NZ620100B2 - Rspo binding agents and uses thereof - Google Patents

Abstract

Disclosed is an isolated monoclonal antibody that specifically binds human R-spondin2 (RSPO2), which comprises: (a) a heavy chain CDR1 comprising SSYAMS (SEQ ID NO:29), a heavy chain CDR2 comprising SISSGGSTYYPDSVKG (SEQ ID NO:30), and a heavy chain CDR3 comprising RGGDPGVYNGDYEDAMDY (SEQ ID NO:31); and (b) a light chain CDR1 comprising KASQDVSSAVA (SEQ ID NO:32), a light chain CDR2 comprising WASTRHT (SEQ ID NO:33), and a light chain CDR3 comprising QQHYSTP (SEQ ID NO:34). ; and (b) a light chain CDR1 comprising KASQDVSSAVA (SEQ ID NO:32), a light chain CDR2 comprising WASTRHT (SEQ ID NO:33), and a light chain CDR3 comprising QQHYSTP (SEQ ID NO:34).

Description

RSPO G AGENTS AND USES THEREOF BACKGROUND OF THE ION Field of the Invention The field of this invention generally relates to antibodies and other agents that bind R-Spondin proteins (RSPO), particularly human R-Spondin proteins ing RSP01, RSP02 and RSPO3, as well as to s of using the dies or other agents for the treatment of diseases such as cancer.

Background of the Invention The R-Spondin (RSPO) family of ns is conserved among vertebrates and comprises four members, RSPOI, RSP02, RSPO3 and RSPO4. These proteins have been referred to by a variety of names, including roof plate-specific spondins, hPWTSR (hRSPO3), THSZD (RSPO3), Cristin 1-4, and Futrin 1-4. The RSPOs are small ed proteins that overall share approximately 40-60% sequence homology and domain organization. All RSPO proteins contain two like cysteine-rich domains at the N— terminus followed by a thrombospondin domain and a basic charged C-terminal tail (Kim et al., 2006, Cell Cycle, 5:23-26).

Studies have shown that RSPO proteins have a role during vertebrate development (Kamata et al., 2004, m. Biophys Acta, 1676:51-62) and in Xenopus myogenesis (Kazanskaya et al., 2004, Dev. Cell, 7:525-534). RSPOl has also been shown to function as a potent mitogen for gastrointestinal lial cells (Kim et al., 2005, Science, 309:1256-1259). RSPO proteins are known to activate B-catenin signaling similar to Wnt signaling, however the relationship between RSPO proteins and Wnt signaling is still being investigated. It has been reported that RSPO proteins possess a positive modulatory activity on Wnt ligands (Nam et al., 2006, JBC 281:13247-57). This study also reported that RSPO proteins could function as Frizzled8 and LRP6 receptor ligands and induce B-catenin ing (Nam et al., 2006, JBC 281:13247-57). Recent studies have identified an interaction between RSPO proteins and LGR (leucine-rich repeat containing, G protein-coupler receptor) proteins, such as LGRS (US. Patent Publication Nos. 2009/0074782 and 2009/0191205), and these data present an ative pathway for the activation of B-catenin signaling.

The Wnt signaling pathway has been identified as a potential target for cancer therapy. The Wnt signaling pathway is one of several critical regulators of embryonic n formation, post-embryonic tissue maintenance, and stem cell biology. More specifically, Wnt signaling plays an important role in the generation of cell polarity and cell fate specification including self-renewal by stem cell populations. Unregulated activation of the Wnt pathway is associated with us human s where it is believed the activation can alter the developmental fate of cells. The activation of the Wnt pathway may maintain tumor cells in an undifferentiated state and/or lead to uncontrolled proliferation. Thus carcinogenesis can proceed by king homeostatic mechanisms which control normal development and tissue repair (reviewed in Reya & Clevers, 2005, Nature, 434:843-50; Beachy et al., 2004, Nature, 432:324-31).

The Wnt signaling pathway was first elucidated in the Drosophila developmental mutant Wingless (wg) and from the muréne proto-oncogene int-1, now Wntl (Nusse & Varmus, 1982, Cell, 31299-109; Van Ooyen & Nusse, 1984, Cell, -40; Cabrera et al., 1987, Cell, 50:659-63; ijk et al., 1987, Cell, 50:649-57). Wnt genes encode secreted lipid-modified glycoproteins of which 19 have been identified in mammals.

These secreted ligands activate a receptor complex ting of a Frizzled (FZD) receptor family member and low-density lipoprotein (LDL) receptor-related protein 5 or 6 (LRP5/6). The FZD receptors are seven transmembrane domain proteins of the ein coupled receptor (GPCR) amily and contain a large extracellular N-terminal ligand binding domain with 10 conserved cysteines, known as a cysteine-rich domain (CRD) or Fri domain. There are ten human FZD receptors, FZDl, FZD2, FZD3, FZD4, FZDS, FZD6, FZD7, FZD8, FZD9, and FZDIO. Different FZD CRDs have different g ies for specific Wnt proteins (Wu & Nusse, 2002, J. Biol. Chem, 277:41762-9), and FZD receptors have been grouped into those that activate the canonical B-catenin pathway and those that activate nonical pathways (Miller et al., 1999, Oncogene, 18:7860- 72).

A role for Wnt signaling in cancer was first uncovered with the identification of Wntl (originally intl) as an oncogene in mammary tumors transformed by the nearby insertion of a murine virus (Nusse & Varmus, 1982, Cell, 31:99-109). Additional evidence for the role of Wnt signaling in breast cancer has since accumulated. For instance, transgenic over-expression of B-catenin in the mammary glands results in hyperplasias and adenocarcinomas (lmbert et al., 2001, J Cell Biol, 153:555-68; Michaelson & Leder, 2001, Oncogene, 20:5093-9) whereas loss of Wnt signaling disrupts normal mammary gland development (Tepera et al., 2003, J. Cell Sci, 37-49; Hatsell et al., 2003, J. Mammary Gland Biol. Neoplasia, 8:145-58). In human breast cancer, B-catenin accumulation implicates ted Wnt signaling in over 50% of omas, and though c mutations have not been identified, up-regulation of Frizzled receptor expression has been observed an & Brown, 2004, .1 Mammary Gland Biol. Neoplasia, 91119—31; novic et al., 2004, Int. J Oncol, 25:1337-42).

Activation of the Wnt pathway is also associated with colorectal cancer. imately 5-10% of all colorectal cancers are hereditary with one of the main forms being al adenomatous polyposis (FAP), an autosomal dominant disease in which about 80% of affected individuals contain a germline mutation in the adenomatous polyposis coli (APC) gene. Mutations have also been fied in other Wnt pathway components including Axin and B—catenin. Individual adenomas are clonal outgrowths of lial cells containing a second inactivated allele, and the large number of FAP adenomas inevitably results in the pment of adenocarcinomas through additional mutations in oncogenes and/or tumor suppressor genes. Furthermore, activation of the Wnt ing pathway, including loss—of—funct‘ion mutations in APC and stabilizing mutations in B-catenin, can induce hyperplastic development and tumor growth in mouse models (Oshima et al., 1997, Cancer Res., 57:1644-9; Harada et al., 1999, EMBO J., 18:5931-42).

Similar to breast cancer and colon cancer, melanoma often has constitutive activation of the Wnt pathway, as indicated by the nuclear lation of B-catenin.

Activation of the Wnt/B-catenin pathway in some melanoma tumors and cell lines is due to modifications in pathway components, such as APC, ICAT, LEFl and B-catenin (see e.g., Larue et al. 2006, Frontiers Biosci., 112733-742). However, there are conflicting reports in the literature as to the exact role of Wnt/B-catenin signaling in ma. For example, one study found that elevated levels of nuclear B-catenin correlated with improved survival from melanoma, and that activated Wnt/β-catenin signaling was associated with decreased cell proliferation (Chien et al., 2009, PNAS, 106:1193- 1198).

The focus of cancer drug research is shifting toward targeted therapies aimed at genes, proteins, and pathways involved in human cancer. There is a need for new agents targeting signaling pathways and new combinations of agents that target le pathways that could provide therapeutic benefit for cancer patients. Thus, ecules (e.g., anti-RSPO antibodies) that disrupt β-catenin signaling are a potential source of new eutic agents for cancer, as well as other β-catenin- ated diseases.

BRIEF SUMMARY OF THE INVENTION [0009a] In a first aspect, the present invention provides an isolated monoclonal antibody that specifically binds human R-spondin2 (RSPO2), which comprises: (a) a heavy chain CDR1 comprising SSYAMS (SEQ ID , a heavy chain CDR2 comprising SISSGGSTYYPDSVKG (SEQ ID NO:30), and a heavy chain CDR3 comprising RGGDPGVYNGDYEDAMDY (SEQ ID NO:31); (b) a light chain CDR1 comprising KASQDVSSAVA (SEQ ID NO:32), a light chain CDR2 comprising WASTRHT (SEQ ID NO:33), and a light chain CDR3 comprising QQHYSTP (SEQ ID NO:34).

] In a second aspect, the present invention provides an isolated onal antibody that competes with a second antibody for specific binding to RSPO2, wherein the second antibody comprises (a) a heavy chain variable region sing SEQ ID NO:27 and a light chain le region comprising SEQ ID NO:28; (b) a heavy chain variable region comprising SEQ ID NO:63 and a light chain variable region comprising SEQ ID NO:67; or (c) a heavy chain variable region comprising SEQ ID NO:63 and a light chain variable region comprising SEQ ID NO:76. 11282561_1 [0009c] In a third aspect, the present invention provides an isolated monoclonal antibody that binds the same epitope on RSPO2 as the antibody according to the first or second aspect. [0009d] In a fourth aspect, the present invention provides a monoclonal antibody produced by the hybridoma cell line having ATCC deposit number PTA-12021. [0009e] In a fifth aspect, the present ion es a monoclonal humanized antibody comprising the same heavy chain CDR1, CDR2, and CDR3, and light chain CDR1, CDR2, and CDR3 as the antibody produced by the hybridoma cell line having ATCC deposit number 021. [0009f] In a sixth aspect, the present invention provides a cell comprising or ing the antibody ing to any of the first to fifth s, wherein the cell is not within a human body. [0009g] In a seventh aspect, the present ion provides a hybridoma cell line having ATCC deposit number PTA-12021. [0009h] In an eighth aspect, the present invention provides an isolated polynucleotide molecule comprising a nucleotide sequence that encodes an antibody according to any of the first to fifth aspects. [0009i] In a ninth , the present invention provides a cell comprising the polynucleotide of the eighth aspect, n the cell is not within a human body. [0009j] In a tenth aspect, the present invention provides a pharmaceutical composition comprising the antibody according to any of the first to fifth aspects and a ceutically acceptable carrier. [0009k] In an eleventh aspect, the present invention provides use of an antibody according to any of the first to fifth aspects in the preparation of a medicament for inhibiting growth of a tumor. [0009l] In a twelfth aspect, the present invention provides use of an antibody according to any of the first to fifth aspects in the preparation of a medicament for inhibiting β-catenin signaling in a cell. [0009m] In a enth aspect, the present invention es use of an antibody according to any of the first to fifth aspects in the ation of a medicament for the treatment of cancer.

AH26(11355570_1):EOR [0009n] In a enth aspect, the present invention provides use of an antibody ing to any of the first to fifth aspects in the preparation of a medicament for ent of a disease associated with activation of β-catenin. [0009o] In a fifteenth aspect, the present invention provides an isolated monoclonal antibody that specifically binds human din2 (RSPO2), which comprises a heavy chain amino acid ce of SEQ ID NO:41 and a light chain amino acid sequence of SEQ ID NO:42. [0009p] In a sixteenth aspect, the present invention provides an isolated monoclonal antibody that specifically binds human R-spondin2 (RSPO2), which comprises a heavy chain amino acid sequence of SEQ ID NO:70 and a light chain amino acid sequence of SEQ ID NO:71. [0009q] In a seventeenth aspect, the present invention provides an isolated monoclonal antibody that specifically binds human R-spondin2 (RSPO2), which comprises a heavy chain amino acid sequence of SEQ ID NO:70 and a light chain amino acid sequence of SEQ ID NO:74.

The t invention provides binding agents, such as antibodies, that bind RSPO proteins, as well as itions, such as pharmaceutical compositions, comprising the binding agents. In n embodiments, the RSPO-binding agents are novel polypeptides, such as antibodies, antibody fragments, and other polypeptides d to such antibodies. In certain embodiments, the binding agents are dies that specifically bind human RSPO1, RSPO2, and/or RSPO3. The invention further provides methods of inhibiting the growth of a tumor by administering the RSPO- g agents to a subject with a tumor. The invention further provides methods of treating cancer by administering the RSPO-binding agents to a subject in need thereof.

In some embodiments, the methods of treating cancer or inhibiting tumor growth comprise targeting cancer stem cells with the RSPO-binding agents. In certain embodiments, the methods comprise ng the frequency of cancer stem cells in a tumor, reducing the number of cancer stem cells in a tumor, reducing the tumorigenicity of a tumor, and/or reducing the tumorigenicity of a tumor by reducing the number or frequency of cancer stem cells in the tumor.

