consisting of colorectal cancer, pancreatic cancer, lung cancer, ovarian cancer, liver cancer, breast cancer, kidney cancer, prostate cancer, gastrointestinal cancer, melanoma, cervical cancer, bladder cancer, glioblastoma, and head and neck cancer. In certain embodiments, the cancer is pancreatic cancer. In certain embodiments, the cancer is colorectal cancer. In certain embodiments, the subject is a human.

The invention also provides methods of inhibiting tumor growth using the antibodies or other agents described herein. In certain embodiments, the method of inhibiting tumor growth comprises contacting a cell in vitro with a FZD-binding agent (e.g., an antibody). For example, an immortalized cell line or cancer cell line expressing the target FZD is cultured in a culture medium, to which the antibody or other agent is added to inhibit tumor growth. In some embodiments, tumor cells are isolated from a patient sample, such as a tissue biopsy, pleural effusion, or blood sample, and cultured in a culture medium to which a FZD-binding agent is added to inhibit tumor growth.

In some embodiments, a method of inhibiting tumor growth comprises contacting a tumor or tumor cell with an FZD-binding agent (e.g., an antibody) in vivo. In certain embodiments, contacting the tumor or tumor cell with the FZD-binding agent is performed in an animal model. For example, FZD-binding agents can be administered to xenografts expressing one or more FZD that have been grown in immunocompromised mice (e.g., NOD/SCID mice) to inhibit tumor growth. In some embodiments, cancer stem cells are isolated from a patient sample such as a tissue biopsy, pleural effusion, or blood sample and injected into an immunocompromised mouse, which is subsequently administered an FZD-binding agent to inhibit tumor growth. In some embodiments, the FZD-binding agent is administered simultaneously with or immediately after the introduction of the tumorigenic cells into the animal, thereby inhibiting tumor growth. In some embodiments, the FZD-binding agent is administered as a therapeutic agent after the tumorigenic cells have grown to a predetermined size.

In certain embodiments, a method of inhibiting tumor growth comprises administering to a subject a therapeutically effective amount of an FZD-binding agent. In certain embodiments, the subject is a human. In certain embodiments, the subject has a tumor or its tumor has been removed.

In certain embodiments, the tumor is a tumor with active Wnt signaling. In certain embodiments, the active Wnt signaling is canonical Wnt signaling. In certain embodiments, the tumor is a Wnt-dependent tumor. For example, in some embodiments, the tumor is sensitive to overexpression of axin. In certain embodiments, the tumor does not comprise an inactivating mutation (e.g., a truncation mutation) in the adenomatous polyposis coli protein (APC) tumor suppressor gene or an activating mutation in the β -catenin gene. In certain embodiments, the tumor expresses one or more genes in a Wnt gene signature. In certain embodiments, the cancer being treated in the subject involves such a tumor.

In certain embodiments, the tumor expresses one or more human frizzled receptors that bind to the FZD-binding agent or antibody. In certain embodiments, the tumor overexpresses a human frizzled receptor.

In certain embodiments, the tumor is a tumor selected from the group consisting of colorectal tumor, pancreatic tumor, lung tumor, ovarian tumor, liver tumor, breast tumor, kidney tumor, prostate tumor, gastrointestinal tumor, melanoma, cervical tumor, bladder tumor, glioblastoma, and head and neck tumor. In certain embodiments, the tumor is a colorectal tumor. In certain embodiments, the tumor is a pancreatic tumor.

The invention also provides a method of inhibiting Wnt signaling in a cell, comprising contacting the cell with an effective amount of a FZD-binding agent. In certain embodiments, the cell is a tumor cell. In certain embodiments, the method is an in vivo method, wherein the step of contacting the cell with the agent comprises administering to the subject a therapeutically effective amount of the agent. In some alternative embodiments, the method is an in vitro or ex vivo method. In certain embodiments, the Wnt signaling that is inhibited is canonical Wnt signaling. In certain embodiments, Wnt signaling is signaling through Wnt1, Wnt2, Wnt3, Wnt3A, Wnt7a, Wnt7b, and/or Wnt 10B. In certain embodiments, Wnt signaling is signaling through Wnt1, Wnt3A, Wnt7b, and/or Wnt 10B.

Furthermore, the present invention provides a method of reducing the tumorigenicity of a tumor in a subject, the method comprising administering to the subject a therapeutically effective amount of an FZD-binding agent. In certain embodiments, the tumor comprises cancer stem cells. In certain embodiments, the frequency of cancer stem cells in the tumor is reduced by administering the agent.

Accordingly, the present invention also provides a method of reducing the frequency of cancer stem cells in a tumor comprising contacting the tumor with a therapeutically effective amount of a FZD-binding agent (e.g., an anti-FZD antibody).

The present invention also provides methods of differentiating a tumorigenic cell into a non-tumorigenic cell, the methods including contacting the tumorigenic cell with a FZD-binding agent (e.g., by administering the FZD-binding agent to a subject that has a tumor containing the tumorigenic cell or that has been removed). In certain embodiments, the tumorigenic cell is a pancreatic tumor cell. In certain alternative embodiments, the tumorigenic cell is a colon tumor cell.

Also provided is the use of a FZD-binding agent, polypeptide, or antibody described herein to induce differentiation of cells, including but not limited to tumor cells. For example, contemplated are methods of inducing differentiation of a cell comprising contacting the cell with an effective amount of a FZD-binding agent described herein (e.g., an anti-FZD antibody). Also provided are methods of inducing differentiation of cells in a tumor in a subject, the method comprising administering to the subject a therapeutically effective amount of a FZD-binding agent, polypeptide, or antibody. In certain embodiments, the tumor is a pancreatic tumor. In certain embodiments, the tumor is a colon tumor.

Further provided are methods of treating a disease or disorder in a subject, wherein the disease or disorder is associated with activation of Wnt signaling and/or is characterized by elevated stem cell and/or progenitor cell levels. In some embodiments, the method of treatment comprises administering to the subject a therapeutically effective amount of an FZD-binding agent, polypeptide, or antibody. In certain embodiments, the Wnt signaling is canonical Wnt signaling.

The invention also provides a method of reducing myofibroblast activation in a solid tumor matrix comprising contacting the matrix with a therapeutically effective amount of a FZD-binding agent, polypeptide, or antibody.

The present invention also provides pharmaceutical compositions comprising one or more FZD-binding agents described herein. In certain embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. These pharmaceutical compositions find use in inhibiting tumor growth and treating cancer in human patients.

In certain embodiments, formulations for storage and use are prepared by combining a purified antibody or pharmaceutical agent of the invention with a pharmaceutically acceptable carrier (e.g., vehicle, excipient) (Remington, the science and Practice of Pharmacy 20th Edition mach Publishing, 2000). Suitable pharmaceutically acceptable carriers include, but are not limited to: non-toxic buffers such as phosphates, citrates and other organic acids; salts such as sodium chloride; antioxidants including ascorbic acid and methionine; preservatives (for example octadecyl dimethyl benzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; phenethylammonium chloride; phenol, butanol or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); small molecular weight polypeptides (e.g., less than about 10 amino acid residues); proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; saccharides such as monosaccharides, disaccharides, glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and nonionic surfactants such as TWEEN or polyethylene glycol (PEG).

The pharmaceutical compositions of the present invention can be administered in a variety of routes for topical or systemic treatment. Administration may be: topical administration (e.g., mucosal administration including vaginal and rectal delivery), such as transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders; pulmonary administration (e.g., by inhalation or insufflation of powders or aerosols, including the use of nebulizers; intratracheal administration, intranasal administration, epidermal administration, and transdermal administration); orally taking; or parenteral administration, including intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial (e.g., intrathecal or intracerebroventricular) administration.

The therapeutic formulation may be in unit dosage form (unit dosage form). Such formulations include tablets, pills, capsules, powders, granules, solutions or suspensions in aqueous or non-aqueous media, or suppositories for oral, parenteral, rectal or administration by inhalation. In solid compositions such as tablets, the principal active ingredient is mixed with a pharmaceutical carrier. Conventional tableting ingredients include corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums and other diluents (e.g. water) used to form solid preformulation compositions comprising a homogeneous mixture of a compound of the present invention or a pharmaceutically acceptable non-toxic salt thereof. The solid preformulation composition is then subdivided into unit dosage forms of the type described above. Tablets, pills, and the like of the novel compositions can be coated or compounded to provide a dosage form having the advantage of long-term action. For example, the tablet or pill may comprise an inner composition coated with an outer ingredient. In addition, the two components may be separated by an enteric layer (enteric layer) which serves to resist disintegration and allows the inner component to pass intact through the stomach or to be delayed in release. A variety of materials may be used for such enteric layers or coatings, including polymeric acids and mixtures of polymeric acids with materials such as shellac, cetyl alcohol and cellulose acetate.

The antibody or agent may also be encapsulated in a microcapsule. Such microcapsules are prepared, for example, hydroxymethylcellulose or gelatin microcapsules and poly- (methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or macroemulsions described in Remington, The Science and Practice of pharmacy 20 th edition Mack Publishing (2000) by, for example, coacervation techniques or interfacial polymerization.

In certain embodiments, the pharmaceutical formulation comprises an antibody or other agent of the invention complexed with liposomes (Epstein, et al, 1985, Proc. Natl. Acad. Sci. USA 82: 3688; Hwang et al, 1980, Proc. Natl. Acad. Sci. USA 77: 4030; and U.S. Pat. Nos. 4,485,045 and 4,544,545). Liposomes having increased circulation time are disclosed in U.S. patent No. 5,013,556. Some liposomes can be produced by reverse phase evaporation using a liquid composition containing phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through a filter having a set pore size to produce liposomes having the desired diameter.

In addition, sustained release formulations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels such as poly (2-hydroxyethyl-methacrylate) or poly (vinyl alcohol), polylactide (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and 7-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT (TM) (microspheres for injection composed of lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose isobutyl acetate, and poly-D- (-) -3-hydroxybutyric acid.

In certain embodiments, the methods or treatments further comprise (prior to, concurrently with, and/or after administration of the FZD-binding agent) administration of a second anti-cancer agent in addition to the FZD-binding agent. Also provided are pharmaceutical compositions comprising the FZD-binding agent and the second anticancer agent.

It will be appreciated that the combination of FZD-binding agent and second anticancer agent can be administered in any order or simultaneously. In selected embodiments, the FZD-binding agent will be administered to a patient that has been previously treated with the second anticancer agent. In certain other embodiments, the FZD-binding agent and the second anticancer agent are administered substantially simultaneously or concurrently. For example, the subject may receive the FZD-binding agent while undergoing a treatment course with a second anti-cancer agent (e.g., a chemotherapeutic agent). In certain embodiments, the FZD-binding agent is administered within 1 year after treatment with the second anticancer agent. In certain alternative embodiments, the FZD-binding agent is administered within 10, 8, 6, 4, or 2 months after any treatment with the second anticancer agent. In certain other embodiments, the FZD-binding agent is administered within 4, 3, 2, or 1 weeks after any treatment with the second anticancer agent. In some embodiments, the FZD-binding agent is administered within 5, 4, 3, 2, or 1 days after any treatment with the second anticancer agent. It will also be appreciated that the two agents or treatments may be administered to the subject in as little as a few hours or minutes (i.e., substantially simultaneously).

Useful classes of anticancer agents include, for example, tubulin inhibitors, auristatins (auristatins), DNA minor groove binders, DNA replication inhibitors, alkylating agents (e.g., platinum complexes such as cisplatin, monoplatin, biplatin, and trinuclear platinum complexes, and carboplatin), anthracyclines, antibiotic antifolates, antimetabolites, chemosensitizers, duocarmycins, etoposide, fluorinated pyrimidines, ionophors, distamycin (lexicin), nitrosoureas, cisplatin (platinol), preformed compounds (purine antimetabolites), puromycin, radiosensitizers, steroids, taxanes (taxanes), topoisomerase inhibitors, or vinca alkaloids, and the like. In certain embodiments, the second anti-cancer agent is an antimetabolite, antimitotic, topoisomerase inhibitor, or angiogenesis inhibitor.

Anti-cancer agents that may be administered in combination with FZD-binding agents include chemotherapeutic agents. Thus, in some embodiments, the methods or treatments involve the combined administration of a chemotherapeutic agent or a mixture of multiple different chemotherapeutic agents and an antibody or agent of the invention. Treatment with the antibody may be performed before, simultaneously with, or after the administration of chemotherapy. Chemotherapeutics for use in the present invention include chemicals or drugs known and commercially available in the art, such as gemcitabine, irinotecan, doxorubicin, 5-fluorouracil, cytarabine ("Ara-C"), Cyclophosphamide (Cyclophosphamide), thiophosphine triamide, busulfan, Cyclophosphamide (Cytoxin), paclitaxel, methotrexate, cisplatin, melphalan, vinblastine, and carboplatin. Combined administration may include co-administration of a single pharmaceutical agent or using separate multiple agents, or sequential administration in any order but typically over a period of time such that all active agents exert their biological activity simultaneously. The formulation and dosage regimen for such chemotherapeutic agents may be used according to the manufacturer's instructions or as determined empirically by one skilled in the art. Formulations and dosage regimens for such Chemotherapy are also described in Chemotherapy Service ed, m.c. perry, Williams & Wilkins, Baltimore, Md, (1992).

Chemotherapeutic agents useful in the present invention also include, but are not limited to: alkylating agents such as thiophosphine triamine and carcinostat (cycloxan); alkyl sulfonates such as busulfan, amisulide, and propionyl piperazine dimethane sulfonate (piposulfan); aziridines such as benzodidopa, carboquone, methurodopa (meteedopa), and urodopa (uredopa); ethyleneimine and methyl melamine including triethylmelamine, trivinyl melamine, trivinyl phosphoramide, trivinyl thiophosphoramide and trimethylol melamine; nitrogen mustards such as chlorambucil, chlorophosphamide (cholophosphamide), estramustine, ifosfamide, mechlorethamine (mechlorethamine), oxydichloromethyldiethylamine hydrochloride, melphalan, neomustard, cholesterol chlorambucil, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorouramicin, fotemustine, lomustine, pyriminosulfenamide, and ranolazine; antibiotics such as aclacinomycin, actinomycin, amtricin, azaserine, bleomycin, actinomycin C, calicheamicin, carrubicin, carminomycin, carcinotropic, chromomycin, actinomycin D, orthopyricin, ditetracycline, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, isorubicin, idarubicin, sisomicin, mitomycin, mycophenolic acid, nogomycin, olivomycin, pelomycin, mitomycin methyl, puromycin, trirubicin, roxobicin, streptonigrin, streptozotocin, tubercidin, ubenimex, setastin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate, and the like; purine analogs such as fludarabine, 6-mercaptopurine, thiomiaurine, thioguanine; pyrimidine analogs such as Cytidine, azacitidine, 6-azauridine, carmofur, Cytarabine, dideoxyuridine, deoxyfluorouridine, enocitabine, fluorouridine, 5-FU, and the like; androgens such as carroterone, prasterone androsterone propionate, epithioandrostanol, meindrotane, testolactone, and the like; anti-adrenal agents such as aminoglutethimide, mitotane, trostane, and the like; folic acid supplements such as folinic acid; acetic acid glucurolactone; an aldehydic phosphoramide glycoside; (ii) aminolevulinic acid; amsacrine; (ii) a bittubacil (betrabucil); a bisantrene group; edatrexae; defofamine (decafamine); dimecorsine; diazaquinone; aifensin (elformithine); ammonium etiolate; etoglut; gallium nitrate; a hydroxyurea; (ii) mushroom polysaccharides; lonidamine; propionylaminohydrazone; mitoxantrone; mopidanol; diamine nitracridine (nitrarine); gustatostatin; methionine; a pyran doxorubicin; podophyllinic acid (podophyllic acid); 2-ethyl hydrazide; procarbazine; PSK; lezoxan; sisofilan (sizofuran); a germanium spiroamine; alternarionic acid; a tri-imine quinone; 2, 2', 2 "-trichlorotriethylamine; a carbamate; vindesine; (ii) azotemidine; mannomustine; dibromomannitol; dibromodulcitol; pipobroman; glycocytosine (glycocytosine); arabinoside ("Ara-C"); cyclophosphamide; thiophosphine triamine; taxanes (taxoids), such as paclitaxel (TAXOL, Bristol-myers squibb Oncology, Princeton, NJ.) and docetaxel (TAXOTERE, Rhone-Poulenc ror, antonyx, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine (vinorelbine); navelbine (navelbine); norfloxacin (novantrone); (ii) teniposide; daunomycin; aminopterin; (xiloda); ibandronate (ibandronate); CPT 11; topoisomerase inhibitor RFS 2000; difluoromethyl ornithine (DMFO); retinoic acid; esperamicin (esperamicin); capecitabine (capecitabine); and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing. Chemotherapeutic agents also include anti-hormonal agents that can modulate or inhibit the effects of hormones on tumors, such as anti-estrogens, including, for example, tamoxifen (tamoxifen), raloxifene (raloxifene), aromatase that inhibits 4(5) -imidazole, 4-hydroxyttamoxifen, trioxifene (trioxifene), raloxifene hydrochloride (keoxifene), LY 117018, onapristone (onapristone), and toremifene (toremifene) (Fareston); and antiandrogens, such as flutamide (flutamide), nilutamide (nilutamide), bicalutamide (bicalutamide), leuprolide, and goserelin (goserelin); and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing.

In certain embodiments, the chemotherapeutic agent is a topoisomerase inhibitor. Topoisomerase inhibitors are chemotherapeutic agents that interfere with the action of a topoisomerase enzyme (e.g., topoisomerase I or II). Topoisomerase inhibitors include, but are not limited to, doxorubicin hydrochloride, nordstrandycin citrate, mitoxantrone hydrochloride, actinomycin D, etoposide, topotecan hydrochloride, teniposide (VM-26), and irinotecan. In certain embodiments, the second anticancer agent is irinotecan. In certain embodiments, the tumor to be treated is a colorectal tumor and the second anticancer agent is a topoisomerase inhibitor, e.g., irinotecan.

In certain embodiments, the chemotherapeutic agent is an antimetabolite. An antimetabolite is a chemical substance that is structurally similar to the metabolites required for normal biochemical reactions, but differs sufficiently to interfere with one or more normal functions of a cell, such as cell division. Antimetabolites include, but are not limited to, gemcitabine, fluorouracil, capecitabine, methotrexate sodium, raltitrexed, Pemetrexed, tegafur, cytarabine, thioguanine (GlaxoSmithKline), 5-azacytidine, 6-mercaptopurine, azathioprine, 6-thioguanine, pentostatin (pentostatin), fludarabine phosphate (fludarabine phosphate), and cladribine (cladribine), as well as pharmaceutically acceptable salts, acids, or derivatives of any of these. In certain embodiments, the second anticancer agent is gemcitabine. In certain embodiments, the tumor to be treated is a pancreatic tumor and the second anticancer agent is an antimetabolite (e.g., gemcitabine).

In certain embodiments, the chemotherapeutic agent is an antimitotic agent, including but not limited to agents that bind to tubulin. As a non-limiting example, the agent comprises a taxane. In certain embodiments, the agent comprises paclitaxel or docetaxel, or a pharmaceutically acceptable salt, acid or derivative of paclitaxel or docetaxel. In certain embodiments, the agent is paclitaxel (TAXOL), docetaxel (TAXOTERE), albumin-bound paclitaxel (e.g., ABRAXANE), DHA-paclitaxel, or PG-paclitaxel. In certain alternative embodiments, the antimitotic agent comprises a vinca alkaloid, such as vincristine, vinblastine, vinorelbine, or vindesine, or a pharmaceutically acceptable salt, acid, or derivative thereof. In some embodiments, the anti-mitotic agent is an inhibitor of Eg5 kinesin or an inhibitor of a mitotic kinase (e.g., Aurora a or Plk 1). In certain embodiments, when the chemotherapeutic agent administered in combination with the FZD-binding agent or polypeptide or antibody comprises an anti-mitotic agent, the cancer or tumor treated is breast cancer or a breast tumor.

In certain embodiments, treatment involves administration of radiation therapy in combination with an antibody (or other agent) of the invention. Treatment with the antibody (or other agent) may be performed before, simultaneously with, or after the radiation therapy. Any dosing regimen for such radiation therapy as determined by the skilled person may be used.

In some embodiments, the second anti-cancer agent comprises an antibody. Thus, treatment can involve the combined administration of an antibody (or other agent) of the invention and other antibodies against additional tumor-associated antigens, including but not limited to antibodies that bind EGFR, ErbB2, HER2, DLL4, Notch, and/or VEGF. Exemplary anti-DLL 4 antibodies are described, for example, in U.S. patent application No. 2008/0187532 (incorporated herein by reference in its entirety). In certain embodiments, the second anti-cancer agent is an antibody that is an angiogenesis inhibitor (e.g., an anti-VEGF antibody). Additional anti-DLL 4 antibodies are described, for example, in international patent nos. WO2008/091222 and WO2008/0793326, U.S. patent application nos. 2008/0014196, 2008/0175847, 2008/0181899, and 2008/0107648, each of which is incorporated herein by reference in its entirety. Exemplary anti-Notch antibodies are described, for example, in U.S. patent application No. 2008/0131434 (incorporated herein by reference in its entirety). In certain embodiments, the second anti-cancer agent is an antibody that is an angiogenesis inhibitor (e.g., an anti-VEGF antibody). In certain embodiments, the second anti-cancer agent is an inhibitor of Notch signaling. In certain embodiments, the second anti-cancer agent is AVASTIN (bevacizumab), Herceptin (trastuzumab), VECTIBIX (panitumumab), or Erbitux (cetuximab). Combined administration may include co-administration of a single pharmaceutical agent or using separate multiple agents, or sequential administration in any order but typically over a period of time such that all active agents exert their biological activity simultaneously.

In addition, treatment may include administration of one or more cytokines (e.g., lymphokines, interleukins, tumor necrosis factors, and/or growth factors) or any other therapy that may be accompanied by surgical removal of cancer cells or deemed necessary by the treating physician.

For the treatment of a disease, the appropriate amount of the antibody or agent of the invention will depend on the type of disease to be treated, the severity and course of the disease, the responsiveness of the disease, whether the antibody or agent is administered for therapeutic or prophylactic purposes, previous therapy, patient clinical history, etc., and is determined by the treating physician. The antibody or agent can be administered at one time, or in a series of treatments over a period of days to months, or until a cure is achieved or the disease state is reduced (e.g., tumor size is reduced). The optimal dosage regimen can be calculated from measurements of drug accumulation in the patient and will vary with the relative potency of each antibody or agent. The optimum amount, method of administration and repetition rate can be readily determined by the administering physician. In certain embodiments, the amount is 0.01 μ g to 100mg/kg body weight and may be administered more than once per day, week, month or year. In certain embodiments, the antibody or other FZD-binding agent is administered once every two weeks or once every three weeks. In certain embodiments, the amount of antibody or other FZD binding agent is from about 0.1mg to about 20mg per kg of body weight. The treating physician can estimate the repetition rate based on the measured residence time and the concentration of the drug in the body fluid or tissue.

Wnt gene tag and application thereof

The invention also provides Wnt gene signatures that are gene signatures indicative of Wnt signaling activity in tumors and can be used to select tumors, patients, and/or therapies.

The Wnt gene signature includes the differential expression of a set of genes in tumors in which Wnt signaling is activated (and/or dependent on Wnt signaling) compared to tumors in which Wnt signaling is not activated. In certain embodiments, the Wnt signaling is canonical Wnt signaling. The Wnt gene signature can be used to identify tumors and/or patients that are likely to respond to treatment with a Wnt signaling inhibitor (e.g., a FZD-binding agent that is at least one human frizzled receptor antagonist and/or Wnt signaling inhibitor).

In certain embodiments, the Wnt gene signature comprises one or more genes (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 genes) listed in table 3 below. ' Probe setID "serial number isProbe set ID numbers for the human genome U133 Plus 2.0 array ("HG _ U133_ Plus _ 2"; Affymetrix, Santa Clara, Calif.). In tumors in which Wnt signaling was activated (i.e., tumors that showed a positive Wnt gene signature), the expression levels of the genes in table 3 that make up the Wnt gene signature were elevated relative to tumors in which Wnt signaling was not activated. In some embodiments, the Wnt gene signature comprises two or more genes listed in table 3 below. In some embodiments, the Wnt gene signature comprises three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or more, sixteen or more, seventeen or more, eighteen or more, or nineteen of the genes listed in table 3 below. In certain embodiments, the tumor being evaluated for the level of gene expression is a colorectal tumor, wherein the gene is one or more of the genes in table 3. In certain embodiments, the Wnt gene signature comprises AXIN2 and/or FOXQ 1. In certain embodiments, the tumor is a colorectal tumor and the Wnt gene signature comprises AXIN2, LGR5, and/or FOXQ 1.

Table 3: exemplary Wnt Gene signature genes

Also provided are methods of using Wnt gene signatures to select (or identify) patients eligible for treatment with Wnt pathway inhibitors or for evaluation of the utility of a particular therapy. In certain embodiments, the Wnt signaling inhibitor is a FZD-binding agent, such as an antagonistic FZD antibody. For example, a patient may be identified as suitable for treatment with a FZD-binding agent (or FZD-binding agents) by determining whether a tumor in the patient or a tumor that has been removed from the patient exhibits a Wnt gene signature. In certain embodiments, detecting the Wnt gene signature comprises assessing the expression level of one or more genes in table 3 in a tumor. Identifying the patient as suitable for treatment with a FZD-binding agent (e.g., an anti-FZD antibody that inhibits Wnt signaling) if one or more genes in table 3 that make up the Wnt gene signature are elevated in expression level in the tumor (thereby indicating that Wnt signaling is activated in the tumor). Also provided are methods of using Wnt gene signatures to select appropriate therapies for a particular patient.

The present invention provides a method of treating cancer in a patient having a tumour or a tumour thereof which has been removed, the method comprising (a) providing the expression level in the tumour of one or more genes of table 3 (b) selecting a patient for whom treatment with a FZD-binding agent is to be commenced or continued in dependence on the expression level of the one or more genes, and (c) administering the FZD-binding agent to the patient. In certain embodiments, the method comprises measuring the expression level of one or more genes in the tumor. In certain embodiments, the expression level of one or more genes is compared to a control or reference level.

Also provided are methods of identifying tumors that can respond to treatment with a Wnt signaling inhibitor. In certain embodiments, the Wnt signaling inhibitor is a FZD-binding agent. In certain embodiments, the method comprises subjecting the tumor to a Wnt gene signature test. In certain embodiments, the method comprises assessing the expression level of one or more genes in table 3 in the tumor.

Also provided are methods of screening for candidate drugs against tumors that have been identified as exhibiting a Wnt gene signature. In certain embodiments, the candidate drug is a Wnt signaling inhibitor. Such drug candidates are preferably tested for their utility in tumors with active and/or dependent Wnt signaling. The present invention also provides a method of screening for a drug candidate comprising (a) assessing the expression level of one or more genes in table 3 in a tumour, (b) selecting (at least in part) a tumour for testing with a drug candidate based on the expression level of the one or more genes, and (c) testing the effect of the drug candidate on the tumour.

Furthermore, in certain embodiments, the effect of a drug on the Wnt gene signature can be determined and used to assess the therapeutic utility of a tumor with active Wnt signaling. In certain embodiments, this provides a method of monitoring therapy administered to a patient. In some alternative embodiments, this provides a method of assessing the utility of a drug candidate. In certain embodiments, a decrease in the expression level of one or more genes in table 3 (i.e., a decrease or elimination of the Wnt gene signature) indicates the utility of the treatment.

In certain embodiments, assessing the level of one or more genes in the Wnt gene signature comprises determining the expression level of a polynucleotide of the one or more genes. In certain embodiments, detection of a Wnt gene signature comprises detecting mRNA expression of polynucleotides of one or more genes that make up the signature. In some embodiments, the detection of mRNA expression is by northern blot. In some embodiments, the detection of mRNA expression is by RT-PCR, real-time PCR, or quantitative PCR, using a primer set that specifically amplifies the polynucleotides that make up the cancer stem cell signature. In certain embodiments, the detection of mRNA comprises contacting the sample with a nucleic acid probe that is complementary to a polynucleotide that constitutes a cancer stem cell gene signature. In some embodiments, the mRNA of the sample is converted to cDNA prior to detection. In some embodiments, detection of mRNA is by a microarray comprising polynucleotides that hybridize to one or more genes in the Wnt gene signature.

In certain embodiments, assessing the level of one or more genes in the Wnt gene signature comprises detecting polypeptides encoded by the one or more genes. In some embodiments, assessing the level of the polypeptide expression product of the one or more genes comprises contacting the sample with an antibody specific for the polypeptide and detecting binding of the antibody to the polypeptide by, for example, quantitative immunofluorescence or ELISA. Other detection methods are known to those skilled in the art, see, for example, U.S. Pat. No. 6,057,105.

Also provided are arrays comprising polynucleotides that hybridize under stringent conditions to one or more genes in table 3. Kits comprising the arrays are also provided.

Also provided are kits comprising antibodies that bind to the expression product of one or more genes in table 3.

Kit comprising a FZD-binding agent

The present invention provides kits comprising the antibodies or other agents described herein, which can be used to carry out the methods described herein. In certain embodiments, the kit comprises at least one purified antibody against one or more human frizzled receptors in one or more containers. In some embodiments, the kit contains all components necessary and/or sufficient to perform a detection assay, including all controls, instructions for performing the assay, and any necessary software for analyzing and displaying the results. One skilled in the art will readily recognize that the disclosed antibodies or agents can be readily incorporated into a shaped kit format as is known in the art.

Kits comprising a FZD-binding agent (e.g., a FZD-binding antibody) and a second anticancer agent are also provided. In certain embodiments, the second anticancer agent is a chemotherapeutic agent (e.g., gemcitabine or irinotecan). In certain embodiments, the second anti-cancer agent is an angiogenesis inhibitor. In certain embodiments, the second anti-cancer agent is a Notch signaling inhibitor (e.g., anti-DLL 4 or an anti-Notch antibody).