In one , the invention provides a binding agent, such as an antibody, that specifically binds human RSPO1. In certain embodiments, the RSPO1-binding agent binds within amino acids 21-263 of human RSPO1. In certain embodiments, the AH26(11355570_1):EOR RSPO1-binding agent binds within amino acids 34-135 of human RSPO1. In certain embodiments, the RSPO1-binding agent binds within amino acids 91-135 of human RSPO1. In some embodiments, the RSPO1-binding agent (e.g., an dy) specifically AH26(11355570_1):EOR binds at least one other human RSPO selected from the group consisting of RSP02, RSPO3, and RSPO4. In some embodiments, the RSPOl-binding agent or antibody modulates B-catenin ty, is an antagonist of B-catenin signaling, inhibits B-catenin signaling, and/or inhibits activation of B-catenin. In some embodiments, the RSPOl- binding agent inhibits RSPOl signaling. In some embodiments, the RSPOl-binding agent inhibits or interferes with binding of RSPOl to one or more LGR protein (e.g., LGR4, LGRS, and/or LGR6). In some embodiments, the RSPOl-binding agent inhibits binding of RSPOl to LGRS.

In another , the invention provides a binding agent, such as an antibody, that specifically binds human RSP02. In certain embodiments, the RSP02—binding agent binds within amino acids 22—243 of human RSP02. In n ments, the RSP02- binding agent binds within amino acids 22-205 of human RSP02. In certain embodiments, the RSP02-binding agent binds within amino acids 34-134 of human RSP02. In certain embodiments, the RSP02—binding agent binds Within amino acids 90- 134 of human RSP02. In some embodiments, the RSP02-binding agent (e.g., an antibody) specifically binds at least one other human RSPO selected from the group consisting of RSPOl, RSPO3, and RSPO4. In some embodiments, the RSP02-binding agent or antibody modulates B-catenin activity, is an nist of B-cateniri signaling, inhibits B-catenin signaling, and/or inhibits activation of B-catenin. In some embodiments, the binding agent inhibits RSP02 sigrzaling. In some embodiments, the RSP02-binding agent inhibits or interferes with binding of RSP02 to one or more LGR protein (e.g., LGR4, LGRS, and/or LGR6). In some embodiments, the binding agent inhibits binding of RSP02 to LGRS.

In certain embodiments of each of the entioned aspects and embodiments, as well as other aspects and embodiments descrébed herein, the RSPO-binding agent is an dy. In certain embodiments, the antibody is a monoclonal antibody. In n embodiments, the antibody is a humanized antibody. In certain embodiments, the antibody binds human RSPOI. In certain embodiments, the antibody binds human RSPOl and mouse RSPOl. In certain embodiments, the antibody binds human RSPOl with a KB of less than lnM and mouse RSPOl with a KD of less than 1nM.

In certain embodiments, the RSPOl-binding agent is an antibody which comprises a heavy chain CDRl sing TGYTMH (SEQ ID NO:12), a heavy chain CDR2 comprising GINPNNGGTTYNQNFKG (SEQ ID NO:13), and a heavy chain CDR3 comprising KEFSDGYYFFAY (SEQ ID NO:14). In some embodiments, the dy further comprises a light chain CDRl comprising KASQDVIFAVA (SEQ ID NO:15), a light chain CDR2 comprising WASTRHT (SEQ ID NO:16), and a light chain CDR3 comprising QQHYSTPW (SEQ ID NO:17). In certain embodiments, the RSPOl-binding agent is an antibody which comprises a light chain CDRl comprising KASQDVIFAVA (SEQ ID , a light chain CDR2 sing WASTRHT (SEQ ID NO:16), and a light chain CDR3 comprising QQHYSTPW (SEQ ID NO:17). In certain embodiments, the binding agent is an antibody which comprises: (a) a heavy chain CDRl comprising TGYTMH (SEQ ID NO:12), or a variant thereof sing 1, 2, 3, or 4 amino acid substitutions; (b) a heavy chain CDR2 comprising GINPNNGGTTYNQNFKG (SEQ ID NO:13), or a variant thereof comprising 1, 2, 3, or 4 amino acid substitutions; (c) a heavy chain CDR3 comprising KEFSDGYYFFAY (SEQ ID NO:14), or a variant f comprising 1, 2, 3, or 4 amino acid tutions; (d) a light chain CDRl comprising KASQDVIFAVA (SEQ ID NO:15), or a variant thereof comprising 1, 2, 3, or 4 amino acid substitutions; (e) a light chain CDR2 comprising WASTRHT (SEQ ID NO:16), or a variant thereof sing 1, 2, 3, or 4 amino acid substitutions; and (f) a light chain CDR3 sing QQHYSTPW (SEQ ID N0:17), or a variant thereof comprising 1, 2, 3, or 4 amino acid substitutions. In some embodiments, the amino acid substitutions are conservative amino acid substitutions.

In certain embodiments, the RSPOl-binding agent is an antibody which comprises: (a) a heavy chain variable region having at least 80% sequence identity to SEQ ID NOle; and/or (b) a light chain variable region having at least 80% sequence identity to SEQ ID NO:11. In certain embodiments, the RSPOl-binding agent is an antibody that comprises: (a) a heavy chain variable region having at least 90% sequence identity to SEQ ID NO:10; and/or (b) a light chain le region having at least 90% sequence identity to SEQ ID NO:11.

In certain embodiments, the RSPOl—binding agent is an antibody which comprises: (a) a heavy chain variable region having at least 80% sequence identity to SEQ ID NO:55; and/or (b) a light chain variable region having at least 80% sequence identity to SEQ ID NO:59. In certain ments, the RSPOl-binding agent is an antibody that comprises: (a) a heavy chain variable region having at least 90% sequence identity to SEQ ID NO:55; and/or (b) a light chain variable region having at least 90% sequence identity to SEQ ID NO:59.

In some embodiments, the RSPOl-binding agent is monoclonal antibody 89M5 and is produced by the oma cell line 89M5 deposited on June 30, 2011 with ATCC having deposit no. PTA-11970. In some embodiments, the RSPOl-binding agent is a humanized form of dy 89M5. In some embodiments, the RSPOl-binding agent is zed monoclonal antibody h89M5—H2L2.

In certain embodiments, the RSPOZ-binding agent is an antibody which binds human RSP02. In some embodiments, the antibody binds human RSPO2 and mouse RSPO2. In certain embodiments, the antibody comprises a heavy chain CDRl comprising SSYAMS (SEQ ID N0229), a heavy chain CDR2 comprising SISSGGSTYYPDSVKG (SEQ ID N030), and a heavy chain CDR3 comprising RGGDPGVYNGDYEDAMDY (SEQ ID NO:31). In some embodiments, the antibody further comprises a light chain CDRl comprising KASQDVSSAVA (SEQ ID NO:32), a light chain CDR2 comprising WASTRHT (SEQ ID NO:33), and a light chain CDR3 comprising QQHYSTP (SEQ ID NO:34). In certain embodiments, the binding agent is an antibody which comprises a light chain CDRI sing SSAVA (SEQ ID NO:32), a light chain CDR2 comprising WASTRHT (SEQ ID N033), and a light chain CDR3 comprising QQHYSTP (SEQ ID NO:34). In certain embodiments, the RSPOZ-binding agent is an antibody which comprises: (a) a heavy chain CDRl comprising SSYAMS (SEQ ID N0229), or a t f comprising 1, 2, 3, or 4 amino acid substitutions; (b) a heavy chain CDR2 comprising SISSGGSTYYPDSVKG (SEQ ID NO:30), or a variant thereof comprising 1, 2, 3, or 4 amino acid substitutions; (c) a heavy chain CDR3 comprising RGGDPGVYNGDYEDAMDY (SEQ ID NO:31), or a t thereof comprising 1, 2, 3, or 4 amino acid substitutions; (d) a light chain CDRl sing KASQDVSSAVA (SEQ ID NO:32), or a variant thereof comprising 1, 2, 3, or 4 amino acid substitutions; (e) a light chain CDR2 comprising WASTRHT (SEQ ID NO:33), or a variant thereof comprising 1, 2, 3, or 4 amino acid substitutions; and (t) a light chain CDR3 comprising QQHYSTP (SEQ ID NO:34), or a variant thereof comprising 1, 2, 3, or 4 amino acid substitutions. In some embodiments, the amino acid substitutions are conservative amino acid tutions.

In certain embodiments, the RSPO2-binding agent is an antibody Which comprises: (a) a heavy chain variable region having at least 80% sequence identity to SEQ ID NO:27; and/or (b) a light chain variable region having at least 80% sequence identity to SEQ ID NO:28. In certain embodiments, the RSPOZ-binding agent is an antibody that comprises: (a) a heavy chain variable region having at least 90% sequence identity to SEQ ID NO:27; and/or (b) a light chain variable region having at least 90% sequence identity to SEQ ID NO:28.

In n embodiments, the RSPOZ-binding agent is an antibody which comprises: (a) a heavy chain variable region having at least 80% sequence identity to SEQ ID NO:63; and/or (b) a light chair: variable region having at least 80% sequence identity to SEQ ID NO:67. In n embodiments, the RSPOZ-binding agent is an antibody that ses: (a) a heavy chain variable region having at least 90% sequence identity to SEQ ID NO:63; and/or (b) a light chain variable region having at least 90% sequence identity to SEQ ID NO:67.

In certain embodiments, the RSPOZ-binding agent is an antibody which comprises: (a) a heavy chain variable region having at least 80% sequence identity to SEQ ID NO:63; and/or (b) a light chain variable region léaving at least 80% sequence identity to SEQ ID NO:76. In certain embodiments, the RSPOZ-binding agent is an antibody that comprises: (a) a heavy chain variable region having at least 90% sequence identity to SEQ ID NO:63; and/or (b) a light chain variable region having at least 90% sequence identity to SEQ ID NO:76.

In some embodiments, the RSPOZ-binding agent is monoclonal antibody 130M23 and is produced by the hybridoma cell line 130M23 deposited on August 10, 2011 with ATCC having deposit no. PTA-12021. In some ments, the RSPOZ-binding agent is a humanized form of dy 130M23. In some ments, the RSPO2-binding agent is humanized monoclonal antibody h130M23-H1L2. In some ments, the RSPOZ-binding agent is humanized monoclonal antibody hl30M23-HIL6, In r aspect, the invention provides a g agent (e.g., an antibody) that es for specific binding to a human RSPO protein with an antibody of the invention. In some embodiments, the binding agent (e.g., an antibody) competes for specific binding to human RSP01 with an antibody that comprises a heavy chain variable region comprising SEQ ID NO:10, and a light chain variable region comprising SEQ ID NO:11. In some embodiments, the g agent (e.g., an antibody) competes for specific binding to human RSPOl with an antibody that comprises a heavy chain variable region comprising SEQ ID NO:55, and a light chain variable region sing SEQ ID NO:59.

In some embodiments, the antibody with which the RSP01-binding agent competes is 89M5 or h89M5-H2L2. In some embodiments, the g agent competes for specific binding to RSPOI with an antibody of the invention in an in vitro competitive g assay.

In certain embodiments, the antibody binds the same epitope, or essentially the same epitope, on RSPOl as an antibody of the invention (e.g., 89M5).

In still another , the binding agent is an antibody that binds an epitope on RSP01 that overlaps with the epitope on RSPOl bound by an antibody of the invention (e.g., 89M5).

In another aspect, the invention provides a binding agent (e.g., an antibody) that competes for c binding to human RSP02 with an antibody of the invention. In some ments, the binding agent (e.g., an antibody) competes for specific binding to human RSP02 with an antibody that comprises a heavy chain variable region comprising SEQ ID NO:27, and a light chain variable region comprising SEQ ID NO:28. In some embodiments, the binding agent (e.g., an antibody) competes for specific binding to human RSP02 with an antibody that comprises a heavy chain variable region sing SEQ ID NO:63, and a light chain variable region comprising SEQ ID NO:67. In some embodiments, the binding agent (e.g., an antibody) competes for specific binding to human RSP02 with an antibody that comprises a heavy chain variable region comprising SEQ ID NO:63, and a light chain variable region comprising SEQ ID NO:76. In some embodiments, the antibody with which the RSP02-binding agent competes is 130M23, h130M23-H1L2, or h130M23—H1L6. In some embodiments, the binding agent competes for specific binding to RSP02. with an antibody of the invention in an in vitro itive binding assay.

In certain embodiments, the antibody binds the same epitope, or essentially the same epitope, on RSP02 as an antibody of the invention (e.g., ).

In still another aspect, the binding agent is an antibody that binds an epitope on RSP02 that overlaps with the epitope on RSP02 bound by an antibody of the invention (e.g., 130M23).

In certain embodiments of each of the aforementioned aspects, as well as other aspects and/or embodiments described elsewhere herein, the RSPO—binding agent or antibody is isolated.