Also provided is a kit comprising a FZD-binding agent and one or more agents that assess the expression of one or more genes in table 3 above ("exemplary Wnt gene signature genes").

Embodiments of the present disclosure can be further defined by reference to the following non-limiting examples that detail the preparation of certain antibodies of the present disclosure and methods of using the antibodies of the present disclosure. It will be apparent to those skilled in the art that various modifications, both to materials and methods, may be practiced without departing from the scope of the present disclosure.

Examples

It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application.

Example 1 identification/preparation of anti-FZD antibody

Phage display can be used to isolate human antibodies that specifically recognize one or more human frizzled receptors. For example, a synthetic antibody library comprising human antibody variable domains can be panned to screen for specific and high affinity recognition of the extracellular domain of the human FZD7 receptor. Once a particular Fab with the desired properties was identified, the human variable region of the Fab was then cloned into an Ig expression vector containing the heavy and light chains (κ or λ) of human IgG2, which was used to express human antibodies in CHO cells.

Specific fabs that bind to the extracellular domain of FZD7 were identified using phage display: 18R 8. 2X 10 from human Fab phage library¹³Individual Fab-displaying phage particles were incubated with passively immobilized recombinant FZD7 ECD Fc protein. Non-specific phages were washed off, followed by elution of specific phages with DTT. The TG 1F + bacteria were infected with the eluted product and rescued with helper phage. Fab display was then induced with IPTG (0.25 mM). To thereby pass throughThe first round of rescue products served as starting points for other rounds of selection. Selection was continued through the third round, followed by screening of the products in ELISA to obtain fabs specific for the recombinant FZD7 ECD Fc protein. Fab that specifically binds to human FZD7 was identified.

The variable region sequences of the identified fabs were obtained. The N-linked glycosylation site is removed from the parent sequence by site-directed mutagenesis. The N-linked glycosylation site Asn in the heavy chain CDR1 was changed to His. This mutation was made to prevent glycosylation upon expression in mammalian systems. The Fab obtained was designated 18R 8. The heavy and light chain CDR sequences of 18R8 are shown in table 4 below. The VH and VL sequences of 18R8 are set forth in SEQ ID NOs: 10 and SEQ ID NO: 12 is provided.

Table 4: CDRs of 18R8 and 18R5 human antibodies

The site-directed changes used to remove the N-linked glycosylation sites are underlined.

The VH-CH1 chain of the 18R8 Fab was linked to multiple VL-CL chains from the original Fab phage library (i.e., the library from which 18R8 was identified) to produce an FZD-resistant Fab 18R 5. After three rounds of panning with the immobilized recombinant FZD7 ECD Fc protein, 18R5 was isolated from the library. The CDR sequences of 18R5 are shown in table 4 above. The VL of the 18R5 antibody has the amino acid sequence of SEQ ID NO: 14, or a sequence shown in fig. 14. The heavy chain CDRs and VH of the 18R5 antibody are the same as for the 18R8 antibody.

The human variable regions of 18R8 and 18R5Fab were cloned into Ig expression vectors containing human IgG2 heavy and light chains (λ) for expression in CHO cells. The amino acid sequences (including signal sequences) of the heavy and light chains of the 18R8IgG antibody are set forth in SEQ ID NOs: 11 and SEQ ID NO: 13 is provided. The signal sequence at the N-terminus of the amino acid sequence of each chain is cleaved off upon secretion. The nucleic acid sequences encoding the heavy and light chains of the 18R8IgG antibody are set forth in SEQ ID NOs: 18 and SEQ ID NO: 20 is provided. The amino acid sequences of the heavy and light chains of the 18R5IgG antibody are set forth in SEQ ID NOs: 11 and SEQ ID NO: 15 is provided. (similarly, the signal sequence at the N-terminus of each chain amino acid sequence is cleaved off upon secretion.) the nucleic acid sequences encoding the heavy and light chains of the 18R5IgG antibody are set forth in SEQ ID NO: 18 and SEQ ID NO: 22. These antibodies were purified using protein a purification.

K determination of 18R8 and 18R5 antibodies using the Biacore 2000 System from Biacore Lifescience (GE Healthcare)_D. Specifically, the purified anti-Fzd 7 antibody was serially diluted from 100nM to 0.78nM in two-fold increments with HBS-P (0.01M HEPES pH 7.4, 0.15M NaCl, 0.005% (v/v) surfactant P20). Each dilution was tested against recombinant Fzd Fc protein immobilized on CM5 Biacore chips. Measurement of association and dissociation rates and determination of K using the Biaeevaluation software program_DValues (table 5 below).

Table 5: affinity of 18R8 and 18R5IgG antibodies

Example 2 FACS analysis of anti-FZD antibodies demonstrated binding to a variety of cell surface human FZD.

Flow cytometry analysis was used to determine the ability of the antibody to bind to cell surface expressed FZD protein.

In order to enable strong expression of the selected FZD protein on the cell surface, mammalian expression plasmids containing a CMV promoter upstream of the FZD-encoding polynucleotide (such constructs are referred to as "FL-FLAG-free") were generated using standard recombinant DNA techniques. Similar expression plasmids were generated for each of the 10 human frizzled proteins. An alternative version of the FZD expression vector was also prepared in which a polynucleotide encoding an N-terminal signal sequence-FLAG epitope tag fused to the N-terminus of the mature FZD protein was also generated by standard recombinant techniques (such construct is referred to as "FL FLAG"). Furthermore, expression vectors were designed which encode a chimeric protein consisting of: the CRD domain of FZD (also known as "fri" domain) or the entire N-terminal extracellular domain of FZD protein (referred to as "fri FLAG" and "ECD FLAG", respectively) fused to N-terminal signal sequence-FLAG epitope, and the C-terminal portion encoding transmembrane and cytoplasmic domains of human CD4 protein.

To measure binding of antibodies to FZD by flow cytometry, HEK293 cells were co-transfected with FZD expression vector and transfection marker GFP. After 24-48 hours of transfection, cells were collected in suspension and incubated on ice with anti-FZD antibody (10 μ g/ml unless otherwise indicated) or with control IgG that detected background antibody binding. Cells are washed and primary antibodies are detected with an Fc fragment specific secondary antibody (e.g., anti-human IgG coupled to phycoerythrin) coupled to a fluorescent chromophore. The labeled cells are then analyzed by flow cytometry to identify anti-FZD antibodies that specifically recognize cell surface FZD protein expression. Monoclonal antibodies 18R5 and 18R8 recognize FZD located on transfected cells. As shown in fig. 1 and 2, each of 18R8 and 18R5 may bind a plurality of FZD including FZD1, FZD2, FZD5, FZD7, and FZD 8. To investigate the relative ability of 18R8 and 18R5 to bind to each FZD, titration assays were performed with varying amounts of antibody in the binding reactions (figure 2). This analysis showed that 18R5 showed stronger binding ability to each FZD receptor (FZD1, FZD2, FZD5, FZD7, and FZD8) than 18R 8.

Example 318 inhibition of Wnt signaling by R8 and 18R5

The ability of anti-FZD IgG antibodies 18R8 and 18R5 to block activation of the Wnt signaling pathway was determined in vitro using a luciferase reporter assay.

STF293 cells were cultured in DMEM supplemented with antibiotics and 10% FCS. STF293 cells are 293 cells in which the following are stably integrated: (1) an 8XTCF Luc reporter vector for measuring canonical Wnt signaling levels, comprising 7 copies of TCF binding site linked to a promoter upstream of the firefly luciferase reporter (Gazit et al, 1999, Oncogene 18: 5959-66) and (2) Renilla luciferase reporter as an internal control for transfection efficiency (Promega; Madison, Wis.). Cells were added to the medium plate. FZD antibody to be tested (or no antibody added) was added. Cells were then incubated in the presence or absence of Wnt3A conditioned medium, prepared from L cells stably expressing Wnt3a (ATCC CRL-2647), or control conditioned medium, prepared from L cells that did not overexpress Wnt3A (ATCC cell line CRL-2648). After overnight incubation, luciferase levels were measured using a dual luciferase assay kit (Promega; Madison, Wis.), where firefly luciferase activity was normalized to Renilla luciferase activity.

The ability of the 18R8 and 18R5 antibodies to inhibit Wnt-induced pathway activation was thus determined. STF293 cells were treated with different concentrations of 18R8 or 18R5 IgG antibody, supplemented with Wnt3A conditioned medium. Cells were assayed 18 hours later using a dual luciferase assay kit. The results are shown in fig. 3. A stronger inhibition of TCF signaling of the Wnt3a pathway was observed with anti-FZD antibody 18R 5.

In other experiments, the ability of the 18R8 antibody to antagonize signaling by different Wnt ligands was determined. HEK293 cells were transfected by Fugene 6(Roche) with Wnt1, Wnt2, Wnt2b2, Wnt3, Wnt3a, Wnt7a, Wnt7b, Wnt8a, Wnt9b and Wnt10b for 48 hours. Wnt3A conditioned medium ("Wnt 3 ACM") was used as a positive control for activation. STF293 cells were cultured in DMEM supplemented with antibiotics and 10% FCS and treated with or without 20 μ g/ml 18R8 antibody. Wnt-overexpressing HEK293 cells were subsequently added. Luciferase levels were measured 18 hours after treatment using a dual luciferase assay kit. The results are shown in fig. 4. anti-FZD antibody 18R8 was shown to inhibit signaling of various Wnt, including Wnt1, Wnt2, Wnt3, Wnt7A, Wnt7B, and Wnt10B in addition to Wnt 3A.

Example 418R 8 blocking the binding of FZD to Wnt

To evaluate the ability of 18R8 to block FZD binding to Wnt, 2 μ l of 1.32 μ g/μ l of soluble FZD8-Fc containing Fri domain (FZD 8 amino acids 1-157 linked in the same reading frame to human IgG1 Fc) was added to the culture medium alone or in the presence of 18R8 IgG antibody (with addition of 4 μ l of 3.71 μ g/μ l antibody) to bind Wnt3A (with addition of 20 μ l of Wnt3A conditioned medium). The mixture was incubated at 4 ℃ for 2 hours either alone or with protein A Sepharose microspheres (product of GE Healthcare; 20. mu.l of 50% solution in PBS) for 2 hours. After incubation, the protein a microspheres (and any protein complexed to the protein a microspheres) in each sample were removed by centrifugation and the supernatant was assayed for its ability to induce 8xTCF luciferase activity. The supernatant was added to STF293 cells that stably expressed 8xTCF (8 copies of TCF binding domain located upstream of firefly luciferase reporter) for measurement of canonical Wnt signaling levels and cultured in DMEM supplemented with antibiotics and 10% FCS. Luciferase levels were measured 18 hours after treatment of STF293 cells using a dual luciferase assay kit (Promega; Madison, Wis.).

As seen in figure 5, Wnt3A + protein a induced strong activation of the reporter gene as measured in luciferase units (RLU) (figure 5, column 1). Addition of Fzd8-Fc induced complexation between Wnt3A-Fzd8 and between the Fc fusion protein and protein a sepharose microspheres, and once the protein a-Wnt3A-Fzd8 complex was removed by centrifugation, the supernatant was no longer able to activate the reporter gene (figure 5, column 2). Blocking of the Wnt3A interacting domain of Fzd8 by this antibody presumably results in the breakdown of the complex as described above upon addition of 18R8, because although the protein a-Fzd8-Fc complex is removed along with 18R8 (which can readily interact with protein a through its own Fc moiety), reporter activity can be readily observed, indicating the presence of sufficient levels of uncomplexed Wnt3A (figure 5, column 3). 18R8 also acted on endogenous Fzd present on STF293 cells, as the absence of removal of 18R8 with protein a did not show recovery of Wnt3A activation (figure 5, bar 6).

The data in figure 5 show that Wnt3A activity in the supernatant was retained in the presence of 18R8IgG antibody in the incubation reaction relative to the activity observed with FZD8-Fc alone. These results indicate that 18R8 blocks the ability of FZD8 to bind Wnt3A, and that antibodies that bind to epitopes recognized by 18R8 are able to block Wnt-FZD interactions.

Epitope mapping of examples 518R 8 and 18R5

To identify the FZD epitope recognized by the 18R8IgG antibody, epitope mapping was performed. Antibody binding was measured using flow cytometry analysis of cell surface expressed FZD. Mammalian expression plasmid vectors containing the CMV promoter upstream of the following polynucleotides were produced by standard recombinant techniques: the polynucleotide encodes an N-terminal signal sequence FLAG epitope tag fused to the N-terminus of the Fri domain of FZD8, which is in turn fused to the transmembrane and intracellular domains of the CD4 protein. This expression construct allows the FZD8Fri domain to be expressed on the cell surface and express a FLAG epitope tag that is used to monitor expression. Site-directed mutagenesis was subsequently used to make alterations to selected amino acids in the extracellular domain of FZD. HEK293 cells were co-transfected with expression vectors encoding FZD and the transfection marker GFP. 48 hours after transfection, cells were collected in suspension and incubated with anti-FZD antibody on ice or control IgG that detected binding of background antibody. Cells were washed and for the primary antibody, a secondary antibody against the antibody coupled to a fluorescent chromophore was used for detection. The labeled cells are then analyzed by flow cytometry to measure the binding of anti-FZD antibodies to cell surface FZD.

Thus, specific amino acids in the extracellular domain of FZD important for binding to anti-FZD antibodies were identified. When amino acid residues 82-83 of FZD8 were mutated from PD to SQ, binding of 18R8 to FZD8 was substantially unaffected (fig. 6). Similarly, when amino acid 109S was changed to 109Q, no significant effect on binding was observed. On the other hand, when residues 70 to 71 of FZD8 were changed from HQ to AE, binding of 18R8 to FZD8 on cells was significantly weakened (fig. 6 and 7). When amino acids 66 to 67 were mutated from GL to AA, or amino acids 68 to 69 were mutated from EV to QL, or amino acids 126 to 127 were mutated from FG to NV, binding of the 18R8 antibody to FZD8 on the cell similarly disappeared. When certain amino acids of the FZD extracellular domain are substituted, binding to FZD disappears, revealing the specific recognition site of the antibody. Thus, it was determined that the antibody 18R8 binds to an epitope of FZD8 that comprises amino acids 66-71 GLEVHQ (SEQ ID NO: 25) and amino acids 124-128 GF of human FZD 8. This epitope region is highly conserved among the human frizzled receptors to which FZD8 binds (i.e., FZD1, FZD2, FZD5, FZD7, and FZD8), while this epitope region is not highly conserved among those to which FZD8 does not bind (i.e., FZD3, FZD4, FZD6, FZD9, or FZD 10).

FACS experiments were also performed comparing the binding of 18R5 IgG and 18R8 IgG to wild-type and mutant FZD8 on cells. These experiments showed that the 18R8 antibody and the 18R5 antibody bound similar epitopes on FZD8 (fig. 7).