In another aspect, the invention provides a polypeptide comprising SEQ ID NO:lO and/or SEQ ID NO:1 1. In another , the invention provides a polypeptide comprising SEQ ID NO:55 and/0r SEQ ID NO:59. In another aspect, the invention provides a polypeptide sing SEQ ID NO:27 and/or SEQ ID NO:28. In another aspect, the invention provides a polypeptide comprising SEQ ID NO:63 and/or SEQ ID NO:67. In another aspect, the invention provides a polypeptide comprising SEQ ID NO:63 and/or SEQ ID NO:76. In some embodiments, a polypeptide that binds RSPOl comprises a polypeptide comprising SEQ ID NO:25 and/or SEQ ID NO:26. In some embodiments, a ptide that binds RSPOI ses a polypeptide sing SEQ ID NO:68 and/or SEQ ID NO:69. In some embodiments, a polypeptide that binds RSP02 comprises a polypeptide comprising SEQ ID NO:41 and/or SEQ ID NO:42. In some embodiments, a ptide that binds RSP02 comprises a polypeptide comprising SEQ ID NO:70 and/or SEQ ID NO:71. In some embodiments, a polypeptide that binds RSP02 comprises a polypeptide comprising SEQ ID NO:70 and/or SEQ ID NO:74. In some embodiments, the polypeptide is isolated. In certain embodiments, the polypeptide is substantially pure. In certain embodiments, the ptide is an dy.

In another aspect, the invention provides isolated polynucleotide molecules comprising a polynucleotide that encodes the antibodies and/or ptides of each of the aforementioned aspects, as well as other aspects and/or embodiments described . In some embodiments, the polynucleotide comprises a sequence selected from the -ll— group consisting of SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, and SEQ ID NO:58. In some embodiments, the polynucleotide comprises a sequence selected from the group consisting of SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:39, SEQ ID NO:40, SEQ ID N0260, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:72, and SEQ ID NO:75. The invention further provides sion vectors that se the polynucleotides, as well as cells that comprise the expression vectors and/or the polynucleotides. In some embodiments, the cell is a hybridoma cell line. In certain embodiments, the cell is a hybridoma cell line having the ATCC deposit number PTA- 11970. In certain embodiments, the cell is a hybridoma cell line having the ATCC deposit number PTA-12021.. [0032} In other s, the invention provides methods of inhibiting growth of a tumor, comprising contacting the tumor with an ive amount of a RSPO-binding agent or antibody, ing each of those described herein. {0033} In r aspect, the invention es a method of inhibiting the growth of a tumor in a subject, comprising administering to the subject a therapeutically ive amount of a RSPO-binding agent or antibody, including each of those described herein. {0034} In another aspect, the invention provides a method of inhibiting B-catenin signaling in a cell, sing contacting the cell with an effective amount of a RSPO- binding agent or antibody, including each of those described herein. In some embodiments, the cell is a tumor cell. In some embodiments, the tumor is a colorectal tumor. In some embodiments, the tumor is an n tumor. In some embodiments, the tumor is a pancreatic tumor. In some embodiments, the tumor is a lung tumor. In some embodiments, the tumor expresses elevated levels of at least one RSPO protein. In some embodiments, the tumor expresses elevated levels of RSPOl. In some embodiments, the tumor expresses elevated levels of RSPO2. In some embodiments, the tumor expresses ed levels of RSPO3. In certain embodiments, the RSPO-binding agent inhibits growth of the tumor, for example, by reducing the number and/or frequency of cancer stem cells in the tumor.

In another aspect, the invention provides methods of treating cancer in a subject.

In some embodiments, the method comprises administering to a subject a therapeutically effective amount of any of the RSPO-Einding agents or antibodies described above, as well as those described elsewhere herein. In some embodiments, the cancer is pancreatic cancer. In some embodiments, the cancer is colorectal cancer. In some embodiments, the colorectal cancer comprises an vating mutation in the adenomatous polyposis coli (APC) gene. In some embodiments, the colorectal cancer does not comprise an vating on in the APC gene. In some embodiments, the colorectal cancer comprises a wild-type APC gene. In some embodiments, the cancer is ovarian cancer. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is lung cancer. In some ments, the cancer ses elevated levels of at least one RSPO protein. In some embodiments, the cancer is an ovarian cancer that expresses elevated levels of RSPOl. In some embodiments, the cancer is colon cancer that expresses elevated levels of RSPO2. In some embodiments, the cancer is a pancreatic cancer that expresses elevated levels of RSPOZ. In some embodiments, the cancer is a breast cancer that expresses elevated levels of RSPOZ. In some embodiments, the cancer is a lung cancer that expresses elevated levels of RSPOZ.

In another aspect, the ion provides methods of treating a disease in a subject wherein the disease is associated with activation of B-catenin, and/or aberrant nin signaling comprising administering a therapeutically effective amount of a RSPO—binding agent or antibody, including each of those described herein.

In certain embodiments of each of the entioned aspects, as well as other aspects and/or embodiments described ere herein, the treatment methods comprise stering a RSPO-binding agent in combination with at least one additional therapeutic agent. In some embodiments, the treatment methods se stering a RSPOl-binding agent in combination with a second RSPO-binding agent such as a RSPO2-binding agent, a RSPO3-binding agent, and/or a RSPO4—binding agent. In some embodiments, the treatment methods comprise stering a RSPOZ-binding agent in combination with a second RSPO-binding agent such as a RSPOl-binding agent, a RSPO3-binding agent, and/or a RSPO4-binding agent. In some embodiments, the treatment methods comprise administering a RSPO] -binding agent in combination with a RSPOZ-binding agent. In some embodiments, the treatment methods comprise administering a combination of a RSPOl-binding agent, a RSPOZ-binding agent, and a chemotherapeutic agent. -13, In certain embodiments of each of the aforementioned s, as well as other aspects and/or embodiments described elsewhere herein, the ent methods further comprise a step of determining the level of at least one RSPO n expression in the tumor or cancer.

In another aspect, the invention provides a method of identifying a human subject or selecting a human subject for treatment with a RSPO-binding agent or antibody, including but not limited to, each of those described herein. In some embodiments, the method comprises determining if the subject has a tumor that has an elevated expression level of a specific RSPO (e.g., RSPOl or RSPO2) as compared to the expression of the same RSPO protein in normal tissue. In some embodiments, the method comprises identifying a subject for treatment or selecting a subject for treatment if the tumor has an elevated level of RSPO sion. In some embodiments, the method comprises determining if the subject has a tumor that ses an inactivating mutation in the APC gene. In some ments, the method comprises identifying a subject for ent or selecting a subject for treatment if the tumor comprises an inactivating mutation in the APC gene.

Pharmaceutical compositions comprising a RSPO-binding agent or antibody described herein and a pharmaceutically acceptable carrier are further provided, as are cell lines that produce the RSPO—binding agents. Methods of treating cancer and/or inhibiting tumor growth in a subject (e.g., a human) comprising administering to the subject an ive amount of a composition comprising the RSPO-binding agents are also provided.

Where aspects or ments of the invention are described in terms of a Markush group or other grouping of altematives, the t invention encompasses not only the entire group listed as a whole, but also each member of the group individually and all possible subgroups of the main group, and also the main group absent one or more of the group members. The present invention also envisages the it exclusion of one or more of any of the group members in the claimed invention.

BRIEF DESCRIPTION OF THE FIGURES Figure l. RSPO expression in tumors and normal tissues. Shown is a summary of microarray data from normal, benign, and malignant tissue human samples. Individual tick marks indicate the expression level of RSPO mRNA. A) RSP01 B) RSP02 C) RSPO3 Figure 2. Binding studies of RSPO proteins and LGRS. FACS analysis of HEK- 293 cells expressing LGRS. HEK-293 cells were transiently transfected with a cDNA expression vector encoding FLAG-LGRS-CD4TM-GFP and then subsequently mixed with soluble RSPOl-Fc, RSP02—PC, RSPO3—Fc, or Fc fusion proteins. An anti- FLAG antibody was used as a positive control, and e FZD8-Fc was used as a negative control. Specific binding is indicated by the presence of signal within the dark lined box overlay on each FACS plot. [0044} Figure 3. Identification of inhibitory activity in lung tumor cell-conditioned medium. A 6xTCF-luciferase reporter assay was used to measure B-catenin signaling in HEK—293 cells. 3 cells were exposed to control medium (DMEM media) containing Wnt3a L cell-conditioned medium or medium containing lung tumor cell- ioned medium and Wnt3a L onditioned medium in the presence of soluble LGRS-Fc. e Jag-Fe and antibody LZl were used as negative controls. Soluble FZD8-Fc and an anti-FZD antibody which blocks Wnt3a were used as positive controls. e LGRS-Fc, Jag-Fc and FZD8—Fc fusion proteins were used at l. Anti-FZD antibody and LZl antibody were used at 40ug/ml.

Figure 4. Inhibition of ion of B-catenin signaling. A 6xTCF-luciferase er assay was used to measure B-catenin signaling in HEK-293 cells. HEK-293 cells were exposed to medium ning lOng/ml RSP02 and 25% Wnt3a L cell-conditioned medium (“RSP02”) or medium containing 25% lung tumor cell-conditioned medium and % Wnt3a L cell-conditioned medium (“LT”) in the presence of soluble LGRS-Fc at 4- fold dilutions from l to 0.02ug/ml; RSP02 with LGRS-Fc () and LT with LGRS-Fc (1-). Soluble Jag-Fc was used as a negative control at 20ug/ml with RSP02 (- A-) or with LT (-A-). Soluble FZD8—Fc which blocks Wnt3a was used as a positive control at 20ug/ml with RSP02 (—O—) or with LT (41-). [0046} Figure 5. Identification of antibodies that bind RSPOl. A) A diagram of the fiJsion protein FLAG-RSPOlfurin—CD4TM-GFP. B) FACS analyses of dies generated to human RSPOl. Relative antibody g is sleown on the y-axis and expression of the FLAG-RSPOlfurin-CD4TM-GFP fusion protein is indicated on the x- axis. Positive binding is indicated by the presence of signal within the dark lined'box overlay on each FACS plot. An anti-FLAG antibody was used as a positive control. An anti-PE antibody was used as a negative control.

Figure 6. Identification of SPOI antibodies that inhibit B-catenin signaling induced by RSPOl. A TOPflash luciferase reporter assay was used to measure B-catenin signaling in HEK—293 cells after exposure to a combination of Wnt3a l) and RSPOl (lOng/ml) and in the presence of increasing concentrations of anti—RSPOl antibodies (89M2, 89M4, 89M5, 89M7, 89M19 or 89M25) or irrelevant control antibodies (254M14 or 254M26). Antibodies were used as 2-fold dilutions from lOug/ml to 0.625 ug/ml. Controls included exposure to control medium (no Wnt3a and no RSPO), Wnt3a alone, or a ation of Wnt3a and RSPO in the absence of antibody.

Figure 7. Identification of SPOl antibodies that block RSPOl/LGRS binding. FACS analysis of HEK-293 cells expressing LGRS. HEK-293 cells were transiently transfected with a cDNA expression vector ng FLAG-LGRS-CD4TM- GFP and then subsequently mixed with soluble RSPOl-Fc fusion protein in ation with individual anti-RSPOl antibodies. Binding was detected wéth a PE-conjugated anti- human Fc secondary antibody. Relative RSPOl-Fc binding is shown on the y-axis and expression of the FLAG-LGRS-CD4TM—GFP fusion protein is indicated on the x—axis.

Positive binding is indicated by the ce of signal within the dark lined box overlay on each FACS plot. An anti-PE antibody was used as a negative control.

Figure 8. Inhibition of tumor growth With anti-RSPOl antibodies. OV19 ovarian tumor cells were ed subcutaneously into NOD/SCID mice. Mice were treated with 89M5 (), 89M25 (), taxol (), a combination of antibody 89M5 and taxol (), a combination of antibody 89M25 and taxol , or control antibody 137.11 (-I-). Data is shown as tumor volume (mm3) over days post—treatment.

Figure 9. Epitope mapping of anti-RSPOl antibody. A) A diagram of fusion proteins constructed that contain a deletion series of RSPOl s. These constructs all comprise a CD4TM domain which allows for cell surface expression of the ns.

B) FACS analyses of anti-RSPOI dy binding to cells transfected with the fusion proteins. Relative antibody binding is shown on the y—axis and expression of the é'usion protein is indicated on the x-axis. An anti-FLAG antibody was used as a positive control.

An anti-PE antibody was used as a negative control.

Figure 10. Identification of antibodies that bind RSP02. FACS analyses of antibodies generated to human RSP02. Relative antibody binding is shown on the y-axis and sion of the FLAG-RSP02furin-CD4TM-GFP fusion protein is indicated on the x-axis. An anti—FLAG dy was used as a positive control. An anti-PE antibody was used as a negative control.

Figure 11. Identification of anti-RSP02 antibodies that inhibit induction of B— catenin signaling by RSP02. A TOPflash luciferase er assay was used to measure B-catenin signaling in HEK-293 cells after exposure to a combination of Wnt3a (Sng/ml) and human RSP02 (lOng/ml) or Wnt3a l) and human RSPO3 (10ng/ml) and in the presence of antibodies to RSP02 (mAbs 130M23, 130M24, 130M25, 130M26, 130M27, and 130M28). Controls included exposure to control medium (no added Wnt3a and no RSPO - d “cells”), Wnt3a alone (labeled , or a combination of Wnt3a and RSPO in the absence of antibody.

[OESS] Figure 12. fication of anti—RSP02 antibodies that block RSP02/LGR5 binding. FACS is of HEK-293 cells expressing LGRS. 3 cells were transiently transfected with a cDNA expression vector encoding FLAG-LGRS-CD4TM- GFP and then subsequently mixed with soluble RSP02-fc fusion protein in combination with individual anti-RSP02 antibodies. Binding was detected with a PE-conjugated anti— human Fc secondary antibody. Relative RSP02-Fe binding is shown on the y-axis and expression of the GRS-CD4TM-GFP fusion protein is indicated on the x-axis.