Example 6 identification of biological binding sites (BBS of FZD receptor)

Based on the discovery of antibodies that inhibit Wnt signaling and the discovery of epitopes in FZD proteins bound by these antibodies, it has now been possible to analyze which regions of FZD protein structure are important for Wnt signaling. To investigate this, the crystal structure of the Fri domain of mouse Fzd8 was investigated. The present inventors have identified that the 18R8 and 18R5 binding epitopes are located in a region of the FZD structure that has never been previously recognized as having a specific function. In addition the epitope comprises two separate surface elements of Fzd (referred to as "upper edge" and "lower edge") separated by a cleft. A comparison of the 10 human frizzled receptors also found that there was a striking conservation in identity of the amino acids arranged at the base of the cleft. The region to which 18R8 and 18R5 bind, which comprises the cleft and the "upper edge" and "lower edge" has been designated as the biological binding site of FZD (BBS). Structural images of the FZD Fri domain based on analysis of the previously reported crystal structure of mouse FZD8 (Dann CE et al, Nature 412(6842)86-90, 2001) and analysis done using the software program Pymol are shown in figure 9. In the upper left panel, a surface view of the FZD Fri domain is shown, with the region of the FZD protein containing the Biological Binding Site (BBS) marked with white circles. Each region of the BBS designated "upper edge", "lower edge" and "crack" is highlighted with darker surface coloration in the single image at the bottom of the figure. Conserved residues in 9 or 10 of the human Fzd family members are highlighted with darker surface coloration in the upper right image of figure 9.

Example 718 inhibition of tumor growth in vivo by R5

18R5 preventing Wnt-dependent tumor growth

50,000 Mouse Mammary Tumor Virus (MMTV) -WNT1 tumor-derived cells were injected into the upper right mammary fat pad of 5-7 week old female NOD/SCID mice. Transgenic (MMTV) -Wnt-1 mice display discrete stages of mammary tumorigenesis, including hyperplasia, invasive ductal carcinoma, and distant metastasis, and therefore this mouse model of breast cancer provides a powerful tool for analyzing the role of Wnt in tumor formation and growth (Nusse and Varmus (1982) Cell 31: 99-109). Tumors from these mice were isolated and these isolated tumor cells were used for tumor propagation purposes. Mice implanted with tumor cells in the mammary fat pad were monitored twice weekly. Once tumors became palpable, tumors were measured twice a week using the formula 1/2(a × b)²) To determine tumor volume, where a is length and b is width. Data are expressed as mean and mean ± s.e.m. Group means were compared using two-tailed unpaired student's t-test. Will be provided withA probability (p) value of less than 0.05 was considered significant. On day 19, the mean tumor volume was 44mm³The mice were randomly divided into 2 groups of 10 animals each. Animals were injected with either control antibody or 18R5IgG antibody (10 mg/kg). The antibody was administered by injection into the peritoneal lumen twice weekly. Treatment with antibody 18R5 completely abolished tumor growth compared to tumors treated with the control antibody (fig. 10; p ═ 0.002).

Combination treatment of 18R5 with irinotecan reduces growth of OMP-C28 xenograft tumors

In another embodiment, the ability of anti-FZD antibodies to reduce the growth of OMP-C28 colon tumor xenografts was analyzed. Isolated human OMP-C28 cells (10,000 per animal) were injected subcutaneously into 6-8 week old male NOD/SCID mice. Tumor growth was monitored weekly and tumor measurements were initiated once tumors became palpable. On day 24, the mean tumor volume was 129mm³The mice were randomly divided into 4 groups of 10 animals each. Animals were injected with either a control antibody or an 18R5IgG antibody (10mg/kg) or irinotecan (7.5mg/kg) or a combination of 18R5 and irinotecan. The antibody and irinotecan were administered by injection into the peritoneal lumen twice weekly. Tumors were measured twice a week using formula 1/2(a × b)²) To determine tumor volume, where a is length and b is width. Data are expressed as mean and mean ± s.e.m. Group means were compared using two-tailed unpaired student's t-test. A probability (p) value of less than 0.05 was considered to be significantly different. As shown in fig. 11, treatment with 18R5 reduced tumor growth by 40% (p ═ 0.02). Furthermore, treatment with 18R5 and irinotecan reduced tumor growth by 53% compared to irinotecan alone (p ═ 0.0002 relative to irinotecan alone) (fig. 11). Thus, 18R5 as a single agent and 18R5 in combination with irinotecan both showed anti-tumor growth activity in the OMP-C28 colon tumor model.

Combination treatment of 18R5 with gemcitabine reduces the growth of OMP-Pn4 xenograft tumors

In another embodiment, the ability of anti-FZD antibodies to reduce the growth of OMP-Pn4 pancreatic tumor xenografts was analyzed. NOD/SCID mice were purchased from Harlan (Indianapolis, Indiana) and acclimated for several days prior to study. The establishment and characterization of a model for Cancer stem cell-promoted pancreatic xenografts in vivo has been previously described (Li et al, Cancer Res., 67: 1030-7, 2007). For utility studies, OMP-Pn4 human pancreatic tumor cells were dispersed into single cell suspensions and resuspended in FACS buffer (Hank's Balanced salt solution [ HBSS ] supplemented with 2% heat-inactivated fetal bovine serum and 20mM Hepes]) And Matrigel (BD Bioscience, San Jose, Calif.) in a 1: 1 (vol/vol) mixture and subcutaneously implanted into the right flank of 6-7 week old male NOD/SCID mice using a 25G needle (25-gauge needle) containing 50,000 cells/100. mu.l. Tumors were monitored weekly and tumor measurements were initiated once tumors became palpable. On day 36, the mean tumor volume reached about 120mm³Tumor bearing animals were randomly divided into 4 groups of 9 animals each. Treatment was started two days later. Animals were injected with control antibody, 18R5IgG antibody (10mg/kg), gemcitabine (40mg/kg) or a combination of 18R5IgG antibody and gemcitabine. The antibody and/or gemcitabine is administered by injection into the peritoneal cavity once a week. Tumor growth was measured by using an electronic caliper (Coast Tools Company, San leiando, CA). Tumors were measured once a week using the formula 1/2(a × b) ²) To determine tumor volume, where a is length (tumor longest axis) and b is width (tumor shortest axis). Animals were weighed daily if their body weight was reduced by more than 15% and euthanized if their body weight was reduced by 20%. Data are expressed as mean ± s.e.m. Mean differences between groups were analyzed using a non-maternal (non-parimetric) t-test. Multiple comparisons one-way ANOVatest with post hoc t-test comparisons was used. A difference value of p < 0.05 was considered as a significant difference. The Software used for statistical analysis was GraphPad Prism4(GraphPad Software inc., San Diego, CA). At the end of this study, CO was used₂Ventricular and subsequent cervical dislocation methods were used to euthanize mice. Tumors were collected for RNA and histological analysis. Remaining tumorTransferred to cold medium 199 for processing into single cell suspensions for use in cancer stem cell frequency analysis.

The results of the OMP-Pn4 xenograft study are shown in FIG. 12. Treatment with the 18R5 antibody as monotherapy did not result in a significant reduction in tumor growth in this experiment. However, treatment with 18R5 and gemcitabine reduced tumor growth by 42% compared to treatment with gemcitabine (fig. 12; p ═ 0.001 relative to gemcitabine alone). Thus, 18R5 used in combination with gemcitabine, a recognized standard-of-care chemotherapeutic agent, showed synergistic anti-tumor growth activity in the OMP-Pn4 pancreatic tumor model.

Combination treatment of 18R5 with paclitaxel reduces the growth of PE-13 breast tumors

10,000 PE-13 human breast tumor cells (HER2 negative) were implanted into NOD-SCID mice and allowed to grow for 22 days until they reached approximately 120mm³Average volume of (d). The animals were then randomized into 4 groups of 10 animals each and treated with control antibody, anti-FZD 18R5, paclitaxelOr 18R5 in combination with paclitaxel. Figure 37 shows the mean tumor volume of mice administered control antibody, anti-FZD 18R5, paclitaxel, or a combination of 18R5 and paclitaxel. Antibodies were administered Intraperitoneally (IP) at a dose of 10mg/kg twice weekly. Paclitaxel was administered Intraperitoneally (IP) at a dose of 10mg/kg once a week. Tumors were measured on days shown in fig. 37. Fig. 38 shows tumor growth of individual animals in the 18R5 plus paclitaxel group. Treatment with 18R5 in combination with paclitaxel showed antitumor activity and resulted in the depletion of molded breast tumors.

Example 8 assay to determine the Effect on cancer Stem cell frequency

Limiting Dilution Assay (LDA) can be used to assess the effect of FZD-binding agents or antibodies on solid tumor stem cells and the tumorigenicity of tumors containing cancer stem cells. This assay can be used to determine the frequency of cancer stem cells in a tumor from an animal treated with an FZD-binding antibody or other agent, and compare this frequency to the frequency of cancer stem cells in tumors of control animals.

Effect of combination treatment of 18R5 with irinotecan on cancer stem cells in OMP-C28 tumors

At the end of the OMP-C28 xenograft study (example 7) described above (day 48), control tumors and post-treatment tumors from this study were harvested. These tumors were treated and dispersed into single cells. Tumor cells were then incubated with biotinylated mouse antibody (α -mouse CD 45-biotin 1: 200 dilution, and rat α -mouse H2 Kd-biotin 1: 100 dilution, BioLegend, San Diego, CA) on ice for 30 minutes, followed by addition of streptavidin (streptavidin) labeled magnetic beads (Invitrogen, Carlsbad, CA) to remove mouse cells with the aid of a magnet.

For LDA, human cells in suspension were collected, counted, and the appropriate cell dose (2, 25 and 125 cells) in FACS buffer was mixed with Matrigel at a 1: 1 ratio and injected subcutaneously into NOD/SCID mice (10 mice/cell dose/treatment group). Tumors were allowed to grow for up to 4 months. At the desired time point, the percentage of mice with detectable tumor was determined in all groups injected with tumor cells treated with anti-FZD antibody and compared to the percentage of mice with detectable tumor in the control. For example, the number of detectable tumor bearing mice injected with 125 control-treated tumor cells was determined and compared to the number of detectable tumor bearing mice injected with 125 FZD antibody-treated tumor cells. The frequency of cancer stem cells was then calculated using L-CalcTM software (StemShell Technologies Inc.). Briefly, based on poisson statistics, if 37% of the animals do not develop a tumor, there is just one cancer stem cell in a known number of injected cells.

For analysis of cell surface markers, single tumor cell suspensions were stained with anti-ESA antibodies (Biomeda) and anti-CD 44 antibodies (BD Biosciences) directly conjugated with fluorescent dyes. Dead cells were excluded using the survival dye DAPI. Flow cytometry was performed using FACS Aria (Becton Dickinson). The cell mass was removed using side scatter and forward scatter plots. Analysis of tumors treated with control antibodies revealed that 64% of the large tumor population expressed ESA and CD44 at high levels (fig. 39). As shown in figure 39, treatment with irinotecan alone did not have a significant effect on the double positive population (55%), but treatment with 18R5 or with a combination of 18R5 and irinotecan reduced the double positive population (40% and 32%, respectively).

Effect of combination treatment of 18R5 with Gemcitabine on cancer stem cells in OMP-Pn4 tumors

At the end of the 41 days of treatment in the OMP-Pn4 xenograft study described above (example 7), control tumors and post-treatment tumors from this study were collected. These tumors were treated and dispersed into single cells. The tumor cells were then incubated with biotinylated mouse antibody (alpha-mouse CD 45-biotin 1: 200 dilution, and rat alpha-mouse H2 Kd-biotin 1: 100 dilution, BioLegend, San Diego, Calif.) on ice for 30 minutes, followed by addition of streptavidin-labeled magnetic beads (Invitrogen, Carlsbad, Calif.) to remove the mouse cells. The remaining human cells in suspension were collected, counted and diluted to the appropriate cell dose (30, 90, 270 and 810 cells), mixed into a 1: 1 (volume/volume) mixture of FACS buffer and Matrigel, and injected subcutaneously into NOD/SCID mice (10 mice/cell dose/treatment group). As shown in fig. 40, tumors were allowed to grow for 75 days. Each point in figure 40 represents the tumor volume of an individual mouse. In all groups injected with tumor cells treated with anti-FZD antibody, the percentage of mice with detectable tumor was determined and compared to the percentage of mice with detectable tumor in the control. For example, the number of detectable tumor bearing mice injected with 810 control-treated tumor cells was determined and compared to the number of detectable tumor bearing mice injected with 810 FZD antibody-treated tumor cells. Tumor growth frequency and L-CalcTM software were used to calculate cancer stem cell frequency. The calculated cancer stem cell frequency for each treatment group is shown in fig. 41. Treatment with 18R5 alone and with a combination of 18R5 and gemcitabine reduced the frequency of cancer stem cells, while treatment with gemcitabine alone had no effect.

Example 9 production of FZD antibodies

Antigen production

Recombinant polypeptide fragments of the extracellular domain (ECD) or Fri domain (Fri) of the human FZD receptor (FZD) are produced as antigens for the production of antibodies. Polynucleotides encoding the amino acids of the above domains of the desired human frizzled receptor or receptors are isolated using standard recombinant DNA techniques. These polynucleotides are linked in a unified reading frame to the N-terminus of a human Fc-tag or histidine-tag and cloned into a transfer plasmid vector for baculovirus-mediated expression in insect cells. Standard transfection, infection and cell culture protocols were used to generate recombinant insect cells expressing the corresponding FZD polypeptide (O' Reilley et al, Baculoviral expression vectors: A Laboratory Manual, Oxford: Oxford University Press (1994)).

Using protein A and Ni²⁺Chelate affinity chromatography to purify the antigenic protein from insect cell conditioned media. The purified antigenic protein was dialyzed against PBS (pH 7) and concentrated to about 1mg/ml, followed by sterile filtration into the immunization preparation.

Immunization

Mice were immunized with purified FZD antigen protein using standard techniques. Approximately 70 days after initial immunization, blood from each mouse was screened for antigen recognition using ELISA and FACS analysis (described in detail below). The two animals with the highest antibody titer were selected for final antigen boosting (antigen boost) followed by isolation of splenocytes for hybridoma production. Hybridoma cells were plated at a density of 1 cell/well in 96-well plates, and supernatants from each well were subsequently screened for antigenic proteins by ELISA and FACS analysis. 7 hybridomas with high antibody titers were selected and propagated in static flask medium. Protein a or protein G sepharose chromatography was used to purify the antibody from the hybridoma supernatant. The purified monoclonal antibodies were again tested by FACS and isotyping was performed to select IgG and IgM antibodies.

Epitope mapping

Epitope mapping was performed in order to identify antibodies that recognize specific regions of the FZD extracellular domain (including the cysteine-rich domain). A mammalian expression vector plasmid containing the CMV promoter upstream of the polynucleotide encoding the fragment of the extracellular FZD domain was generated using standard recombinant DNA techniques. The recombinant protein is then expressed in cultured mammalian cells by transient transfection. 24-48 hours after transfection, cells were harvested and cell lysate proteins were separated on SDS-PAGE acrylamide gels for western blotting using antibodies from mice immunized with FZD antigen. Antibodies that recognize the ligand binding domain of FZD can be further analyzed for their binding that competes with Wnt proteins by ELISA.

To identify a specific epitope in The extracellular domain recognized by The anti-FZD antibody, The SPOT system (Sigma Genosys, The Woodlands, TX) was used. By the SPOT synthesis technique, a series of 10-residue linear peptides overlapping by one amino acid and covering the entire FZD extracellular domain were synthesized and covalently bound to a cellulose membrane. The membrane was pre-incubated with blocking buffer for 8 hours at room temperature and allowed to hybridize with the antibody overnight at 4 ℃. The membrane was then washed, incubated with a secondary antibody conjugated to horseradish peroxidase (HRP) (Amersham Bioscience, Piscataway, NJ), washed again, and visualized with a signal developing solution containing 3-amino-9-ethylcarbazole. Thereby identifying the particular epitope recognized by the antibody.

FACS analysis

In order to select a monoclonal antibody recognizing the FZD protein on the surface of natural cells produced by hybridoma clones, FACS analysis was used. HEK293 cells were transfected with expression vectors encoding full-length cDNA clones of the corresponding FZD alone or co-transfected with vectors expressing GFP. A Flag epitope tag may be introduced at the amino terminus, so that the expression of the tagged FZD receptor on the cell surface can be confirmed. 24-48 hours after transfection, cells were harvested in suspension and incubated on ice with anti-FZD antibodies, FLAG antibodies, immune serum (for FZD5 expressing cells), or with control IgG for detection of background antibody binding. Cells were washed and primary antibodies were detected with anti-mouse secondary antibodies conjugated to fluorescent chromophores. The labeled cells are then sorted by FACS to identify anti-FZD antibodies expressed on the cell surface that specifically recognize the corresponding FZD receptor. Antibodies that recognize the desired human frizzled receptor were identified.

Chimeric antibodies

After monoclonal antibodies that specifically recognize FZD receptors have been identified, these antibodies are modified to overcome human anti-mouse antibody (HAMA) immune responses when rodent antibodies are used as therapeutics. The variable regions of the heavy and light chains of the selected monoclonal antibodies were isolated from hybridoma cells by RT-PCR and linked in the same reading frame to the human IgG1 heavy and kappa light chain constant regions, respectively, in a mammalian expression vector. Alternatively, a human Ig expression vector such as TCAE 5.3 is used which contains human IgG1 heavy and kappa light chain constant region genes on the same plasmid (Preston et al, 1998, Infection & Immunity 66: 4137-42). Expression vectors encoding the chimeric heavy and light chains were then co-transfected into Chinese Hamster Ovary (CHO) cells to produce chimeric antibodies. Immunoreactivity and affinity of the chimeric antibody was compared to murine parent antibody by ELISA and FACS.

Humanized antibodies

Since chimeric antibody therapeutics are still often antigenic and generate human anti-chimeric antibody (HACA) immune responses, chimeric antibodies against FZD receptors can be further humanized. To produce humanized antibodies, the critical parts of the antibody specificity determining motifs, which potentially include elements from three short hypervariable sequences or Complementarity Determining Regions (CDRs), and/or the framework regions required for the correct positioning of the CDR regions of the heavy and light chain variable domains of the above-described antibodies, were engineered into the germline DNA sequences of the human heavy and light chain antibody genes, respectively, using recombinant DNA technology, and subsequently cloned into mammalian expression vectors for expression in CHO cells. Immunoreactivity and affinity of the humanized antibody was compared to the parent chimeric antibody by ELISA and FACS. In addition, site-directed mutagenesis or high-density mutagenesis of the variable region can be used to optimize the specificity, affinity, etc., of the humanized antibody.

Human antibodies

In some embodiments, phage display technology can be used to isolate human antibodies that specifically recognize the extracellular domain of the FZD receptor. Phage display antibody libraries containing human antibody variable regions displayed as single chain Fv or fab domains are screened for specific and high affinity recognition of the FZD receptor antigens described above. The identified variable domain antibody sequences were then rearranged into Ig expression vectors containing human IgG1 heavy and kappa light chains for expression of human antibodies in CHO cells.

Example 10 Generation of antibodies recognizing specific epitopes

Monoclonal antibodies from hybridomas

In certain embodiments, antibodies that recognize functional epitopes of FZD receptors are generated by immunizing mice with one or more FZD receptor antigens. Mice were immunized with purified FZD antigen protein using standard techniques. In certain embodiments, the mice are immunized sequentially with different FZD receptor antigens. Approximately 70 days after the initial immunization, the blood of each mouse was screened. Animals with high antibody titers to FZD antigen were selected for final antigen boosting, followed by isolation of splenocytes for hybridoma production. Hybridoma cells were plated at a density of 1 cell/well in 96-well plates, and supernatants from each well were subsequently screened by ELISA and flow cytometry. To identify monoclonal antibodies that recognize specific epitopes, including epitopes within or overlapping the Biological Binding Site (BBS), hybridoma supernatants are screened for antibodies that bind the desired FZD and antibodies that do not bind FZD having a specific amino acid substitution within the specific epitope of interest (e.g., BBS).

Human antibodies

Phage display libraries can be used to identify antibodies that recognize a desired epitope of the FZD receptor (e.g., an epitope shared by multiple FZD and/or an epitope within or overlapping the BBS or a portion thereof). For example, the Fri domain of a selected FZD is expressed as a recombinant protein and coated on a suitable surface at a concentration of 10 μ g/ml. The human phage library was then panned by two more rounds of enrichment (see, e.g., Griffiths et al, EMBO J.12: 715-34). Optionally, a subsequent round of panning may be optionally performed using a different FZD protein. Alternatively, each round of panning may be performed in the presence of a water-soluble decoy FZD protein having specific amino acid substitutions within the desired target epitope region (e.g., including epitopes located in the Biological Binding Site (BBS)). Each clone in the panning results was then screened for its ability to bind the desired FZD protein by ELISA or flow cytometry analysis and binding to the desired epitope was assessed by the lack of binding to FZD proteins having specific amino acid substitutions within the desired target epitope. The gene encoding the antigen binding domain is then recovered from the phage and used to construct a complete human antibody molecule by binding the antigen binding domain to the constant region for expression in a suitable host cell line. Antibodies were identified and tested for their ability to prevent tumor cell growth as described elsewhere herein.

Example 11 other in vitro assays to evaluate antibodies to FZD receptors

This example describes representative in vitro assays to test the activity of antibodies raised against FZD receptors in cell proliferation, pathway activation, and cytotoxicity.

Proliferation assay

Expression of FZD receptors in different cancer cell lines was quantified using Taqman assay. Cells identified as expressing FZD receptors were plated at a density of 104 cells/well in 96-well tissue culture microplates and allowed to proliferate for 24 hours. The cells were then cultured in fresh DMEM with 2% FCS for a further 12 hours, at which time the anti-FZD antibody and the control antibody against it were added to the culture medium in the presence of 10 μmol/l BrdU. After BrdU labeling, the media was removed and the cells were fixed in ethanol for 30 min at room temperature and subsequently reacted with a monoclonal anti-BrdU antibody (clone BMG 6H8, Fab fragment) conjugated to peroxidase for 90 min. The substrate was developed in a solution containing tetramethylbenzidine and terminated after 15 minutes with 25. mu.l of 1mol/L H2SO 4. The color reaction was measured with an automatic ELISA plate reader (UV microplate reader; Bio-Rad laboratories, Richmond, Calif.) using 450nm filters. All experiments were performed in triplicate. The ability of anti-FZD antibodies to inhibit cell proliferation (compared to control antibodies) was determined.

Pathway activation assay

In certain embodiments, the ability of an anti-FZD receptor antibody to block activation of the Wnt signaling pathway is determined in vitro. For example, HEK293 cells cultured in DMEM supplemented with antibiotics and 10% FCS were co-transfected with the following (1), (2) and (3): (1) wnt7B and FZD10 expression vectors used to activate the Wnt signaling pathway; (2) TCF/Luc wild-type or mutant reporter vectors comprising 3 or 8 copies of the TCF binding domain upstream of the firefly luciferase reporter gene for use in measuring levels of canonical Wnt signaling (Gazit et al, 1999, Oncogene 18: 5959-66); and (3) Renilla luciferase reporter gene (Promega; Madison, Wis.) as an internal control for transfection efficiency. The anti-FZD 10 antibody and control antibody were then added to the cell culture medium. Luciferase levels were measured 48 hours post-transfection using a dual luciferase assay kit (Promega; Madison, Wis.), where firefly luciferase activity was normalized to Renilla luciferase activity. Three independent experiments were performed in parallel three times. Thereby determining the ability of the FZD antibody to inhibit Wnt pathway activation.

Complement dependent cytotoxicity assay

In certain embodiments, Complement Dependent Cytotoxicity (CDC) mediated by anti-FZD receptor antibodies is measured using FZD receptor expressing cancer cell lines or cancer stem cells isolated from patient samples and passaged as xenografts in immunocompromised mice. Cells were suspended at a density of 106 cells/ml in 200 μ l RPMI1640 medium supplemented with antibiotics and 5% FBS. The suspended cells were then mixed in triplicate with 200 μ l serum or heat-inactivated serum carrying anti-FZD receptor antibody or control antibody. At 37 ℃ in 5% CO₂And incubating the cell mixture for 1-4 hours. The treated cells were then collected, resuspended in 100. mu.l of FITC-labeled annexin V diluted in culture medium, and incubated for 10 minutes at room temperature. Mu.l of a solution of propidium iodide diluted in HBSS (25. mu.g/ml) was added and incubated at room temperature for 5 minutes. Cells were collected, resuspended in culture medium and analyzed by flow cytometry. Flow cytometry on FITC stained cells yielded total cell counts; cell death was measured using propidium iodide uptake by a percentage of dead cells of the total number of cells in the presence of serum and anti-FZD antibodies as compared to heat inactivated serum and control antibodies. The ability of the anti-FZD antibody to mediate complement dependent cytotoxicity was thus determined.

Antibody-dependent cell-mediated cytotoxicity assay

Can useFZD receptor-expressing cancer cell lines or cancer stem cells isolated from patient samples and passaged as xenografts in immunocompromised mice were measured for antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by anti-FZD receptor antibodies. Cells were suspended at a density of 106 cells/ml in 200 μ l phenol red free RPMI1640 medium supplemented with antibiotics and 5% FBS. Peripheral Blood Mononuclear Cells (PBMC) were isolated from heparinized peripheral blood by Ficoll-Paque density gradient centrifugation and used as effector cells. Target cells (T) are then mixed with PBMC effector cells (E) in a 96-well plate at E/T ratios of 25: 1, 10: 1 and 5: 1 in the presence of at least one FZD receptor antibody or control antibody. Control experiments included incubation of target cells alone and effector cells alone in the presence of antibodies. At 37 ℃ in 5% CO₂And incubating the cell mixture for 1-6 hours. The released Lactate Dehydrogenase (LDH), a stable cytosolic enzyme released upon cell lysis, was then measured by colorimetric assay (CytoTox96 non-radioactive cytotoxicity assay; Promega; Madison, Wis.). Absorbance data was collected at 490nm with a standard 96-well plate reader and background corrected. The percentage of specific cytotoxicity was calculated according to the following formula: % cytotoxicity × (experimental LDH release-effector cell spontaneous LDH release-target cell spontaneous LDH release)/(target cell maximum LDH release-target cell spontaneous LDH release). The ability of antibodies against FZD receptors to mediate antibody-dependent cell-mediated cytotoxicity was thus determined.

Example 12 prevention of tumor growth in vivo Using an antibody against FZD receptor

This example describes the use of anti-FZD receptor antibodies to prevent tumor growth in a xenograft model. In certain embodiments, tumor cells from a patient sample (solid tumor biopsy or pleural effusion) that have been passaged as a xenograft in mice are prepared for re-passaging to experimental animals. Tumor tissue was removed under sterile conditions, cut into small pieces, completely minced using a sterile razor blade, and single cell suspensions were obtained by enzymatic digestion and mechanical disruption. Specifically, pleural effusion cells or resulting tumor fragments are mixed with ultrapure collagenase III (200-250 units collagenase/ml) in culture medium and incubated at 37 ℃ for 3-4 hours with aspiration with a 10ml pipette every 15-20 minutes. The digested cells were filtered through a 45 μ M nylon mesh, washed with RPMI/20% FBS, and washed twice with HBSS. Dispersed tumor cells were then injected subcutaneously into the mammary fat pad of NOD/SCID mice to initiate tumor growth.

In certain embodiments, prior to injection into the experimental animal, the dispersed tumor cells are first sorted into tumorigenic cells and non-tumorigenic cells according to cell surface markers. Specifically, the dispersed tumor cells were washed twice with a Hepes-buffered saline solution (HBSS) containing 2% heat-inactivated calf serum (HICS) and resuspended at 106 cells/100. mu.l. Antibodies were added and cells were incubated on ice for 20 minutes followed by two washes with HBSS/2% HICS. Antibodies include anti-ESA (Biomeda, Foster City, Calif.), anti-CD 44, anti-CD 24 and lineage markers anti-CD 2, anti-CD 3, anti-CD 10, anti-CD 16, anti-CD 18, anti-CD 31, anti-CD 64, and anti-CD 140b (collectively referred to as Lin; PharMingen, San Jose, Calif.). The antibodies are conjugated directly to fluorescent chromophores, allowing positive or negative selection of cells expressing these markers. Mouse cells were removed by selection against H2Kd + cells and dead cells were removed by using the survival dye 7 AAD. Flow cytometry was performed on a FACSVantage (Becton Dickinson, Franklin Lakes, NJ). Side scatter and forward scatter plots were used to eliminate cell clumps. The isolated ESA +, CD44+, CD24-/low, Lin-tumorigenic cells were then injected subcutaneously into NOD/SCID mice to initiate tumor growth.

For example, the ability of anti-FZD antibodies to reduce tumor cell growth was analyzed. Dispersed tumor cells (10,000 per animal) were injected subcutaneously into the flank of 6-8 week old NOD/SCID mice. Two days after injection of tumor cells, the anti-FZD antibody was injected into the animals intraperitoneally (i.p.) at 10mg/kg twice a week. Tumor growth was monitored weekly until growth was detected, after which tumor growth was measured twice weekly for 8 weeks. FZD-binding antibodies were thereby identified that significantly reduced tumor growth compared to PBS-injected controls.

Example 13 treatment of tumors in vivo Using antibodies against FZD receptor

This example describes the use of antibodies against FZD receptors to treat cancer in a xenograft model. In certain embodiments, tumor cells from a patient sample (solid tumor biopsy or pleural effusion) that have been passaged as a xenograft in mice are prepared for re-passaging to experimental animals. Tumor tissue was removed, cut into small pieces, completely minced using a sterile razor blade, and single cell suspensions were obtained by enzymatic digestion and mechanical disruption. Dispersed tumor cells were then injected subcutaneously into the mammary fat pad (for breast tumors) or the costal region (for non-breast tumors) of NOD/SCID mice to initiate tumor growth. Alternatively, ESA +, CD44+, CD24-/low, Lin-tumorigenic cells were isolated and injected as described above.