Positive binding is indicated by the presence of signal within the dark-lined box overlay on each FACS plot. An anti-FLAG antibody was used as a positive control and an anti- PE antibody was used as a negative control.

Figure 13. Identification of inhibitory activity in tumor cell-conditioned medium.

STF-293 cells were exposed to control medium (DMBM media), medium containing Wnt3a L cell-conditioned medium, medium containing tumor cell-conditioned medium> or medium containing tumor cell—conditioned medium and Wnt3a L cell-conditioned medium in the presence of soluble LGRS-Fc, FZD8-Fc, or a control fusion Fc n.

Soluble LGRS-Fc, FZD8-Fc, and control-Fe fusion ns were used at 10ug/ml.

Tumor cell-conditioned medium was prepared from lung tumor LU2 (Fig. 13A), lung tumor LU25 (Fig. 13B), and ovarian tumor OV38 (Fig. 13C).

Figure 14. Inhibition of induction of B-catenin signaling. STF-293 cells were incubated with LU2 cells plus 25% lung tumor cell-conditioned medium plus 25% Wnt3a-L cell-conditioned . Antibody 130M23 (-i-) and soluble LGRS-Fc (-I-) were added to the cells in 5—fold serially dilutions from 50ug/m1 to ug/ml. An irrelevant monoclonal antibody (-EI-), similarly diluted, and a control Fc fusion protein (- A-, 50ug/ml) were used as ve controls.

Figure 15. Inhibition of tumor growth with anti-RSPO antibodies. PN3l pancreatic tumor cells were injected subcutaneously into NOD/SCID mice. Mice were treated with anti-RSPOl antibody 89M5 (-U-), anti—RSPOZ antibody 1.30M23 (-i-), gemcitabine (-I-), a combination of antibody 89M5 and gemcitabine (-Y-), a combination of antibody 130M23 and gemcitabine (), or control dy 1B7.11 (-O- ). Data is shown as tumor volume (mm3) over days post-implantation.

Figure 16. tion of tumor growth with SPO antibodies. PN7 pancreatic tumor cells were injected subcutaneously into NOD/SCID mice. Mice were treated with anti-RSPO2 antibody 130M23 (-V-), anti-FZD antibody 18R5 (-A—), gemcitabine (4-), a combination of 130M23 and 18R5 (), a combination of 130M23 and gemcitabine (43-), a combination of 18R5 and gemcitabine (-A—), a combination of 130M23, 18R5, and gemcitabine (), or control antibody 1B7.ll (). Data is shown as tumor volume (mm3) over days post-treatment (Fig. 16A). Mice were treated with a combination of a Wnt pathway inhibitor FZD8-Fc and gemcitabine (-A-), a combination with 130M23 and gemcitabine (), combination of 130M23, FZD8-Fc, and gemcitabine (), gemcitabine (4-), or control dy 1B7. 11 (-i-). Data is shown as tumor volume (mm3) over days post—treatment (Fig. 16B). The resulting tumors were processed to single cell sions, and ly transplanted into mice. 90 cells from tumors obtained from each treatment group were injected subcutaneously into NOD/SCID mice. Tumors were allowed to grow with no treatment. Data is shown as tumor volume (mm3) on day 40 (Fig. 16C).

Figure 17. FACS analysis of humanized RSPO antibodies. A) FACS analyses of humanized 89M5 antibody (h89M5-H2L2) and parental 89M5 antibody. Five-fold serial dilutions of each antibody were tested. Relative antibody binding is shown on the y-axis and expression of the SPOIfurin-CD4TM-GFP fusion protein is indicated on the x-axis. B) FACS analyses of zed 130M23 antibody (h13OM23-H1L2) and parental 130M23 antibody. Five-fold serial dilutions of each antibody were tested.

Relative antibody binding is shown on the y-axis and expression of the FLAG- RSPOqurin-CD4TM—GFP fusion protein is indicated on the x-axis.

Figure 18. Inhibition of tumor growth with anti-RSPOl and anti-RSP02 antibodies. B39 triple ve breast cancer tumor cells were injected subcutaneously into NOD/SCID mice. Mice were treated with a combination of anti-RSPOl antibody 89M5 and anti-RSP02 antibody 130M23 (), cisplatin (-V—), a combination of 89M5, 130M23 and cisplatin (), or control antibody lB7.11 (-l-). Data is shown as tumor volume (mm3) over days post-treatment.

DETAILED DESCRIPTION OF THE INVENTION The present invention es novel agents, ing, but not limited to polypeptides such as antibodies, that bind RSPO proteins (e.g., human RSPOl, RSPOZ, and/or RSPO3). The RSPO-binding agents include antagonists of B-catenin signaling.

Related polypeptides and polynucleotides, compositions comprising the RSPO-binding agents, and methods of making the inding agents are also provided. s of using the novel RSPO-binding agents, such as methods of inhibiting tumor growth, methods of ng cancer, methods of reducing the frequency of cancer stem cells in a tumor, methods of inhibiting B-catenin signaling, and/or s of identifying and/or selecting subjects for treatment, are further provided.

Monoclonal dies that specifically bind human RSPOl have been identified - monoclonal antibodies 89M2, 89M4, 89M5, 89M7, 89Ml9 and 89M25 (Example 5, Fig.

). Anti-RSPOI antibodies 89M2, 89M4, 89M5, and 89M25 inhibit B-catenin signaling (Example 6, Fig. 6). SPOI antibodies 89M2, 89M4, 89M5, and 89M25 block soluble RSPOl binding to LGRS (Example 7, Fig. 7). Sequence data subsequently demonstrated that dies 89M2, 89M4, 89M5, and 89M25 contain the same heavy chain and light chain variable regions, and it was concluded that these dies would comprise the same antigen-binding site. Anti-RSPOI antibodies 89M4, 89M5, 89M7 and 89M25 have binding affinities for both human and mouse RSPOl of less than 0.1nM (Example 8). A humanized version of 89M5 was produced, h89M5-H2L2 (Example 19) and has a binding affinity for human RSPOI of less than 0.1nM (Example 20). Anti- RSPOl antibodies 89M5 and 89M25 have been found to inhibit tumor cell growth in vivo in an ovarian tumor aft model as single agents and in combination with a chemotherapeutic agent (Example 9, Fig. 8). Anti-RSPOI antibody 89M5 has been shown to inhibit tumor cell growth in vivo in a pancreatic tumor aft model in combination with a chemotherapeutic agent (Example 17, Fig. 15). Preliminary epitope mapping studies suggest that amino acids within the furin2 domain of RSPOI are involved in the binding site for anti-RSPOI antibody 89M5 (Example 10, Fig. 9).

In addition, monoclonal antibodies that specifically bind human RSP02 have been identified monoclonal antibodies 130M23, , 130M25, , 130M27, and 130M28 (Example 11, Fig. 1.0). Anti—RSP02 antibodies 130M23, 130M24, 130M25, 130M26, 130M27, and 130M28 were shown to reduce or completely block B-catenin signaling (Example 12, Fig. 11). Anti—RSP02 antibodies 130M23 and 130M24 block soluble RSP02 binding to LGRS (Example 13, Fig. 12). Anti-RSP02 dy 130M23 has a binding affinity for human RSP02 of 0.14nM and mouse RSP02 of 0.35nM (Example 15). Humanized versions of 130M23 were produced, h130M23-H1L2 and 3-H1L6 (Example 19). Anti-RSP02 antibody h130M23-H1L2 has a binding affinity for human RSP02 of 0.13nM and hl30M23-H1L6 has a binding affinity for human RSP02 of 0.15nM le 20). Anti-RSP02 antibody 130M23 has been shown to t tumor cell growth in vivo in a pancreatic tumor aft model as a single agent and in combination with additional therapeutic agents (Examples 17 and 18, Figs. and 16). 1. Definitions To facilitate an understanding of the present invention, a number of terms and phrases are defined below.

The terms “antagonist” and “antagonistic” as used herein refer to any le that partially or fully blocks, inhibits, reduces or neutralizes a biological ty of a target and/or signaling pathway (e.g., the B—catenin signaling). The term “antagonist” is used herein to include any molecule that lly or fully blocks, inhibits, reduces or neutralizes the activity of a protein (e.g., a RSPO protein). Suitable antagonist molecules specifically include, but are not limited to, antagonist antibodies or antibody fragments.

The terms “modulation” and “modulate” as used herein refer to a change or an alteration in a biological activity. Modulation includes, but is not limited to, stimulating or inhibiting an activity. Modulation may be an increase or a se in activity (e. g., a decrease in RSPO signaling; a decrease in B-catenin signaling), a change in g characteristics, or any other change in the biological, functional, or immunological properties associated with the activity of a protein, pathway, or other biological point of interest.

The term “antibody” as used herein refers to an immunoglobulin molecule that recognizes and specifically binds a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combinations of the foregoing, through at least one n recognition site within the variable region of the immunoglobulin le. As used herein, the term asses intact polyclonal antibodies, intact monoclonal antibodies, antibody feagments (such as Fab, Fab', F(ab')2, and Fv nts), single chain Fv (scFv) dies, multispecific antibodies such as bispecific dies generated from at least two intact dies, monospecific antibodies, monovalent dies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising an antigen determination portion of an antibody, and any other modified immunoglobulin molecule comprising an antigen recognition site as long as the antibodies exhibit the desired biological activity. An antibody can be any of the five major s of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or sses (isotypes) thereof (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2), based on the identity of their heavy-chain constant domains referred to as alpha, delta, epsilon, gamma, and mu, respectively. The different classes of immunoglobulins have different and well-known subunit structures and three-dimensional configurations. Antibodies can be naked or conjugated to other molecules, including but not limited to, toxins and radioisotopes.

The term “antibody fragment” refers to a portion of an intact antibody and refers to the antigenic determining variable regions of an intact antibody. Examples of dy fragments e, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, single chain antibodies, and multispecific dies formed from antibody fragments. “Antibody fragment” as used herein comprises an antigen-binding site or e binding site.

The term “variable ” of an antibody refers to the variable region of the antibody light chain, or the variable region of the antibody heavy chain, either alone or in combination. The variable regions of the heavy and light chain each consist of four framework regions (FR) connected by three complementarity determining regions (CDRs), also known as “hypervariable regions”. The CDRs in each chain are held together in close proximity by the framework regions and, with the CDRs from the other chain, bute to the formation of the antigen-binding sites of the antibody. There are at least two techniques for determining CDRs: (1) an approach based on cross-species sequence ility (i.e., Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Edition, National Institutes of Health, Bethesda MD.), and (2) an approach leased on crystallographic studies of antigen-antibody complexes (Al-Lazikani et al., 1997, J. Mol. Biol, 273:927-948). In on, combinations of these two approaches are sometimes used in the art to determine CDRs.

The term “monoclonal antibody” as used herein refers to a homogenous dy population involved in the highly specific recognition and binding of a single antigenic determinant or epitope. This is in contrast to polyclonal antibodies that typically include a mixture of different antibodies directed t different antigenic determinants. The term lonal antibody” encompasses both intact and full—length onal antibodies as well as antibody fragments (e.g., Fab, Fab’, F(ab')2, Fv), single chain (scFV) antibodies, fusion proteins comprising an antibody portion, and any other modified immunoglobulin molecule comprising an antigen recognition site (antigen-binding site).

Furthermore, “monoclonal antibody” refers to such antibodies made by any number of techniques, including but not limited to, hybridoma production, phage ion, recombinant expression, and transgenic animals.

The term “humanized antibody” as used herein refers to forms of non-human (e. g., murine) antibodies that are specific immunoglobulin chains, chimeric immunoglobulins, or nts thereof that contain minimal non-human sequences.

- Typically, humanized antibodies are human immunoglobulins in which residues of the CDRs are replaced by es from the CDRs of a non-human species (e.g., mouse, rat, rabbit, or hamster) that have the desired specificity, y, and/or binding capability (Jones et al., 1986, Nature, 321:522-525; Riechmann et al., 1988, Nature, 332:323—327; Verhoeyen et al., 1988, Science, 239:1534-1536). In some instances, the Fv framework region residues of a human immunoglobulin are replaced with the corresponding residues in an antibody from a non-human species that has the desired specificity, affinity, and/or binding capability. The humanized antibody can be further modified by the substitution of onal residues either in the Fv framework region and/or wéthin the replaced non- human residues to refine and optimize antibody specificity, affinity, and/or binding capability. In general, the humanized antibody will comprise substantially all of at least one, and typically two or three, variable domains containing all or substantially all of the CDRs that correspond to the non-human immunoglobulin whereas all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence.

The humanized antibody can also comprise at least a portion of an immunoglobulin nt region or domain (Fc), lly that of a human immunoglobulin. Examples of methods used to te humanized antibodies are described in, for example, US. Pat. ,225,539. {0071} The term “human antibody” as used herein refers to an dy produced by a human or an antibody having an amino acid sequence corresponding to an antibody produced by a human made using any of the techniques known in the art. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.

The term “chimeric dy” as used herein refers to an antibody wherein the amino acid sequence of the globulin molecule is derived from two or more species. Typically, the variable region of both light and heavy chains corresponds to the variable region of antibodies d from one s of s (e.g., mouse, rat, , etc.) with the desired specificity, affinity, and/or binding capability, while the constant regions are homologous to the sequences in antihedies derived from another species (usually human) to avoid eliciting an immune respense in that species.

The phrase “affinity matured antibody” as used herein refers to an antibody with one or more alterations in one or more CDRs thereof that result in an ement in the affinity of the antibody for antigen, compared to a parent antibody that does not s those alterations(s). Preferred affinity matured antibodies will have nanomolar or even picomolar ies for the target antigen. Affinity matured antibodies are ed by procedures known in the art. For example, Marks et al., 1992, Bio/Technology 10:779- 783, describes affinity maturation by VH and VL domain shuffling. Random mutagenesis of CDR and/or framework residues is described by Barbas et al., 1994, PNAS, 91:3809-3813; Schier et al., 1995, Gene, 1691147-155; Yelton et al., 1995, J Immunol. 155:1994-2004; n et al., 1995, J. Immunol., 154:3310-9; and Hawkins et al., 1992, J. M01. Biol., 226:889-896.

The terms “epitope” and “antigenic determinant” are used interchangeably herein and refer to that portion of an antigen capable of being recognized and specifically bound by a particular antibody. When the antigen is a polypeptide, es can be formed both from contiguous amino acids and noncontiguous amino acids osed by tertiary folding of a protein. Epitopes formed from contiguous amino acids (also referred to as linear epitopes) are typically retained upon protein denaturing, whereas epitopes formed by tertiary folding (also referred to as conformational epitopes) are typically lost upon protein denaturing. An epitope lly includes at least 3, and more usually, at least 5 or 8—10 amino acids in a unique spatial conformation.

The terms “selectively binds” or “specifically binds” mean that a binding agent or an antibody reacts or ates more frequently, more rapidly, with greater duration, with greater affinity, or with some combination of the above to the e, protein or target molecule than with alternative substances, including unrelated proteins. In certain embodiments “specifically binds” means, for instance, that an antibody binds a n with a KB of about 0.1mM or less, but more usually less than about luM. In certain embodiments, “specifically binds” means that an antibody binds a target at times with a KB of at least about 0.1uM or less, at other times at least about 0.01uM or less, and at other times at least about lnM or less, Because of the sequence identity between homologous proteins in different species, specific binding can include an antibody that recognizes a protein in more than one species (e.g., human RSPOl and mouse RSPOl).

Likewise, because of homology within certain regions of polypeptide sequences of different proteins, specific binding can include an antibody (or other polypeptide or binding agent) that recognizes more than one protein (e.g., human RSFOl and human RSPOZ). It is understood that, in certain embodiments, an antibody or g moiety that specifically binds a first target may or may not specifically bind a second target. As such, “specific binding” does not necessarily require (although it can include) exclusive binding, i.e. binding to a single target. Thus, an antibody may, in certain ments, specifically bind more than one target. In certain ments, multiple targets may be bound by the same antigen—binding site on the antibody. For example, an antibody may, in certain instances, comprise two identical antigen—binding sites, each of which specifically binds the same epitope on two or more proteins (e.g., RSPOl and RSPO2).

In certain alternative embodiments, an antibody may be bispecific or multispecific and comprise at least two n-binding sites with differing specificities. By way of non- limiting example, a bispecific antibody may comprise one antigen—binding site that recognizes an epitope on one protein (e.g., human RSPOl) and r comprise a second, different antigen-binding site that recognizes a different e on a second protein.

Generally, but not necessarily, reference to binding means specific binding.

WE’RE} The terms eptide” and “peptide” and “protein” are used hangeably herein and refer to rs of amino acids of any length. The polymer may be linear or ed, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond ion, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides ning one or more s of an amino acid (including, for example, unnatural amino acids), as well as other modifications known in the art. It is tood that, because the polypeptides of this invention may be based upon antibodies, in certain embodiments, the polypeptides can occur as single chains or associated chains. {@377} The terms “polynucleotide” and “nucleic acid” are used interchangeably herein and refer to polymers of tides of any length, and include DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase.

“Conditions of high stringency” may be identified by those that: (1) employ low ionic strength and high temperature for washing, for example 15mM sodium chloride/15mM sodium citrate/0.1% sodium dodecyl sulfate at 50°C; (2) employ during ization a denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0.1% bovine serum n/0.1% Ficoll/0.1% polyvinylpyrrolidone/SOmM sodium phosphate buffer at pH 6.5 with 750mM sodium chloride, 75mM sodium citrate at 42°C; or (3) employ 50% formamide, 5x SSC (0.75M NaCl, 75mM sodium citrate), 50mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5x Denhardt's solution, sonicated salmon sperm DNA (50ug/ml), 0.1% SDS, and 10% dextran sulfate at 42°C, with washes at 42°C in 0.2x SSC and 50% formamide at 55°C, followed by a high- stringency wash consisting of 0.1x SSC containing EDTA at 55°C.

The terms “identical” or t “identity” in the context of two or more nucleic acids or polypeptides, refer to two or more sequences or subsequences that are the same or have a specified percentage of tides or amino acid residues that are the same, when compared and aligned (introducing gaps, if ary) for maximum correspondence, not considering any conservative amino acid substitutions as part of the sequence identity. The percent identity may be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that may be used to obtain alignments of amino acid or nucleotide sequences are well-known in the art. These e, but are not limited to, BLAST, ALIGN, gn, BestFit, GCG Wisconsin Package, and variants thereof. In some embodiments, two nucleic acids or polypeptides of the invention are substantially identical, g they have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and in some embodiments at least 95%, 96%, 97%, 98%, 99% nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using a ce comparison thm or by visual inspection. In some embodiments, identity exists over a region of the sequences that is at least about 10, at least about 20, at least about 40-60 residues, at least about 60-80 residues in length or any al value therebetween. In some embodiments, identity exists over a longer region than 60—80 residues, such as at least —26- about 80-100 es, and in some embodiments the sequences are substantially cal over the full length of the sequences being compared, such as the coding region of a nucleotide sequence.

A “conservative amino acid substitution” is one in which one amino acid residue is replaced with another amino acid residue having a r side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, ine), 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., tyrosiEe, alanine, tryptophan, histidine). For e, substitution of a alanine for a tyrosine is a conservative substitution. Preferably, conservative substitutions in the sequences of the polypeptides and antibodies of the ion do not abrogate the binding of the polypeptide or antibody ning the amino acid sequence, to the antigen(s), i.e., the one or more RSPO protein(s) to which the polypeptide or antibody binds. Methods of identifying tide and amino acid vative substitutions which do not eliminate antigen binding are nown in the art.

The term “vector” as used herein means a construct, which is capable of delivering, and usually expressing, one or more gene(s) or sequence(s) of interest in a host cell. Examples of vectors e, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid, or phage vectors, DNA or RNA sion vectors associated with cationic condensing agents, and DNA or RNA expression vectors encapsulated in liposomes.

A polypeptide, antibody, polynucleotide, vector, cell, or composition which is “isolated” is a polypeptide, antibody, polynucleotide, vector, cell, or composition which is in a form not found in nature. Isolated polypeptides, antibodies, polynucleotides, vectors, cells or compositions include those which have been purified to a degree that they are no longer in a form in which they are found in nature. In some embodiments, a polypeptide, antibody, polynucleotide, vector, cell, or composition which is isolated is substantially pure.

The term “substantially pure” as used herein refers to material which is at least 50% pure (i.e., free from contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure. {0084] The terms “cancer” and “cancerous” as used herein refer to or be the physiological condition in mammals in which a population of cells are terized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, blastoma, sarcoma, and hematologic s such as lymphoma and leukemia.

The terms “tumor” and “neoplasm” as used herein refer to any mass of tissue that results from excessive cell growth or proliferation, either benign ncerous) or malignant (cancerous) including pre-cancerous lesions. {0086} The term tasis” as used herein refers to the process by which a cancer spreads or transfers from the site of origin to other regions of the body with the development of a similar cancerous lesion at the new location. A tatic” or “metastasizing” cell is one that loses adhesive contacts with neighboring cells and migrates via the bloodstream or lymph from the primary site of disease to invade neighboréng body structures. {0087} The terms “cancer stem cell” and “CSC” and “tumor stem cell” and “tumor initiating cell” are used interchangeably herein and refer to cells from a cancer or tumor that: (1) have extensive proliferative capacity; 2) are capable of asymmetric cell division to generate one or more types of differentiated cell progeny wherein the differentiated cells have reduced erative or developmental potential; and (3) are capable of symmetric cell divisions for self-renewal or self-maintenance. These properties confer on the cancer stem cells the ability to form or establish a tumor or cancer upon serial lantation into an immunocompromised host (e.g., a mouse) compared to the majority of tumor cells that fail to form tumors. Cancer stem cells undergo self-renewal versus differentiation in a chaotic manner to form tumors with abnormal cell types that can change over time as mutations occur. {008$} The terms “cancer cell” and “tumor cell” refer to the total population of cells derived from a cancer or tumor e-cancerous lesion, including both non-tumorigenic cells, which se the bulk of the cancer cell populationr and tumorigenic stem cells r stem cells). As used herein, the terms “cancer cell” or “tumor cell” will be modified by the term “non-tumorigenic” when referring solely to those cells lacking the capacity to renew and differentiate to distinguish those tumor cells from cancer stem cells.

The term “tumorégenic” as used herein refers to the functional features of a cancer stem cell including the properties of self-renewal g rise to additional tumorigenic cancer stem cells) and proliferation to generate all other tumor cells (giving rise to differentiated and thus non-tumorigenic tumor cells). {0090] The term “tumorigenicity” as used herein refers to the ability of a random sample of cells from the tumor to form palpable tumors upon serial transplantation into immunocompromised hosts (e.g., mice).

The term “subject” refers to any animal (e.g., a mammal), including, but not limited to, humans, non—human primates, canines, felines, rodents, and the like, which is to be the recipient of a particular treatment. Typically, the terms “subject” and “patient” are used interchangeably herein in reference to a human subject.

The term “pharmaceutically acceptable” refers to approved or approvable by a regulatory agency of the Federal or a state government or listed in the US. Pharmacopeia or other generally recognized pharmacopeia for use in s, including humans.

The terms aceutically acceptable excipient, carrier or adjuvant” or “acceptable pharmaceutical r” refer to an ent, carrier or adjuvant that can be administered to a subject, together with at least one binding agent (e.g., an antibody) of the present disclosure, and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses ent to deliver a therapeutic effect.

The terms “effective amount” or peutically effective amount” or “therapeutic effect” refer to an amount of a binding agent, an antibody, polypeptide, polyaucleotide, small organic molecule, or other drug effective to ” a disease or er in a subject or mammal. In the case of cancer, the therapeutically effective amount of a drug (e.g., an antibody) has a therapeutic effect and as such can reduce the number of cancer cells; se tumorigenicity, tumorigenic ncy or tumorigenic capacity; reduce the number or frequency of cancer stem cells; reduce the tumor size; reduce the cancer cell tion; inhibit or stop cancer cell ation into peripheral organs including, for example, the spread of cancer into soft tissue and bone; inhibit and stop tumor or cancer cell metastasis; inhibit and stop tumor or cancer cell growth; relieve to some extent one or more of the ms associated with the cancer; reduce morbidity and mortality; improve quality of life; or a combination of such effects. To the extent the agent, for example an antibody, prevents growth and/or kills existing cancer cells, it can be referred to as cytostatic and/or cytotoxic.

The terms “treating” or “treatment” or “to treat” or “alleviating” or “to alleviate” refer to both 1) therapeutic measures that cure, slow down, lessen symptoms of, and/or halt progression of a diagnosed pathologic condition or disorder and 2) prophylactic or preventative es that prevent or slow the development of a targeted pathologic condition or disorder. Thus those in need of ent include those already with the disorder; those prone to have the disorder; and those in. whom the disorder is to be prevented. In some embodiments, a subject is successfully “treated” according to the methods of the t inVention if the patient shews one or more of the following: a ion in the number of or complete absence of cancer cells; a reduction in the tumor size; inhibition of or an absence of cancer cell infiltration into perépheral organs including the spread of cancer cells into soft tissue and bone; tion of or an absence of tumor or cancer cell metastasis; inhibition or an absence of cancer growth; relief of one or more symptoms associated with the specific cancer; reduced morbidity and mortality; improvement in quality of life; reduction in tumorigenicity; reduction in the number or frequency of cancer stem cells; or some combination of effects.

As used in the t disclosure and claims, the singular forms 6‘ 39 65 a an” and “the” include plural forms unless the t clearly dictates otherwise.

It is understood that wherever embodiments are described herein with the language “comprising” otherwise ous embodiments described in terms of “consisting of’ and/or “consisting essentially of” are also provided.

The term “and/or” as used in a phrase such as “A and/or B” herein is ed to include both A and B; A or B; A (alone); and B (alone). Likewise, the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to encompass each of the followéng ments: 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 (I; A (atone); )8 {alone}; and {3 (atone).