Following injection of the tumor cells, the animals were monitored for tumor growth. Antibody treatment was started once the average tumor size reached about 150-200 mm. Each animal received 100. mu.g of FZD receptor antibody or control antibody intraperitoneally at a frequency of 2-5 times per week for 6 weeks. Tumor size was evaluated 2 times per week over this 6 week period. The ability of the FZD receptor antibody to prevent further tumor growth or reduce tumor size compared to control antibodies was thereby determined.

At the end of the antibody treatment, tumors were collected for further analysis. In some embodiments, a portion of a tumor is analyzed by immunofluorescence to assess the penetration and tumor response of the antibody to the tumor. For each tumor collected from mice treated with anti-FZD receptor and control antibody, a portion of it was freshly frozen in liquid nitrogen, embedded within o.c.t., and cut into 10 μm sections on a cryostat and placed on glass slides. In some embodiments, a portion of each tumor is fixed with formalin, paraffin embedded, and cut into 10 μm sections on a microtome and placed on a slide. The sections were post-fixed (post-fix) and incubated with a chromophore-labeled antibody that specifically recognized the injected antibody, thereby detecting the presence of anti-FZD receptor antibody or control antibody in the tumor biopsy. In addition, antibodies that detect different tumors and cell types recruited by tumors can be used to assess the effect of antibody therapy on, for example, angiogenesis, tumor growth, and tumor morphology, e.g., anti-VE cadherin (CD144) or anti-PECAM-1 (CD31) antibodies to detect vascular endothelial cells, anti-smooth muscle alpha-actin antibodies to detect vascular smooth muscle cells, anti-Ki 67 antibodies to detect proliferating cells, TUNEL assays to detect dead cells, anti-beta-catenin antibodies to detect Wnt signaling, and anti-intracellular domain (ICD) Notch fragment antibodies to detect Notch signaling.

In certain embodiments, the effect of treatment with an anti-FZD receptor antibody on tumor cell gene expression is also evaluated. For each collected tumor from FZD antibody treated and control antibody treated mice, total RNA was extracted from a portion thereof and used for quantitative RT-PCR. Expression levels of FZD receptors, components of the Wnt signaling pathway (including, e.g., Wnt1 and β -catenin), and other previously identified cancer stem cell markers (e.g., CD44) were analyzed relative to housekeeping gene GAPDH as an internal control. Thereby determining the change in the gene expression of the tumor cells upon the FZD receptor antibody treatment.

Furthermore, the effect of treatment with anti-FZD receptor antibodies on the frequency of cancer stem cells in tumors was evaluated. Tumor samples from FZD-treated and control antibody-treated mice compared thereto were cut into small pieces, completely minced using a sterile razor blade, and single cell suspensions were obtained by enzymatic digestion and mechanical disruption. Subsequently, dispersed tumor cells were analyzed for the presence of tumorigenic cancer stem cells by FACS analysis based on ESA +, CD44+, CD24-/low, Lin-surface cell marker expression as detailed above.

Subsequently, the cells isolated from the expression of ESA +, CD44+, CD24-/low, Lin-after anti-FZD antibody treatment can be evaluated for tumorigenicity. ESA +, CD44+, CD24-/low, Lin-cancer stem cells isolated from mice treated with FZD antibody and control antibody in comparison thereto were re-injected subcutaneously into the mammary fat pad of NOD/SCID mice. The tumorigenicity of cancer stem cells based on the number of injected cells required for stable tumor formation was subsequently determined.

Example 14 identification of Wnt Gene signature

Experiments were performed to identify a set of genes that can be specifically expressed for the activation of the Wnt signaling pathway in human colon tumors.

Overexpression of Axin abrogates tumor growth

Axin is an important regulator of the canonical Wnt pathway. It is part of a polyprotein complex that triggers β -catenin degradation, thus silencing this pathway in the absence of Wnt. Wnt can reverse this effect, removing axin from the degradative complex, thereby allowing β -catenin translocation and TCF-mediated activation of specific target genes. Overexpression of exogenous axin and expression of dominant-negative truncated form of TCF (DNTCF4) represent well-characterized ways to block the Wnt signaling pathway.

Lentivirally mediated axin overexpression has been shown to completely abolish the growth of UM-PE13 and UM-T3 breast tumors and OMP-C11 and OMP-C17 colon tumors in NOD/SCID mice. The stable expression of DNTCF4 in UM-T3 tumor cells had the same effect. Taken together, these data suggest that intracellular Wnt blockade can negatively impact the development of different tumor types, providing support for the Wnt pathway as a relevant target for the treatment of breast and colon cancer.

The Wnt signaling pathway is constitutively active in many tumor types. In most colon tumors, this activation is due to a truncation mutation of APC or an activating mutation of β -catenin. Such mutations have not been reported in other tissues where the Wnt signaling pathway may be activated by another set of mutations or autocrine mechanisms. In those tumors where the Wnt signaling pathway remains responsive to autocrine stimulation, it should be feasible to block this pathway using extracellular means such as antibodies or other soluble protein inhibitors, and would affect tumor development. The identification of such Wnt-dependent tumors would aid in the development of anti-Wnt agents and in defining the tumor types to be targeted in the clinic.

Immunohistochemical data showed that most OMP-C11 tumor cells expressed high levels of cytoplasmic/nuclear β -catenin, indicating that the Wnt signaling pathway is constitutively active in this tumor. This was confirmed by detection of high levels of β -catenin in OMP-C11 by Western blotting. The combination of Wnt pathway activation and sensitivity to axin overexpression made OMP-C11 a suitable tumor for studying the regulation of gene expression therein in response to Wnt and Wnt blockade and deriving Wnt gene signatures therefrom.

Microarray analysis of differential Gene expression in response to axin overexpression

Differential gene expression was determined by microarray analysis when OMP-C11 colon tumor cells were treated with axin.

Human colon OMP-C11 tumor freshly removed from NOD/SCID mice (xenograft tumor model) was used as a source of colon tumor cells. Two lentiviral vectors were generated for delivery of constitutive axin-IRES-GFP expression cassettes and control IRES-GFP expression cassettes, designated LOM91 and LOM92, respectively.

OMP-C11 tumors were processed as single cell suspensions and depleted from mouse lineage cells. Lin-depleted cells were infected with either LOM91(axin) or LOM92 (control) lentiviral vectors at a multiplicity of infection of 2.5, maintained in culture for 3-4 days, and sorted for GFP expression. Total RNA was extracted from each cell sample sorted. In that The RNA was analyzed on a human genome U133 Plus 2.0 microarray (Affymetrix, Santa Clara, Calif.). The experiment was repeated twice.

By analyzing genes that are differentially expressed after axin treatment (these genes also show a correlation with axin2 expression between a set of normal and malignant colon tumor samples), a gene signature was obtained that contains a set of core genes that are regulated by the Wnt pathway. Genes that show downregulation in response to axin overexpression were identified from the axin microarray experiments described above. The thresholds for this selection are: axin1 was downregulated by more than 50% in the over-expressed sample compared to the control sample (log 2 value of the ratio of Axin1 over-expressed sample relative to the control sample must be-1 or less) and the T-test p-value was less than 0.1. Since Axin1 is a known Wnt pathway inhibitor, overexpression of Axin1 down-regulated genes would be direct or indirect Wnt pathway targets. This selection was subsequently further refined by identifying genes highly correlated with aixin2 (correlation > 0.3) in a panel of colon/intestine/other digested tissue malignancy samples (232 samples). Since Axin2 is a known Wnt target, genes with expression patterns similar to Axin2 may also be Wnt targets. This analysis yielded a gene signature for Wnt pathway activity (table 6). The expression levels of the genes in this signature can be used to assess whether each tumor sample or different types of tumors show evidence of altered Wnt pathway signaling.

Table 6: wnt Gene signature List derived from Colon tumors

Example 15 treatment of human cancer with anti-FZD receptor antibody

This example describes methods of treating cancer using antibodies to FZD receptors to target tumors containing: cancer stem cells, and/or tumor cells in which FZD receptor expression has been detected, and/or tumor cells having a Wnt gene signature (e.g., the Wnt gene signature of example 14) that indicates that they can respond to inhibition of Wnt signaling.

The presence of a cancer stem cell marker or the presence of FZD receptors or the expression of one or more genes in the Wnt gene signature can be first determined from the tumor biopsy. Tumor cells are removed under sterile conditions from a biopsy from a patient diagnosed with cancer. In some embodiments, tissue biopsies are freshly frozen in liquid nitrogen, embedded in o.c.t., and cut into 10 μm sections on a cryostat and placed on glass slides. In some embodiments, the tissue biopsy is fixed with formalin, paraffin embedded, and cut into 10 μm sections on a microtome and placed on a slide.

The slice is incubated with an antibody against the FZD receptor, thereby detecting the expression of the FZD protein. Alternatively, sections can be analyzed to determine the presence of one or more genes in the Wnt gene signature described in example 14.

The presence of cancer stem cells can also be determined. Tissue biopsy samples were cut into small pieces, completely minced using a sterile razor blade, and the cells were subjected to enzymatic digestion and mechanical disruption to obtain single cell suspensions. Subsequently, the dispersed tumor cells were incubated with anti-ESA, anti-CD 44, anti-CD 24, anti-Lin, and anti-FZD antibodies to detect cancer stem cells, and the presence of ESA +, CD44+, CD24-/low, Lin-, FZD + cancer stem cells was determined by flow cytometry as described in detail above.

Treating a cancer patient whose tumor is diagnosed to express an FZD receptor and/or one or more genes in a Wnt gene signature with an antibody to the FZD receptor. In certain embodiments, the humanized or human monoclonal antibody to the FZD receptor produced as described above is purified and formulated with a suitable pharmaceutical carrier for injection. In some embodiments, the patient is treated with the FZD antibody at least once a month for at least 10 weeks. In some embodiments, the patient is treated with the FZD antibody at least once per week for at least 14 weeks. Each administration of the antibody should be a pharmaceutically effective dose. In some embodiments, about 2mg/ml to about 100mg/ml of the anti-FZD antibody is administered. In some embodiments, about 5mg/ml to about 40mg/ml of the anti-FZD antibody is administered. The antibody may be administered prior to, concurrently with, or subsequent to administration of a standard radiation therapy regimen or administration of a chemotherapy regimen using one or more chemotherapeutic agents (e.g., octoplatin, fluorouracil, leucovorin, or streptozotocin). Patients are monitored to determine whether such treatment has elicited an anti-tumor response, for example, in terms of tumor regression, a decrease in the incidence of new tumors, lower tumor antigen expression, a decrease in the number of cancer stem cells, or other methods of assessing disease prognosis.

Example 16 differentiation of pancreatic tumor cells following treatment with 18R5 and Gemcitabine

Analysis of Gene expression by quantitative PCR (Q-PCR) on treated pancreatic tumor cells

Analysis of chromogranin a (CHGA) expression was performed by quantitative PCR analysis on PN4 xenograft tumors treated with control Ab, 18R5 IgG antibody, gemcitabine, or a combination of gemcitabine and 18R5 IgG antibody as described above (example 7). CHGA is known to be a neuroendocrine differentiation marker for a variety of tumours, including breast, colon, lung and pancreatic tumours, and increased expression of CHGA in pancreatic tumours has been found to be associated with increased survival (Tezel et al, 2000.Cancer 89, 2230-6).

Total RNA was prepared from 5 tumors per group in the PN4 xenograft study and Applied Biosystems were used according to standard protocolsThe listed probes were evaluated by one-step Reverse Transcription (RT) -PCR. The probe-primer set (Hs00154441_ ml) used for CHGA analysis included a probe labeled with FAM-dye and the following primers: 5 '-CGCTCTCCAAGGCGCCAAGGAGAGG-S' (SEQ ID NO: 75). GusB was used as an internal control. In short,RT was performed at 48 ℃ for 30 min, at 95 ℃ for 10 min for initial denaturation, followed by 40 cycles: denaturation at 95 ℃ for 15 seconds, and extension at 60 ℃ for 1 minute, the amplification/incorporation of the fluorescent probe was observed in real time.

The islet β cell marker CHGA was only significantly elevated in samples from mice treated with gemcitabine and 18R5 in combination. Tumors from control Ab, 18R5 and gemcitabine-only group expressed similar levels of CHGARNA, while tumors from the combination group showed clearly elevated CHGA expression. In tumors treated with the combination of 18R5 and gemcitabine, CHGA levels were increased 10-fold and 7-fold in both experiments. The results of a representative experiment are shown in fig. 42.

Gene expression analysis of treated pancreatic tumor cells by immunohistochemistry

Elevated CHGA expression was also observed at the protein level by immunohistochemistry on tissue sections prepared from treated tumors (data not shown). Tumors treated with control Ab showed dense staining of a small population of cells scattered in the tumor. Tumors treated with 18R5 alone or gemcitabine alone expressed similar levels of CHGA as controls. In contrast, tumors treated with the combination of 18R5 and gemcitabine showed an increase in CHGA positive cell number, consistent with increased RNA expression detected by Q-PCR.

Staining with alcian blue and anti-ki 67 antibody

Another feature of endocrine, secretory or ductal cells is the production of mucin, which can be detected by alcian blue staining (van Es et al, 2005.Nature 435959-63). During tumor collection and treatment, the 18R 5-treated tumors were observed to be more mucinous than the control-treated tumors. Therefore, PN4 tumor sections from mice treated with control antibody, gemcitabine only, 18R5 only or a combination of 18R5 and gemcitabine were stained with alcian blue. Tumors treated with 18R5 in the group with 18R5 only and the group using 18R5 and gemcitabine in combination showed significantly increased alcian blue staining relative to the control group and the gemcitabine only group (data not shown).

The second pancreatic tumor line PN13 also noted increased mucinous cells following treatment with 18R5 or a control antibody (fig. 43). Mice bearing PN13 tumors in this experiment received treatment as described above (example 7). Tumor sections were stained with alcian blue to reveal mucinous cells, and with antibodies against ki67 by immunohistochemistry to reveal proliferating cells. The results show that treatment with 18R5 resulted in a significant increase in the number of alcian blue positive mucinous cells. In addition, 18R5 treatment reduced the frequency of ki67 positive cells. Interestingly, there was no overlap between myxoid cells and ki67 positive cells, indicating that the myxoid cells were non-proliferative. This provides evidence that treatment with 18R5 promotes differentiation of tumor cells into non-proliferative progeny.

In conclusion, elevated CHGA expression, increased production of mucin as demonstrated by alcian blue staining, and production of non-proliferative progeny as demonstrated by anti-ki 67 antibody staining are consistent with the following model: inhibition of Wnt-FZD signaling by treatment with 18R5 promotes differentiation of pancreatic tumor cells into a variety of different cell types that are characterized as non-proliferating differentiated cells.

Example 17 additional in vivo efficacy Studies with 18R5 alone and/or in combination with 18R5 and other anti-cancer Agents

Effect on growth of OMP-LU24 xenograft tumors

For anti-FZD antibody 18R5 alone or withThe utility of the anti-FZD antibody 18R5 used in combination (paclitaxel) in inhibiting the growth of OMP-LU24 human lung tumors in vivo was evaluated.