II. inding agents $8th The present invention provides agents that hind human RSPQ proteins. These agents are referred to herein as “RSPQ—hinding agents”, in some embodiments, the RSPO—binding agents are antibodies. In some emhodiments, the RSPO-hinding agents are poiypeptides. in. certain ments, the RSihiiuhinding agents hind RSPOI. in certain embodiments, the inding agents hind RSPG’Z. In certain embodiments, the RS?O~hinding agents bind RS390? to certain embodiments, the RSPQegents speeifieaiiy hind at tens: ene other human RSPO. in some embodiments, the at least ene other human RSPO hound by e REPOEvhinding agent is seieeted from the group; eonsisting of RSFGZ, RSPOS‘I, and RSPO4. in some ments, the at least one other human RSPQ bound by a KSPOZ—hinding agent is seieeted from the group consisting of RSPOL RSPOE, and RSP04. in some embodiments, the at. least ene other human RSPQ bound by a RSPGfidiindihg agent is selected from the group consisting of RSPOE, RSE’OZ, and RSPQ4. The thit—Eength amino acid (as) sequences for human RSPGL R3992, RS 993, and RSPG‘ét are known in the art and. are provided herein as SEQ ID Nth} (IRSFOE), 5:2th 11) N02 (RSPQ2), SEQ ID N03 (RSI-‘03), and SEQ 1D N024 (RSF04).

In. certain ernhodimentsf, the antigen-binding site of a. RSPO~binrting agent (e.g., antibody) described herein is eapabie of g {or binds) one, two, three, or tour RSPOS, In certain embodiments, the antigen—binding site of a RSPOE-binding agent (eg, antibody) described herein is capable of binding {or hinds) RSPDE as weii as one, two, or three other RSi‘Os, For example, in certain embodiments, the antigen-binding site of a. RSZP()t~hindi.ng agent is eepahie of specitieetiy binding RSE’QE as wet} as at least one other ESE-’0 seieeted from the group consisting of RSPOZ, RSPQS, and RSPO4, in n embodiments, the RSPOE ng agent speeitieahy hihds R3130} and RSPOZ. in certain embodiments, the R.SPO1~hinding agent speeificaiiy binds RSPOE and RSE’03. in n emhodiments, the RSPOi«hintiint.g agent speeifieeily binds RSPO’E and RSPOd. in certain embodiments, the RSPQEvhinding agent speeifieeily binds RSPOI, RSPQZZ, and RSI-363. In next-air; embodiments, the RSPOluhinding agent speeifieaiiy binds RSPOE, R3902, and RSFOIE. in eettain embodiments, the RSPOEuhinding agent specifically binds RSPOl, RSPO3, and RSPO4. In some embodiments, the RSPUI- binding agent cally binds human RSPOl. In some embodiments, the RSPOl- binding agent (e.g., antibody) specifically binds both human RSPOI and mouse RSPOl.

In certain embodiments, the agent-binding agent is an antibody that cally binds within amino acids 21—263 of human RSPOl. In certain embodiments, the agent- binding agent is an antibody that specifically binds within amino acids 31-263 of human RSPOl. In certain embodiments, the antigen-binding agent is an antibody that specifically binds within amino acids 34-135 of human RSPOl. In n embodiments, the n-binding agent is an antibody that specifically binds within amino acids 91-135 of human RSPOl. In certain embodiments, the RSPOl-binding agent binds within SEQ ID NO:5. In some embodiments, the RSPOl-binding agent binds within SEQ ID NO:9.

In certain embodiments, the RSPOl-binding agent or antibody binds a furin-like cysteine- rich domain of RSPOl. In some embodiments, the agent or antibody binds at least one amino acid within a furin-like cysteine-rich domain of KSPOl. In certain embodiments, the RSPOl-binding agent or antibody binds within sequence SEQ ID NO:6 or SEQ ID NO:7. In certain ments, the RSPOl-binding agent or antibody binds within ce SEQ ID NO:6 and SEQ ID NO:7. In some embodiments, the RSPOl-binding agent binds the thrombospondin domain of RSPOl. In some embodiments, the RSPOl- binding agent or antibody binds at least one amino acid within the ospondin domain of RSFOI. In some embodiments, the RSPOl-binding agent or antibody binds within SEQ ID N028. [0103} In certain embodiments, the antigen-binding site of a RSP02-binding agent (e.g., antibody) described herein is capable of binding (or binds) RSP02 as well as one, two, or three other RSPOs. For example, in certain embodiments, the antigen-binding site of a binding agent is capable of specifically binding RSP02 as well as at least one other RSPO selected from the group consisting of RSPOl, RSPO3, and RSPO4. In n embodiments, the RSt1‘02-binding agent specifically binds RSP02 and RSPOl.

In certain embodiments, the RSP02-binding agent specifically binds RSP02 and RSPO3.

In certain embodiments, the RSP02—binding agent specifically binds RSP02 and RSPO4.

In certain embodiments, the RSP02~binding agent speeiiicaily binds LRSFOZ, RSPO3, and RSPO4. in n embcdimeets, the RSPQQ~binding agent specifically binds RSI-“’02, RSPQI, and RSPO3. in certain embodiments, the REESE—binding agent specifically binds RSP02, RSPOl, and RSPO4. In some embodiments, the RSP02- binding agent specifically binds human RSPOZ. In some embodiments, the RSP02- g agent (e.g., dy) specifically binds both human RSP02 and mouse RSP02.

In n embodiments, the agent-binding agent is an antibody that specifically binds within amino acids 22-243 of human RSP02. In certain embodiments, the agent- binding agent is an antibody that specifically binds within amino acids 22-205 of human RSP02. In certain embodiments, the n-binding agent is an antibody that specifically binds within amino acids 31-146 of human RSP02. In certain embodiments, the antigen-binding agent is an antibody that specifically binds within amino acids 31-89 of human RSP02. In certain embodiments, the antigen-binding agent is an antibody that specifically binds within amino acids 90-134 of human RSP02. In certain embodiments, the n-binding agent is an antibody that specifically binds Within amino acids 90-146 of human RSP02. In certain embodiments, the RSP02-binding agent binds within SEQ ID NO:43~. In some embodiments, the RSP02-binding agent binds within SEQ ID NO:44. In certain embodiments, the RSP02-binding agent or antibody binds a furin-like cysteine-réch domain of RSP02. In some embodiments, the agent or dy binds at least one amino acid within a furin-like cysteine-rich domain of RSP02. In certain embodiments, the RSP02-binding agent or antibody binds within sequence SEQ ID NO:45 or SEQ ID NO:46. In certain embodiments, the RSP02-binding agent or antibody binds within sequence SEQ ID NO:45 and SEQ ID NO:46. In some embodiments, the RSP02-binding agent binds the thrombospondin domain of RSP02. In some embodiments, the RSP02-binding agent or antibody binds at least one amino acid within the thrombospondin domain of RSP02. In some embodiments, the RSP02-binding agent or antibody binds within SEQ ID NO:47.

In certain embodiments, the antigen-binding site of a RSPO3-binding agent (e.g., antibody) described herein is capable of binding (or binds) RSPO3 as well as one, two, or three other RSPOs. For e, in n embodiments, the antigen-binding site of a RSPO3-binding agent is capable of cally binding RSPO3 as well as at least one other RSPO selected from the group consisting of RSPOl, RSP02, and RSPO4. In n embodiments, the RSPO3—binding agent specifically binds RSPO3 and RSPOI.

In certain embodiments, the RSPO3-binding agent specifically binds RSPO3 and RSP02.

In certain embodiments, the RSPO3-binding agent specifically binds RSPO3 and RSPO4.

In n embodiments, the RSPO3-binding agent specifically binds RSPO3, RSPOI, and RSPO2. In ceItain ments, the RSPO3-binding agent specifically binds RSPO3, RSPOl, and RSPO4. In certain embodiments, the RSPO3-binding agent specifically binds RSPO3, RSPO2, and RSPO4. In some embodiments, the RSPO3- binding agent cally binds human RSPO3. In some embodiments, the RSPO3- binding agent (e.g., antibody) specifically binds both human RSPO3 and mouse RSPO3.

In certain embodiments, the binding agent is an antibody that specifically binds within amino acids 22-272 of human RSPO3. In certain embodiments, the agent- binding agent is an antibody that specifically binds within amino acids 22-207 of human RSPO3. In certain embodiments, the antigen-binding agent is an antibody that specifically binds within amino acids 35—135 of human RSPO3. In certain embodiments, the antigen-binding agent is an dy that specifically binds within amino acids 35-86 of human RSPO3. In certain ments, the antigen-binding agent is an antibody that specifically binds within amino acids 92-135 of human RSPO3. In certain embodiments, the binding agent binds Within SEQ ID NO:48. In certain embodiments, the RSPO3-binding agent or antibody binds a furin-like cysteine—rich. domain of RSPO3. In some embodiments, the agent or antibody binds at least one amino acid within a furin-like cysteine-rich domain of RSPO3. In certain embodiments, the RSPO3-binding agent or antibody binds within sequence SEQ ID NO:49 or SEQ ID NO:50. In certain embodiments, the RSPO3—binding agent or antibody binds Within sequence SEQ ID NO:49 and SEQ ID NO:50. In some embodiments, the RSPO3-binding agent binds the thrombospondin domain of RSPO3. In some embodiments, the RSPO3-binding agent or antibody binds at least one amino acid within the thrombospondin domain of RSPO3. In some embodiments, the RSPO3-binding agent or antibody binds within SEQ ID NO:51.

In certain embodiments, the RSPO-binding agent or antibody binds at least one RSPO protein with a dissociation constant (KD) of about luM or less, about 100nM or less, about 40nM or less, about 20nM or less, about 10nM or less, about 1nM or less, or about 0.1nM or less. In certain embodiments, a RSPOl-binding agent or antibody binds RSPOl with a iation constant (KD) of about luM or less, about 100nM or less, about 40nM or less, about 20nM or less, about 10nM or less, about 1nM or less, or about 0.1nM or less. In some embodiments, a RSPOl-binding agent or dy binds RSPOl with a KD of about lnM or less. In some embodiments, a RSPOl—binding agent or antibody binds RSPOl with a KD of about 0.1nM or less. In certain embodiments, a RSPOl-binding agent or antibody described herein binds at least one other RSPO. In certain ments, a RSPOl-binding agent or antibody described herein that binds at least one other RSPO, binds at least one other RSPO with a KB of about 100nM or less, about 20nM or less, about 10nM or less, about lnM or less or about 0.1nM or less. For example, in some embodiments, a RSPOl—binding agent or dy also binds RSP02, RSPO3, and/or RSPO4 with a KD of about 10nM or less. In some embodiments, a RSPOl-binding agent (e.g., antibody) binds human RSPOl with a KD of about 0.1nM or less. In some embodiments, the RSPO-binding agent binds both human RSPO and mouse RSPO with a KD of about 10nM or less. In some embodiments, a RSPOl-binding agent binds both human RSPOl and mouse RSPOl with a KB of about lnM or less. In some embodiments, a RSPOl-binding agent binds both human RSPOl and mouse RSPOI With a KB of about 0.1nM or less. In certain embodiments, a RSP02-binding agent or antibody binds RSP02 with a dissociation nt (KD) of about luM or less, about 100nM or less, about 40nM or less, about 20nM or less, about 10nM or less, about lnM or less, or about 0.1nM or less. In some embodiments, a RSP02-binding agent or antibody binds RSP02 with a KB of about 10nM or less. In some embodiments, a RSP02-binding agent or antibody binds RSP02 with a KD of about lnM or less. In certain embodiments, a RSP02-binding agent or antibody described herein binds at least one other RSPO. In certain embodiments, a RSP02-binding agent or dy described herein that binds at least one other RSPO, binds at least one other RSPO with a KB of about 100nM or less, about 20nM or less, about 10nM or less, about lnM or less or about 0.1nM or less. For example, in some embodiments, a RSP02-binding agent or antibody also binds RSPOl, RSPO3, and/or RSPO4 with a KD of about 10nM or less. In some embodiments, a RSP02-binding agent (e.g., antibody) binds human RSP02 with a KB of about lnM or less. In some embodiments, the RSPO-binding agent binds both human RSPO and mouse RSPO with a KB of about 10nM or less. In some embodiments, a RSP02-binding agent binds both human RSP02 and mouse RSP02 with a KB of about lnM or less. In some embodiments, a RSP02-binding agent binds both human RSP02 and mouse RSP02 with a KD of about 0.1nM or less. In some embodiments, the dissociation constant of the g agent (e.g., an dy) to a RSPO protein is the dissociation constant detemtined using a RSPQ fusion protein comprising at ieast a portion of the RSPG protein immobiiized on a Biaeore chip. b13138} in n embodiments, the RSPO~binding agent (e.g., an antibody) binds to at ieast one human RSPO protein with a bait maximai effective concentration {EC-50) of about inM or iess, about itittnM or Hess, about 4011M or less, about 2913M or less, about itinM or iess, about tnM or teas? or about dink/i or less in. certain embodiments, a inding agent (eg, an antibody) binds to human RSPOE with a half maximal etiiective concentration (Eng) of about 133M or less, about 1001th or less, about 4911M or iess, about 2011M or less, about thM or less, about tnM or iess, or about 0.111M or less. in certain embodiments, a RSPO‘t—binding agent {egg an antibody) also binds to human RSPOZ, R8903, and/or RSP04 with an £27ng of about fiftieth/t or iess, about ZtEnM or iess, about tbnM or less, about inb’i or iess or about Gan or less In eettain embodiments, a RSP02~binding agent tag, an antibody) binds to human RSPG'Z with a bait maximai efbctive concentration ) ofaboitt 1 itM. or less, about EQGnM or iess, about dbnM or iess, about anM or iess, about 1013M or toes, about inM or iessi or about 0. inM or less. in certain embodiments, a RSPOfibintting agent {es}, an dy) also binds to human 'RSPOE, RSPOBE and/or RSP04 with an £535.; of about Minty/i oi" tess, about 2GnM or iess, about iQnM or iess, about inM oi" Eess or about 0.1nM or iess.

In certain embodiments, the RSPO~binding agent is an antibody. in some embodiments, the antibody is a recombinant antibody. in some embodiments, the antibody is a inonoeionai antibody. in some embodiments, the antibody is a chimeric antibody. in some embodiments, the antibody is a humanized antibody. in some embodiments, the antibody is a human dy. in certain embodiments, the antibody is an igtfi‘ri antibody. in certain embodiments, the antibody is an igGQ antibody. in eeitain embodiments, the antibody is an antibody fragment comprising an n—binding site. in some embodiments, the antibody is monovaient, monospeeitie, bivaient, iiispeeitic, or moitispeeific. In some ments, the antibody is conjugated to a cytotoxic: moiety: in some embodiments, the antibody is isoiatett in some embodiments, the antibody is sobstantiaiiy pine.

{Mitt} The RSPOnbinding agents (on, antibodies) of the t ion tan be assayed for specific binding by any method known in the art. The iintnnnoassays that can be used e, but are not limited to, itive and noncompetitive assay systems using techniques such as Biacore analysis, FACS analysis, immunofluorescence, immunocytochemistry, Western blots, radioimmunoassays, ELISA, “sandwich” immunoassays, immunoprecipitation assays, precipitation ons, gel diffusion precipitin reactions, immunodiffusion assays, agglutination , complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, and protein A immunoassays. Such assays are routine and well-known in the art (see, e.g., Ausubel et al., Editors, 1994-present, Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, NY).

For example, the specific binding of an antibody to human RSPOl may be ined using ELISA. An ELISA assay comprises preparing antigen, coating wells of a 96 well microtiter plate with antigen, adding the RSPOl-binding dy or other RSPOl-binding agent conjugated to a detectable compound such as an tic substrate (e.g. horseradish dase or ne phosphatase) to the well, incubating for a period of time and detecting the presence of the antibody bound to the antigen. In some ments, the RSPOl-binding antibody or agent is not conjugated to a detectable nd, but instead a second conjugated antibody that recognizes the RSPOl-binding antibody or agent is added to the well. In some embodiments, instead of coating the well with the antigen, the RSPO-binding antibody or agent can be coated to the well and a second dy conjugated to a detectable compound can be added following the addition of the antigen to the coated well. One of skill in the art would be knowledgeable as to the parameters that can be d to increase the signal detected as well as other variations of ELISAs known in the art.

In another example, the specific binding of an antibody to human RSPOl may be determined using FACS. A FACS screening assay may comprise generating a cDNA construct that expresses an antigen as a fusion protein (e.g., RSPOl-Fc or RSPOl- CD4TM), transfecting the construct into cells, expressing the antigen on the surface of the cells, mixing the binding antibody or other RSPOl-binding agent with the transfected cells, and incubating for a period of time. The cells bound by the RSPOl- binding antibody or other RSPO—binding agent may be identified by using a secondary antibody conjugated to a detectable compound (e.g., PE-conjugated anti-Fe antibody) and a flow cytometer. One of skill in the art would be knowledgeable as to the parameters _37_ that can be modified to optimize the signal ed as well as other variations of FACS that may enhance screening (e.g., screening for blocking dies).

The binding affinity of an antibody or other binding-agent to an antigen (e.g., a RSPO protein) and the off-rate of an antibody-antigen interaction can be determined by competitive binding assays. One example of a competitive binding assay is a radioimmunoassay comprising the incubation of labeled antigen (e.g., 3’H or 125I), or fragment or t thereof, with the antibody of interest in the presence of increasing amounts of unlabeled antigen followed by the detection of the antibody bound to the labeled antigen. The affinity of the antibody for an n (e.g., a RSPO n) and the binding off-rates can be determined from the data by ard plot analysis. In some embodiments, Biacore kinetic analysis is used to determine the binding on and off rates of antibodies or agents that bind an antigen (e.g., a RSPO protein). Biacore kinetic analysis comprises analyzing the g and dissociation of antibodies from chips with immobilized antigen (e.g., a RSPO protein) on their surface. 191141 In certain embodiments, the invention provides a RSPOl-binding agent (e.g., an dy) that specifically binds human RSPOl, wherein the RSPOl-binding agent (e.g., an antibody) compréses one, two, three, four, five, and/or six of the CDRs of antibody 89M5 (see Table 1). In some embodiments, the RSPOl-binding agent comprises one or more of the CDRs of 89M5, two or more of the CDRs of 89M5, three or more of the CDRs of 89M5, four or more of the CDRs of 89M5, five or more of the CDRS of 89M5, or all six of the CDRs of 89M5.

E ““““““““““ 89M51£M23 ..........................................................................

TGYTMH SgSYAMS HG -.%-g§99._DN2121._.

.N9.29). .. gHC CDR2 EGINPNNGGTTYNQNFKG SISSGGSTYYPDSVKG (SEQ IDNQB) W,,,,,,,g EKEFSDoYYFFAY RgooDPoVYNoDYEDAMDY HC CDR3 L.....m.m4) (SEQ .19.. N9..31)...W gLL (1112.1 gKASQDVIFAVA KASQDVSSAVA (SEQID 91.91.51- M...

W g(SEQ ID NO32) WASTRHT ‘gWAsrRHT LC CDR2 (SEQ 119No16)______ ID No:33) _____________ (sEQ WMQQHYSTPWW QQHYSTP In certain embodiments, the invention provides a binding agent (e.g., an antibody) that specifically binds human RSPOl, wherein the RSPOl-binding agent comprises a heavy chain CDRI comprising TGYTMH (SEQ ID NO:12), a heavy chain CDR2 comprising GINPNNGGTTYNQNFKG (SEQ ID NO:13), and a heavy chain CDR3 comprising KEFSDGYYFFAY (SEQ ID NO:14). In some embodiments, the RSPOl-binding agent further comprises a light chain CDRl comprising IFAVA (SEQ ID NO:15), a light chain CDR2 comprising WASTRHT (SEQ ID NO:16), and a light chain CDR3 comprising QQHYSTPW (SEQ ID NO:17). In some embodiments, the RSPOl-binding agent comprises a light chain CDRl comprising KASQDVIFAVA (SEQ ID NO:15), a light chain CDR2 comprising WASTRHT (SEQ ID NO:16), and a light chain CDR3 comprising QQHYSTPW (SEQ ID NO:17). In certain embodiments, the binding agent comprises: (a) a heavy chain CDRl comprésing TGYTMH (SEQ ID NO:12), a heavy chain CDR2 comprésing GINPNNGGTTYNQNFKG (SEQ ID NO:13), and a heavy chain CDR3 comprising KEFSDGYYFFAY (SEQ ID NO:14), and (b) a light chain CDRl comprising KASQDVIFAVA (SEQ ID NO:15), a light chain CDR2 comprising WASTRHT (SEQ ID NO:16), and a light chain CDR3 comprising QQHYSTPW (SEQ ID NO:17).

In certain embodiments, the invention provides a RSPOl-binding agent (e.g., an dy) that specifically binds human RSPOI, wherein the RSPOl-binding agent comprises: (a) a heavy chain CDRI comprising TGYTMH (SEQ ID NO:12), or a t thereof comprising 1, 2, 3, or 4 amino acid substitutions; (b) a heavy chain CDR2 sing GINPNNGGTTYNQNFKG (SEQ ID NO:13), or a variant thereof sing 1, 2, 3, or 4 amino acid substitutions; (c) a heavy chain CDR3 comprising KEFSDGYYFFAY (SEQ ID NO:14), or a variant thereof comprising 1, 2, 3, or 4 amino acid substitutions; (d) a light chain CDRl sing IFAVA (SEQ ID , or a variant f comprising 1, 2, 3, or 4 amino acid substitutions; (e) a light chain CDR2 comprising WASTRHT (SEQ ID NO:16), or a variant thereof comprising 1, 2, 3, or 4 amino acid substitutions; and (t) a light chain CDR3 comprésing QQHYSTPW (SEQ ID NO:17), or a variant f comprising 1, 2, 3, or 4 amino acid substitutions. In certain embodiments, the amino acid substitutions are conservative substitutions.

In certain embodiments, the invention es a RSPOl-binding agent (e.g., an antibody) that specifically binds RSPOI, wherein the RSPOl-binding agent comprises a heavy chain variable region having at least about 80% sequence identity to SEQ ID NO:10, and/or a light chain variable region having at least 80% ce identity to SEQ ID NO:11. In certain embodiments, the RSPOl-biriding agent comprises a heavy chain variable region having at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% sequence identity to SEQ ID NO:10. In certain embodiments, the binding agent comprises a light chain le region having at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% sequence identity to SEQ ID NOzll. In certain embodiments, the RSPOl— binding agent comprises a heavy chain variable region having at least about 95% sequence identity to SEQ ID NO:10, and/or a light chain variable region having at least about 95% sequence identity to SEQ ID NO:11. In certain embodiments, the RSPOl- binding agent comprises a heavy chain variable region sing SEQ ID NO:10, and/or a light chain variable region comprising SEQ ID NO:11. In certain embodiments, the RSPOl-binding agent comprises a heavy chain variable region consisting essentially of SEQ ID NO:10, and a light chain variable region consisting essentially of SEQ ID NO:11.

In certain embodiments, the ion provides a RSPOl-binding agent (e.g., an antibody) that specifically binds RSPOI, wherein the RSPOl-binding agent compréses a heavy chain variable region having at least about 80% sequence identity to SEQ ID NO:55, and/or a light chain variable region having at least 80% sequence identity to SEQ ID NO:59. In certain embodiments, the binding agent compréses a heavy chain variable region having at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% sequence identity to SEQ ID NO:55. In certain embodiments, the RSPOl-binding agent ses a light chain variable region having at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% sequence identity to SEQ ID NO:59. In certain embodiments, the RSPOl- binding agent comprises a heavy chain variable region having at least about 95% sequence identity to SEQ ID NO:55, and/or a light chain variable region having at least about 95% sequence identity to SEQ ID NO:59. In certain embodiments, the RSPOl- binding agent comprises a heavy chain variable region comprising SEQ ID NO:55, and/or a light chain vaéable region comprising SEQ ID NO:59. In n embodiments, the binding agent comprises a heavy chain variable region consisting essentially of SEQ ID NO:55, and a light chain le region consisting essentially of SEQ ID NO:59.

In certain embodiments, the invention provides a RSPOl-binding agent (e.g., an antibody) that specifically binds RSPOI, wherein the RSPOl-binding agent comprises: (a) a heavy chain having at least 90% sequence identity to SEQ ID NO:25; and/or (b) a light chain having at least 90% sequence identity to SEQ ID NO:26. In some embodiments, the RSPOl-binding agent comprises: (a) a heavy chain having at least 95% sequence identity to SEQ ID NO:25; and/or (b) a light chain having at least 95% sequence identity to SEQ ID NO:26. In some embodiments, the RSPOl-binding agent comprises a heavy chain comprising SEQ ID NO:25, and/or a light chain comprising SEQ ID NO:26. In some ments, the RSPOl-binding agent comprises a heavy chain consisting essentially of SEQ ID NO:25, and a light chain consisting essentially of SEQ ID NO:26. {812%} In certain embodiments, the invention es a RSPOl-binding agent (e.g., an antibody) that specifically binds RSPOl, wherein the RSPOl-Einding agent comprises: (a) a heavy chain having at least 90% sequence identity to SEQ ID NO:68; and/or (b) a light chain having at least 90% sequence identity to SEQ ID NO:69. In some embodiments, the RSPOl-binding agent ses: (a) a heavy chain having at least 95% sequence ty to SEQ ID NO:68; and/or (b) a light chain having at least 95% sequence identity to SEQ ID NO:69. In some ments, the RSPOl-binding agent comprises a heavy chain comprising SEQ ID NO:68, and/or alight chain comprising SEQ ID NO:69. In some embodiments, the RSPOl-binding agent comprises a heavy chain consisting essentially of SEQ ID NO:68, and a light chain consisting essentially of SEQ ID NO:69.

In certain embodiments, the ion provides a RSPOZ-binding agent (e.g., an antibody) that specifically binds human RSPO2, wherein the RSPO2-binding agent (e.g., an antibody) comprises one, two, three, four, five, and/or six of the CDRs of antibody 130M23 (see Table 1). In some embodiments, the RSPOZ-binding agent comprises one or more of the CDRs of 130M23, two or more of the CDRs of 130M23, three or more of the CDRs of 130M23, four or more of the CDRs of 130M23, five or more of the CDRs of 130M23, or all six of the CDRs of 130M23.