50,000 OMP-LU24 human lung tumor cells were injected subcutaneously into NOD/SCID mice. Growing the tumor for 27 daysUntil the average volume reaches 143mm³. Animals were randomly divided into 4 groups (each group n ═ 9) and treated with a control antibody ("control Ab"), anti-FZD 18R5 ("18R 5"),("Taxol") or 18R5 withIs treated with the combination of (1) ("18R 5+ Taxol"). Tumor measurements were taken on the days shown in figure 44. Administering the antibody Intraperitoneally (IP) at a dose of 10mg/kg once a week; intraperitoneal administration at a dose of 15mg/kgOnce per week.

The results are shown in fig. 44. It was found that anti-FZD treatment reduces tumor growth and is compatible with treatment with FZD aloneThe combination therapy exhibits enhanced anti-tumor activity compared to the treatment of (a).

Effect on growth of OMP-LU33 xenograft tumors

Also for anti-FZD antibody 18R5 alone or withThe utility of the anti-FZD antibody 18R5 used in combination (bevacizumab) in inhibiting the growth of OMP-LU33 human lung tumors in vivo was tested.

10,000 OMP-LU33 human lung tumor cells were injected subcutaneously into NOD/SCID mice. The tumors were allowed to grow for 30 days until their average volume reached 124mm³. Animals were randomized into 4 groups (n-10 per group) and treated with control antibody (squares),(downward-pointing triangle), anti-FZD 18R5 (upward-pointing triangle) or18R5 andthe combination of (a) is treated. Tumor measurements were taken on the days shown in figure 45. The antibody was administered intraperitoneally at a dose of 10mg/kg twice a week.

The results are shown in fig. 45. It was found that anti-FZD treatment reduces tumor growth and is compatible with treatment with FZD aloneThe combination therapy exhibits enhanced anti-tumor activity compared to the treatment of (a).

Effect on the growth of T3 xenograft tumors

Also for anti-FZD antibody 18R5 alone or withThe utility of the anti-FZD antibody 18R5 used in combination (trastuzumab) in inhibiting the growth of T3 human HER2 positive breast tumors in vivo was evaluated.

50,000T 3 human breast tumor cells were injected subcutaneously into NOD/SCID mice. The tumors were allowed to grow for 32 days until their mean volume reached 125mm³. Animals were randomly divided into 4 groups (each group n-10) and treated with control antibody (squares), anti-FZD 18R5 (triangles),(small solid circles) or 18R5 with The combination of (hollow circle) was treated. Tumor measurements were taken on the days shown in figure 46. The antibody was administered intraperitoneally at a dose of 10mg/kg twice a week.

The results are shown in fig. 46. And use onlyTreatment ofFor comparison, 18R5 andthe combination treatment performed showed enhanced antitumor activity.

Example 18 sequence of anti-FZD antibody

The heavy and light chain CDRs of the anti-FZD antibodies are provided in table 7 and table 8 below, respectively. The heavy chain variable region (VH) and light chain variable region (VL) of the anti-FZD antibody and their coding sequences are shown in table 9 below. The amino acid sequences and polynucleotide sequences of the VH and VL listed in table 9 are provided in figures 13-15 or tables 10 and 11 below. Sequences encoding the heavy or light chain of an anti-FZD antibody are provided in figures 14-15 or table 12 below.

Table 7: heavy chain CDR of anti-FZD human antibody

Table 8: light chain CDRs of anti-FZD human antibodies

Table 9: VH and VL of anti-FZD human antibodies

Table 10: other anti-FZD VH and VL amino acid sequences

Table 11: additional nucleotide sequences encoding VH and VL of anti-FZD antibodies

Table 12: additional nucleotides encoding the Heavy Chain (HC) or Light Chain (LC) of an anti-FZD IgG antibody, including the signal sequence

Sequence of

The plasmid encoding anti-FZD IgG antibody 18R4605 (ATCC accession No. PTA-10307), the plasmid encoding 18R4805 (ATCC accession No. PTA-10309), and the plasmid encoding 44R24 (ATCC accession No. PTA-10311), isolated from e.coli, were deposited at the American Type Culture Collection (ATCC) on 26/8/2009, 10801 University Boulevard, Manassas, VA, USA, in accordance with the requirements of the budapest treaty.

Example 19 binding Profile of anti-FZD antibodies

FACS analysis was used to characterize the binding properties of anti-FZD monoclonal antibodies (mabs) to FZD1, 2, 5, 7, and 8.

By expressing the full-length FZD1, 2, 5,The plasmid DNA of 7 or 8 and another plasmid expressing the reporter GFP used as a transfection marker co-transfect HEK293 cells. Fugene 6(Roche) was used as transfection reagent according to the manufacturer's instructions. Transfected cells were incubated at 37 ℃ in 5% CO₂And (4) incubating for 24-48 hours. The anti-FZDmAb was then diluted to a final volume of 50 μ Ι, with 4-fold serial dilutions starting from a concentration of 20 μ g/ml, for a total of 8 dilutions. Each FZD/GFP 293 transient transfection pool was collected in suspension and 100,000 transfected cells were incubated with the diluted anti-FZD mAb to be tested for 30-60 minutes on ice. The cells were washed and bound anti-Fzd antibody was detected with an anti-human secondary antibody coupled to a fluorescent chromophore. The labeled cells were then detected by FACS and counted. The FACS data generated were expressed as Mean Fluorescence Intensity (MFI) units. Data were charted and analyzed using GraphPad Prism software. MFI was plotted as a function of Ab concentration to establish a dose-response curve. Nonlinear regression curve fitting was performed on the numbers and EC50 was calculated.

The binding properties of mabs 18R5 and 44R24 were determined and compared. Dose-response curves representing the binding of each of 18R4 and 44R24 to FZD1, 2, 5, 7, and 8 are shown in figure 47. The calculated EC50(nM) for the two mabs is shown in table 13. 44R24 binds with good affinity to Fzd5 and Fzd 8. Failure to establish sigmoidal dose-response curves for the other 3 Fzd receptors indicates that 44R24 did not bind Fzd1, 2, and 7. For 18R5, high affinity binding to Fzd1, 2, 5, and 7 was determined.

Table 13: EC50(nM) of mAbs 18R5 and 44R24

EC50(nM)	Fzd1	Fzd2	Fzd5	Fzd7	Fzd8
						18R5	0.41	0.62	1.10	0.58	12.00
44R24	117.95	Without bonding	1.89	92.31	1.09

Example 20 evaluation of anti-Wnt Activity of anti-FZD mAb in cell-based assays

The ability of 18R5 and 44R24 to inhibit Wnt signaling in STF-293 cells was determined and compared. STF cells are Human Embryonic Kidney (HEK) -293 cells stably transfected with a Super Top Flash (STF) reporter cassette, in which the expression of the luciferase (Luc) reporter is regulated by multiple copies of the TCF binding site located upstream of the minimal promoter. Low basal Luc expression can be induced 30-60 fold in response to Wnt3a, providing a large window for assessing inhibitory activity of anti-Fzd abs.

To evaluate mAbs, STF-293 cells were cultured in DMEM-10% FBS. On the first day, 10,000 cells were seeded in each well of a 96-well optical bottom white plate (Nunc # 165306). At 37 ℃ in 5% CO ₂Overnight incubation of the cells. On the following day, the Ab to be tested was diluted to a final concentration of 40. mu.g/. mu.l using culture medium. 7 serial 5-fold dilutions were performed. STF-293 cell culture medium was replaced with a mixture containing 50. mu.l Ab dilution, 25. mu.l Wnt3a conditioned medium (from Wnt3a stable L-cells), and 25. mu.l DMEM-10% FBS. The final concentrations tested were 20, 4, 0.8, 0.16, 0.03, 0.006, 0.0013, 0.0003 μ g/ml for each Ab. Three replicates were performed for each Ab concentration. Human anti-hapten Ab, LZ1, was used as a negative control Ab. non-Wnt 3a conditioned medium from parental L-cells was used as a negative control inducer. The plate was returned to the incubator. On day 3, luciferase activity was measured using the Promega Steady Glo kit (VWR # PAE2550-A) according to the manufacturer's instructions. Results are expressed in photons/second. Data were charted and analyzed using GraphPad Prism software. Luciferase activity was plotted as a function of Ab concentration to establish a dose-response curve. Nonlinear regression curve fitting was performed on the numbers and IC50 was calculated.

The ability of 44R24 and 18R5 to inhibit Wnt signaling in STF cells was also determined and compared as described above. The results are shown in fig. 48 and table 14. 44R24 activity was detected only at higher Ab concentrations, reflecting the low activity of the antibody in this assay. The IC50 calculated for 44R24 was 13 times lower than that of 18R 5.

Table 14: IC50 for inhibition of Wnt signaling in ST-293 cells by 18R5 and 44R24

	18R5	44R24
			IC50(nM)	2.73	34.43

The ability of 18R5 and 44R24 to inhibit Wnt signaling in a549 cells was also determined. A549 cells are human lung cancer cells in which Axin2 gene is highly expressed, indicating endogenous activity of Wnt signaling. Axin2 is a well-known Wnt target gene that responds to activation of this pathway by up-regulating its transcription and ultimately down-regulates Wnt signaling through a feedback loop mechanism. This system was used to detect the effect of anti-Fzd Ab on Axin2mRNA levels by qPCR.

In 12-well plates, 30,000 a549 cells per well were seeded and cultured in DMEM + 10% FBS for 3 days. Total RNA was extracted from the cells by adding different concentrations (5, 1, 0.2, 0.04, 0.008. mu.g/ml) of antibody and holding for 24 hours. LZ1 (non-binding antibody) was used only at the highest concentration as a negative control.

In 12-well plates, 30,000 a549 cells were seeded and cultured in DMEM + 10% FBS for 3 days. anti-FZD antibody 18R5 or 44R24 was added at different concentrations (5, 1, 0.2, 0.04, 0.008 μ g/ml) and LZ1 (non-binding antibody) was used only at the highest concentration as a negative control. 24 hours after treatment, RNA was prepared and subsequently treated with DNase.

Axin2 is known to be a robust (robust) target gene in Wnt signaling and its expression level was examined by performing Taqman relative expression (Δ Δ CT) assays using an applied biosystems 7900HT instrument. The GUSB probe was used for internal control using 50ng RNA at each point (performed three times in parallel). All results were normalized to Axin2 levels in LZ1 control samples.

Dose-response curves showing inhibition of axin2 gene expression basal levels by 18R5 and 44R24 and calculated EC50 values for these antibodies are shown in figure 49. 18R5 and 44R24 inhibited Axin2 basal levels at comparable efficiencies relative to the LZ1 control.

Example 21 evaluation of anti-FZD mAb anti-tumor Activity in pancreatic xenograft model

OMP-PN13 pancreatic tumors:

frozen OMP-PN13 tumor cells were obtained from an Onconed tumor bank that had been passaged 2 times in mice. They were thawed and injected subcutaneously into the left flank of NOD/SCID mice immediately after thawing. Approximately 25,000 viable cells were injected per animal. Mice were monitored weekly for tumor growth. After tumor development, tumor size was measured weekly with calipers. Will carry 200-300 mm³The tumor mice of (a) were divided into treatment groups, each group containing 5 animals. The average tumor size in each group was similar. Ab treatment was started the first day after randomization. LZ1 was used as a negative control Ab. During 12 days, 3 doses of Ab, 10mg/kg each, were administered by intraperitoneal injection. Mice were euthanized 24 hours after the last injection. Tumors, duodenum and liver were collected.

Tumor tissue was fixed in formalin for paraffin embedding and sectioning. Muc16 detection was performed by Immunohistochemistry (IHC) to monitor the presence of mucin-producing cells. Mucins are specific for a subset of differentiated cells in the pancreas and are therefore used as differentiation markers in tumor models.

Formalin-fixed paraffin-embedded (FFPE) sections were deparaffinized. Slides were first deparaffinized by treatment with xylene twice in succession (5 minutes each). The tissue was then rehydrated by successive immersions twice in 100% ethanol (3 minutes each), 1 time in 90% aqueous ethanol (1 minute), 1 time in 80% aqueous ethanol (1 minute) and 1 time in 70% aqueous ethanol (1 minute). The tissue was washed with running distilled water for 1 minute.

The mucin 16 antibody (clone X325 from AbCAM, cat No. ab10033) was used for IHC detection of mucin 16 expressing cells in FFPE tissue sections. Heat-induced antigen recovery (heat-induced antigen recovery) was performed in an autoclave with 10mM citrate buffer (pH 6.0). The slides were then placed at room temperature to slowly restore antigenicity to the protein (approximately 2 hours).

Tissue sections were blocked with 3% aqueous hydrogen peroxide, washed, and then blocked again with normal horse serum blocking solution (PBS (38.5ml), 10% NHS (5ml), 1% BSA (5ml), 0.1% gelatin (500. mu.l), 0.1% Tx-100 (500. mu.l), 0.05% NaN3 (500. mu.l) per 50 ml) for 1 hour at room temperature. The sections were then stained with Muc16 primary antibody diluted 1: 200 in Da Vinci Green diluent (pH7.3) (PD 900, Biocare Medical) at room temperature for 1 hour, followed by three washes with phosphate buffered saline containing 0.1% triton X-100. Sections were then stained with 3 drops of ImmPress anti-mouse IgG HRP conjugate (catalog # 101098-260, VWR) for 30 minutes at room temperature, followed by three washes with phosphate buffered saline containing 0.1% triton X-100. Slides were placed in Petri dishes (petridish) and developed with the Vector NovaRed kit (SK4800, Vector labs) for 1-2 minutes. The reaction was stopped by adding distilled water. The slides were rinsed thoroughly under running distilled water. The tissue sections were then counterstained with hematoxylin (catalog No. H3401, Vector Labs Gill's formula) for 1 minute, washed, and then neutralized with bluing solution (blue solution) for 30 seconds. Slides were allowed to dry overnight and subsequently mounted using vectamount (vector labs).

Figure 50 shows representative regions of tumors treated with control Ab (LZ1), 18R5, or 44R 24. LZ1 corresponds to low density staining, with higher levels of Muc16 antibody staining detected in tumors treated with 18R 5. This indicates that 18R5 induces differentiation of tumor cells towards the mucin-producing cell lineage. In this experiment, the level of Muc16 staining in 44R24 treated tumors was weaker than in 18R5 treated tumors, but still slightly higher than in LZ1 treated tumors.

Total RNA was also extracted from tumors, duodenum and liver for providing qPCR for Wnt target gene expression analysis.

Immediately after collection, the tissue was transferred to rnalater (qiagen). RNA was extracted using QIAGEN fibrous tissue RNeasy mini kit according to the manufacturer's instructions. Gene expression analysis was performed on 50ng total RNA using ABI one-step RT-PCR protocols and reagents. The expression of the GUSB gene was used as an endogenous control. Each sample was subjected to three parallel tests. All 5 tumors were analyzed for each treatment group. These experiments were performed using an ABI 7900 TaqMan instrument. ABI SDS 2.2.1 software was used to analyze the data and calculate Δ CT values, which were converted to relative amounts. For each treatment group, three replicates of all 5 tumors were averaged. Fold inhibition factor (fold inhibition factor) was then calculated relative to the control antibody (LZ 1).

The results are shown in Table 15. anti-FZD Ab produced varying degrees of influence on Wnt target genes. 18R5 induced 2.3x and 8x inhibition in tumors and liver, while unaffected in the duodenum. The change induced by 44R24 is weaker.

Table 15: qPCR gene expression analysis of Wnt target genes in 18R5 and 44R24 treated tissues

ND: is not carried out

Separate experiments are shown in italics

OMP-PN4 pancreatic tumors:

the effect of 18R5 on tumor stroma in an OMP-PN4 pancreatic tumor xenograft model was also investigated. Several genes whose expression levels are altered by the treatment are identified by microarrays. Of particular interest is ACTA2 encoding Smooth Muscle Actin (SMA). SMA has been shown to be associated with activated tumor stroma. Down-regulation of SMA can therefore be considered as a marker of a reduction in the tumorigenic phenotype.

NOD/SCID mice bearing OMP-PN4 tumors were treated with control Ab (LZ-1), 18R5, gemcitabine, or a combination of 18R5 and gemcitabine as described above in example 7, once weekly for 6 weeks. The antibody was administered at a concentration of 10 mg/kg. After tumor harvesting, tumors from the above experiments were analyzed for Wnt target gene expression at RNA and protein levels using microarray and IHC, respectively.

Total RNA was extracted from the tumor, amplified, and subjected to microarray analysis. Total RNA was amplified using the Ovation RNA amplification System V2(NuGEN, San Carlos, Calif.). The resulting amplified antisense ss-cDNA was fragmented and biotinylated using FL-Ovation cDNA Biotin Module V2(NuGEN) for use on Affymetrix chips. AffymetrixHG-U133 plus 2 or MG 4302.0 oligonucleotide microarrays were used in these experiments (performed in Almac Diagnostics, Durham, NC). After hybridization, the gene chips were washed, stained and scanned according to the manufacturer's instructions (Affymetrix, Santa Clara, Calif.). The quality of the cDNA and fragmented cDNA was assessed by spectrometer and Bioanalyzer prior to array hybridization. The scanned raw Chip data was quantified and scaled using the GCOS software package (Affymetrix) and subjected to a comprehensive assessment of Gene Chip Quality Control (Gene Chip Quality Control) recommended by Affymetrix to detect any Chip defects and anomalies and to reject them from subsequent data analysis.

Array background adjustment and signal intensity normalization were performed using the GCRMA algorithm in the open source software Bioconductor (www.bioconductor.org). A Bayesian T-test (Cyber-T) was used to identify genes differentially expressed between two groups or time points, incorporating student T-tests and Bayesian assessment of intra-group changes obtained from probe set changes with similar observed expression levels (Baldi P, Long AD. A Bayesian frame for the analysis of micro expression data: standardized analysis-test and statistical information of genes. bioinformatics.2001; 17 (6): 509-19).

For human tumor gene chip analysis, samples were assayed on human and mouse chips to independently assess the effect of treatment on both whole human tumors and on mouse stroma. Affymetrix probe sets without species specificity were omitted from the analysis.

In preparation for smooth muscle actin alpha (SMAa) immunofluorescence, OCT was used to freeze tumor tissue. 4 μm sections were obtained and frozen at-80 ℃. For SMAa staining, the tissue was fixed using ice cold acetone at-20 ℃ for 15 min, then allowed to dry and reach room temperature, then marked with a hydrophobic PAP pen. Slides were then washed with Phosphate Buffered Saline (PBS). Normal horse serum r.t.u. (Vector Labs) was used to seal the tissue for 2 hours at room temperature. Primary antibody staining was performed for 1 hour with FITC-conjugated smooth muscle actin α antibody (cline 1A4, # F3777, SIGMA) diluted 1: 10,000. Sections were washed 3 times with PBS containing 0.1% triton X-100. Slides were then allowed to air dry and subsequently mounted using DAPI-containing robust mounting media (vectashied H-500).

Fig. 51A shows ACTA2 gene expression levels detected by microarray. Tumors treated with anti-FZD antibody 18R5 showed a decrease in expression levels of ACTA 2. FIG. 51B shows smooth muscle actin α (SMAa) immunofluorescence results for OMP-PN4 tumors treated with control mAb (top panel) and 18R5 (bottom panel). A decrease in the amount of SMAa was detected on 18R 5-treated tumors. Expression of ACTA2 and the amount of SMA in the tumor decreased significantly in response to 18R5, indicating that Wnt blockade (i) affected the tumor compartment (tumor component) and (ii) achieved this by decreasing well-established oncogenic markers. These results indicate that a decrease in myofibroblast activation may be one of the mechanisms of 18R5 anti-tumor action.

Example 22 evaluation of the tumorigenic potential of Muc 16-positive OMP-PN13 cells

As described above, 18R5 treatment induces gene expression and cellular phenotypic changes in pancreatic tumors, including elevated mucin expression. In particular, IHC on treated PN-13 tumors showed an increased number of Muc16 positive cells. The Muc16 gene expression level was also higher in the treated tumors than in the control tumors. The tumorigenicity of Muc16 positive cells induced by 18R5 was evaluated to test the following hypothesis: 18R 5-induced Muc 16-positive cells represent a subpopulation of differentiated tumor cells.

Mice carrying OMP-PN13 were treated with 18R5 according to the protocol described in example 21. Mice were euthanized 12 days after Ab treatment was initiated. Tumors were harvested and treated with collagenase III to digest tissue, thereby obtaining single cell suspensions. Mouse stromal cells were stained with biotinylated anti-H-2 Kd antibody and biotinylated anti-CD 45 antibody. It was then incubated with streptavidin coupled magnetic beads (ThermoMagnaBind) and subtracted using Dynal magnets. The resulting lin-depleted tumor cells were stained with anti-Muc 16mAb and detected with PE-conjugated secondary Ab. Muc16 positive cells and Muc16 negative cells were sorted using an ARIAFACS instrument operated by DIVA software. See fig. 52A. The cells were re-injected subcutaneously into the left flank of NOD/SCID mice to compare their tumorigenic potential. Each cell was injected into 10 mice. Each mouse received 75 cells. FIG. 52B shows a representative image of tumors resulting from injection of Muc16- (top panel) and Muc16+ (bottom panel) cells. FIG. 52C shows the growth curves of Muc 16-and Muc16+ tumors after injection in mice. There was no tumor growth after injection of Muc16+ cells in mice, whereas 7 out of 10 mice injected with Muc 16-cells developed tumors. This data indicates that the 18R 5-induced Muc16+ cells are non-tumorigenic, supporting an intrinsic mechanism for the induction of differentiation that is an anti-tumor activity of 18R 5.

Example 23 other in vivo studies with anti-FZD antibodies

Studies of PE13 Breast tumor recurrence with 18R5mAb: PE-13 mammary tumor cells were injected into NOD-SCID mice and allowed to grow until the tumors had reached approximately 100mm³. Animals were randomized into 2 groups (n ═ 10) and either taxol (15mg/kg, 2 times per week) plus control antibody (black squares) or the same dose of taxol plus anti-FZD 18R5 (grey open circles) were administered. The antibody was administered at a dose of 20mg/kg once a week. Taxol treatment was stopped on day 70 and antibody treatment continued. The results are shown in fig. 53. After discontinuation of taxol treatment, 18R5 was observed to increase tumor regression rate and delay tumor recurrence.

PE13 breast tumor Limiting Dilution Assay (LDA) study with 18R5 mAb: animals bearing PE13 breast tumors were treated with control antibodies (grey circles), 18R5 (open triangles), taxol (black circles) or a combination of taxol and 18R5 (open squares). Taxol was administered at a dose of 15mg/kg 2 times per week; the antibody was administered at a dose of 20mg/kg 1 time per week. Tumors were harvested and human tumor cells were purified by lin depletion. 50, 150 or 500 tumor cells were injected into a new group of age-matched mice (n-10/cell dose). The results are shown in fig. 54. Tumor growth frequency was monitored after 59 days and used to calculate CSC frequency (L-calc).

PN4 pancreatic tumor recurrence study with 18R5 mAb: PN4 pancreatic tumor cells were injected into Nod-Scid mice and allowed to grow until the tumors had reached approximately 250mm³. Gemcitabine (75mg/kg, 1 time per week) was administered to the animals for 5 weeks until the tumor had regressed. Animals were randomized into 2 groups and either a control antibody (black squares) or anti-FZD 18R5 (gray open circles) was administered. The antibody was administered at a dose of 10mg/kg once a week. The results are shown in fig. 55. Delay in tumor recurrence was observed with 18R5 following treatment with gemcitabine.

PN4 pancreatic tumor growth study with 44R24 mAb: PN4 pancreatic tumors were injected into Nod-Scid mice. Growing the tumor until its volume has reached about 150mm³. Animals were randomized into 4 groups (n-10 per group) and given control antibody (black squares), 44R24 (grey open triangles) anti-FZD 5/8, gemcitabine (solid open triangles), gemcitabineHeart triangle) or 44R24 in combination with gemcitabine (gray open circle). Gemcitabine was administered at a dose of 15mg/kg 1 time per week; the antibody was administered at a dose of 20mg/kg 2 times per week. The results are shown in fig. 56. 44R24 in combination with gemcitabine was observed to reduce tumor growth relative to gemcitabine alone.

Example 24 epitope mapping of anti-FZD antibody 44R24

Epitope mapping was performed on the anti-FZD antibody 44R24 in a similar manner to that described above for antibodies 18R8 and 18R5 in example 5. The ability of 44R24 to bind to an epitope like the 18R8 epitope was assessed by flow cytometry using a series of amino acid variants of FZD8 previously shown to disrupt binding of 18R8 (see example 5 and fig. 6 and fig. 7). Amino acids 126-127 of FZD8 were found to be required for binding to 44R24 as indicated by a decrease in staining in the co-transfected (GFP positive) cell population. The results of the FACS experiment are shown in fig. 57. These results show that the epitope bound by 44R24 overlaps with the epitope of 18R8 and comprises the common amino acids 126-127.

Example 25C 28 Colon tumor growth Studies with anti-FZD antibodies 18R5, 18R8 and 44R24

C28 tumor cells were injected subcutaneously into Nod/Scid mice. The tumor was allowed to grow until its average volume had reached about 126mm³. Tumor bearing animals were randomized into 4 groups (n-10 mice per group) and treated with control Ab (black squares), 18R8 (grey triangles), 44R24 (black open circles) or 18R5 (grey circles) (fig. 58). The antibody was administered intraperitoneally at a dose of 15mg/kg twice weekly. Tumor volume is indicated. Treatment with antibodies 44R24 and 18R5 reduced growth relative to the control, whereas 18R8 had no effect (fig. 58).

After treatment of the animals in the experiment shown in fig. 58, tumors were harvested, fixed in formalin, paraffin embedded, and cut into 5 μm sections. Tumor sections were analyzed by immunohistochemistry (Vectastain kit, Vector Labs) for expression of cytokeratin 7 (marker for colon cell differentiation). Increased cytokeratin 7 expression was found following treatment with 44R24 or 18R5 (fig. 59).

All publications, patents, patent applications, internet sites, and access number/database sequences (including polynucleotide and polypeptide sequences) cited herein are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent application, internet site, or access number/database sequence was specifically and individually indicated to be incorporated by reference.

Claims (25)

1. An isolated antibody that specifically binds to one or more human frizzled receptors selected from the group consisting of FZD1, FZD2, FZD5, FZD7, and FZD8, wherein the antibody has:

(a) GFTFSHYTLS (SEQ ID NO:1) heavy chain CDR1, VISGDGSYTYYADSVKG (SEQ ID NO:2) heavy chain CDR2, and (c) NFIKYVFAN (SEQ ID NO:3) heavy chain CDR 3; and

(b) SGDNIGSFYVH (SEQ ID NO:7), DKSNRPSG (SEQ ID NO:8) and QSYANTLSL (SEQ ID NO:9) light chain CDR1, DKSNRPSG (SEQ ID NO:8) light chain CDR2 and light chain CDR 3; alternatively, SGDKLGKKYAS (SEQ ID NO:4) light chain CDR1, EKDNRPSG (SEQ ID NO:5) light chain CDR2, and SSFAGNSLE (SEQ ID NO:6) light chain CDR 3.

2. The antibody of claim 1, having a light chain CDR1 of SGDNIGSFYVH (SEQ ID NO:7), a light chain CDR2 of DKSNRPSG (SEQ ID NO:8), and a light chain CDR3 of QSYANTLSL (SEQ ID NO: 9).

3. The antibody of claim 1, having a light chain CDR1 of SGDKLGKKYAS (SEQ ID NO:4), a light chain CDR2 of EKDNRPSG (SEQ ID NO:5), and a light chain CDR3 of SSFAGNSLE (SEQ ID NO: 6).

4. The antibody of claim 1, which specifically binds FZD1, FZD2, FZD5, FZD7, and FZD 8.

5. An isolated antibody that specifically binds to one or more human frizzled receptors selected from the group consisting of FZD1, FZD2, FZD5, FZD7, and FZD8, wherein the antibody comprises:

(a) 10, a polypeptide of SEQ ID NO; and

(b) 12 or 14.

6. The antibody of claim 5, which specifically binds FZD1, FZD2, FZD5, FZD7 and FZD 8.

7. The antibody of claim 5, comprising:

(a) 10, a polypeptide of SEQ ID NO; and

(b) 14, SEQ ID NO.

8. The antibody of any one of claims 1-7, which is a monoclonal antibody, a humanized antibody, a human antibody, an IgG1 antibody, an IgG2 antibody, or an antibody fragment comprising the epitope variable regions of an intact antibody.

9. An antibody encoded by the sequence of a plasmid deposited with the ATCC as accession number PTA-9540, PTA-9541, PTA-10307 or PTA-10309.

10. The antibody of claim 8 conjugated to a cytotoxic agent.

11. An isolated cell that produces the antibody of claim 8.

12. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and the antibody of claim 8.

13. Use of an antibody according to claim 8 in the manufacture of a medicament for the treatment of cancer.

14. Use of the antibody of claim 8 in the manufacture of a medicament for inhibiting Wnt signaling.

15. Use of an antibody according to claim 8 in the manufacture of a medicament for inhibiting tumor growth.

16. The use of claim 13, wherein the medicament further comprises a second anti-cancer agent.

17. The use of claim 16, wherein the second anti-cancer agent is a chemotherapeutic agent or an angiogenesis inhibitor.

18. The use of claim 16, wherein the second anti-cancer agent is an inhibitor of Notch signaling.

19. The use of claim 16, wherein the second anti-cancer agent is an anti-Notch antibody.

20. The use of claim 16, wherein the second anti-cancer agent is an antibody against a Notch ligand.

21. An isolated polypeptide comprising

(a) 10 and 12 SEQ ID NO;

(b) 10 and 14;

(c) 11 and 13 SEQ ID NO; or

(d) SEQ ID NO 11 and SEQ ID NO 15.

22. An isolated polynucleotide comprising:

a polynucleotide encoding the antibody of claim 8.

23. An isolated polynucleotide comprising:

(a) 17 and 19;

(b) 17 and 21 SEQ ID NO;

(c) 18 and 20; or

(d) 18 and 22, respectively.

24. A vector comprising the polynucleotide of claim 22.

25. A vector comprising the polynucleotide of claim 23.