In certain embodiments, the invention provides a RSPOZ-binding agent (e.g., an antibody) that specifically binds human RSPO2, wherein the RSPO2-binding agent comprises a heavy chain CDRl comprising SSYAMS (SEQ ID NO:29), a heavy chain CDR2 comprising SISSGGSTYYPDSVKG (SEQ ID NO:30), and a heavy chain CDR3 comprising RGGDPGVYNGDYEDAMDY (SEQ ID NO:31). In some embodiments, the RSPOZ-binding agent further comprises a light chain CDRl comprising KASQDVSSAVA (SEQ ID NO:32), a light chain CDR2 sing WASTRHT (SEQ ID NO:33), and a light chain CDR3 sing QQHYSTP (SEQ ID NO:34). In some embodiments, the RSPO2-binding agent comprises a light chain CDRl comprising KASQDVSSAVA (SEQ ID NO:32), a light chain CDR2 comprising T (SEQ ID NO:33), and a light chain CDR3 comprising QQHYSTP (SEQ ID NO:34). In certain embodiments, the RSPO2—binding agent comprises: (a) a heavy chain CDRl comprésing SSYAMS (SEQ ID NO:29), a heavy chain CDR2 comprising SISSGGSTYYPDSVKG (SEQ ID NO:30), and a heavy chain CDR3 comprising RGGDPGVYNGDYEDAMDY (SEQ ID NO:31), and (b) a light chain CDRl comprising KASQDVSSAVA (SEQ ID NO:32), a light chain CDR2 sing WASTRHT (SEQ ID N033), and a light chain CDR3 comprising QQHYSTP (SEQ ID .

In certain embodiments, the invention es a RSPO2-binding agent (e.g, an antibody) that specifically binds human RSP02, wherein the RSPOZ-binding agent comprises: (a) a heavy chain CDRl comprising SSYAMS (SEQ ID , or a t thereof sing 1, 2, 3, or 4 amino acid substitutions; (b) a heavy chain CDR2 comprising SISSGGSTYYPDSVKG (SEQ ID NO:30), or a variant thereof sing 1, 2, 3, or 4 amino acid substitutions; (c) a heavy chain CDR3 comprising RGGDPGVYNGDYEDAMDY (SEQ ID NO:31), or a variant thereof comprising 1, 2, 3, or 4 amino acid substitutions; (d) a light chain CDRl comprising KASQDVSSAVA (SEQ ID NO:32), or a variant thereof comprising 1, 2, 3, or 4 amino acid substitutions; (e) a light chain CDR2 comprising WASTRHT (SEQ ID NO:33), or a variant thereof comprising 1, 2, 3, or 4 amino acid substitutions; and (f) a light chain CDR3 comprising QQHYSTP (SEQ ID NO:34), or a variant thereof sing 1, 2, 3, or 4 amino acid substitutions. In certain embodiments, the amino acid substitutions are conservative substitutions.

In certain embodiments, the ion provides a RSPOZ-binding agent (e.g., an antibody) that specifically binds RSPG2, wherein the RSPOZ-binding agent comprises a heavy chain variable region having at least about 80% sequence ty to SEQ ID NO:27, and/or a light chain le region having at least 80% sequence identity to SEQ ID NO:28. In certain embodiments, the RSPOZ-binding agent comprises a heavy chain variable region having at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% sequence identity to SEQ ID NO:27. In certain embodiments, the RSPOZ-binding agent comprises a light chain variable region having at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% sequence identity to SEQ ID NO:28. In certain ments, the RSPOZ— binding agent comprises a heavy chain variable region having at least about 95% sequence identity to SEQ ID NO:27, and/or a light chain variable region having at least about 95% sequence ty to SEQ ID NO:28. In certain embodiments, the RSPOZ- binding agent comprises a heavy chain le region comprising SEQ ID NO:27, and/or a light chain variable region comprising SEQ ID NO:28. In certain embodiments, the RSPOZ-binding agent comprises a heavy chain variable region ting essentially of SEQ ID NO:27, and a light chain variable region consisting ially of SEQ ID NO:28.

In certain embodiments, the invention provides a RSPOZ-binding agent (e.g., an antibody) that specifically binds RSPOZ, wherein the RSPOZ-binding agent comprises a heavy chain variable region having at least about 80% sequence identity to SEQ ID NO:63, and/or a light chain variable region having at least 80% sequence ty to SEQ ID NO:67 or SEQ ID NO:76. In certain embodiments, the RSPOZ-binding agent comprises a heavy chain variable region having at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% sequence identity to SEQ ID NO:63. In certain embodiments, the RSPO2-binding agent compréses a light chain variable region having at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% sequence identity to SEQ ID NO:67 or SEQ ID NO:76.

In certain embodiments, the RSPOZ-binding agent comprises a heavy chain variable region having at least about 95% ce identity to SEQ ID NO:63, and/or a light chain variable region having at least about 95% sequence identity to SEQ ID NO:67 or SEQ ID NO:76. In certain embodiments, the RSPOZ—binding agent comprises a heavy chain variable region comprising SEQ ID NO:63, and/or a light chain variable region comprising SEQ ID NO:67. In certain embodiments, the RSPOZ-binding agent comprises a heavy chain variable region comprising SEQ ID NO:63, and/or a light chain variable region comprising SEQ ID NO:76. In certain embodiments, the binding agent comprises a heavy chain variable region consisting essentially of SEQ ID NO:63, and a light chain variable region consisting essentially of SEQ ID NO:67. In certain ments, the RSPO2-binding agent comprises a heavy chain varéable region consisting essentially of SEQ ID N0263, and a light chain le region consisting essentially of SEQ ID NO:76.

In n embodiments, the invention provides a RSPOZ-binding agent (e.g., an dy) that specifically binds RSPOZ, n the RSPOZ-binding agent comprises: (a) a heavy chain having at least 90% sequence identity to SEQ ID NO:41; and/or (b) a light chain having at least 90% sequence identity to SEQ ID NO:42. In some ments, the RSPOZ-binding agent comprises: (a) a heavy chain having at least 95% sequence identity to SEQ ID NO:41; and/or (b) a light chain having at least 95% sequence identity to SEQ ID NO:42. In some embodiments, the RSPOl-binding agent comprises a heavy chain comprising SEQ ID NO:4l, and/or a light chain comprising SEQ ID NO:42. In some embodiments, the RSPOl-binding agent comprises a heavy chain consisting essentially of SEQ ID NO:41, and a light chain consisting essentially of SEQ ID NO:42. [012?] In certain embodiments, the invention provides a RSPOZ-binding agent (e.g., an antibody) that specifically binds RSPOZ, wherein the binding agent ses: (a) a heavy chain having at least 90% sequence identity to SEQ ID NO:70; and/or (b) a light chain having at least 90% sequence identity to SEQ ID NO:71 or SEQ ID NO:74. In some embodiments, the RSPO2-binding agent comprises: (a) a heavy chain having at least 95% sequence identity to SEQ ID NO:70; and/or (b) a light chain having at least 95% sequence identity to SEQ ID NO:71 or SEQ ID NO:74. In some embodiments, the RSPOZ-binding agent comprises a heavy chain comprising SEQ ID NO:70, and/or a light chain comprising SEQ ID NO:7l. In some embodiments, the RSPO2-binding agent comprises a heavy chain comprising SEQ ID NO:70, and/or a light chain comprising SEQ ID NO:74. In some embodiments, the RSPOZ-binding agent comprises a heavy chain consisting essentially of SEQ ID NO:70, and a light chain consisting essentially of SEQ ID NO:7l. In some embodiments, the RSPO2-binding agent comprises a heavy chain consisting essentially of SEQ ID NO:70, and a light chain consisting essentially of SEQ ID NO:74. [0128} The invention provides polypeptides, including, but not limited to, antibodies that specifically bind human RSPO proteins. In some embodiments, the polypeptides bind human RSPOl. In some embodiments, the polypeptides bind human RSPO2. In some embodiments, the polypeptides bind human RSPO3. [0129} In n embodiments, the ptide compréses one, two, three, four, five, and/or six of the CDRs of antibody 89M5 (see Table 1 ). In n embodiments, the polypeptide comprises one, two, three, four, five, and/or six of the CDRs of antibody 130M23 (see Table 1 herein). In some embodiments, the polypeptide comprises CDRs with up to four Ge, 0, l, 2, 3, 0r 4) amino acid substitutions per CDR. In certain embodiments, the heavy chain CDR(s) are contained within a heavy chain variable region. In certain embodiments, the light chain CDR(s) are contained within a light chain variable .

In some embodiments, the invention provides a polypeptide that specifically binds human RSPOl, wherein the polypeptide comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:10 or SEQ ID NO:55, and/or an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:11 or SEQ ID NO:59. In certain embodiments, the polypeptide comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% sequence identity to SEQ ID NO:10 or SEQ ID NO:55. In certain embodiments, the ptide comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% sequence ty to SEQ ID NOzll or SEQ ID NO:59. In certain embodiments, the polypeptide comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID N0210 or SEQ ID NO:55, and/or an amino acid sequence having at least about 95% sequence identity to SEQ ID NO:11 or SEQ ID NO:59. In certain embodiments, the polypeptide comprises an amino acid sequence comprising SEQ ID NO:10, and/or an amino acid sequence sing SEQ ID NO:1 1. In n embodiments, the polypeptide comprises an amino acid sequence comprising SEQ ID N0255, and/or an amino acid sequence comprising SEQ ID NO:59.

In some embodiments, the invention provides a polypeptide that specifically binds human RSPOZ, wherein the polypeptide comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:27 or SEQ ID NO:63, and/or an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:28, SEQ ID NO:67, or SEQ ID NO:76. In certain embodiments, the polypeptide comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% sequence identity to SEQ ID NO:27 or SEQ ID NO:63.

In n embodiments, the polypeptide comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% sequence identity to SEQ ID NO:28, SEQ ID NO:67, or SEQ ID NO:76. In certain embodiments, the ptide comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO:27 or SEQ ID NO:63, and/or an amino acid sequence having at least about 95% sequence identity to SEQ ID NO:28, SEQ ID NO:67, or SEQ ID NO:76. In certain embodiments, the polypeptide comprises an amino acid ce sing SEQ ID NO:27, and/or an amino acid sequence comprising SEQ ID NO:28. In certain embodiments, the polypeptide comprises an amino acid sequence comprising SEQ ID NO:63, and/or an amino acid sequence comprising SEQ ID NO:67.

In n embodiments, the polypeptide comprises an amino acid sequence comprising SEQ ID NO:63, and/or an amino acid sequence comprising SEQ ID NO:76.

In some embodiments, the invention provides a polypeptide that specifically binds human RSPOl, wherein the polypeptide comprises an amino acid sequence having at least about 80% ce identity to SEQ ID NO:25, and/or an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:26. In n embodiments, the polypeptide comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% sequence identity to SEQ ID NO:25. In certain embodiments, the ptide comprises an amino acid ce having at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% sequence identity to SEQ ID NO:26. In certain embodiments, the polypeptide comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO:25, and/or an amino acid sequence having at least about 95% sequence identity to SEQ ID NO:26. In certain embodiments, the polypeptide comprises an amino acid sequence comprising SEQ IE} NO:25, and/or an amino acid sequence comprising SEQ ID N026. In certain embodiments, the polypeptide ts essentially of SEQ ID NO:25, and/or SEQ ID NO:26.

In some embodiments, the invention es a ptide that specifically binds human RSPOl, wherein the polypeptide comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:68, and/or an amino acid ce having at least about 80% sequence identity to SEQ ID NO:69. In certain embodiments, the polypeptide comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% sequence identity to SEQ ID NO:68. In certain ments, the polypeptide comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% sequence identity to SEQ ID NO:69. In certain embodiments, the polypeptide comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO:68, and/or an amino acid sequence having at least about 95% sequence identity to SEQ ID NO:69. In certain embodiments, the polypeptide comprises an amino acid sequence comprising SEQ ID NO:68, and/or an amino acid sequence comprising SEQ ID NO:69. In certain embodiments, the ptide ts essentially of SEQ ID NO:68, and/or SEQ ID NO:69.

In some embodiments, the invention provides a polypeptide that specifically binds human RSPO2, wherein the polypeptide c0mprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:41, and/or an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:42. In certain embodiments, the polypeptide comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% sequence identity to SEQ ID NO:4l. In certain embodiments, the polypeptide comprises an amino acid ce having at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% sequence identity to SEQ ID NO:42. In certain embodiments, the polypeptide comprises an amino acid ce having at least about 95% sequence identity to SEQ ID NO:41, and/or an amino acid sequence having at least about 95% sequence identity to SEQ ID NO:42. In n embodiments, the polypeptide comprises an amino acid sequence comprising SEQ ID NO:4l, and/or an amino acid sequence comprésing SEQ ID NO:42. In certain embodiments, the polypeptide ts essentially of SEQ ID NO:4l, and/or SEQ ID NO:42.

In some embodiments, the ion provides a polypeptide that specifically binds human RSPOZ, wherein the polypeptide comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:70, and/or an amino acid sequence having at least about 80% ce ty to SEQ ID NO:71 or SEQ ID NO:74. In certain embodiments, the ptide comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% sequence identity to SEQ ID NO:70. In certain embodiments, the polypeptide comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about99% sequence identity to SEQ ID NO:71 or SEQ ID NO:74. In certain embodiments, the polypeptide comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO:70, and/or an amino acid sequence having at least about 95% sequence identity to SEQ ID NO:71 or SEQ ID NO:74. In certain embodiments, the polypeptide comprises an amino acid ce comprising SEQ ID NO:70, and/or an amino acid sequence comprising SEQ ID NO:71.

In certain embodiments, the polypeptide comprises an amino acid sequence comprising SEQ ID NO:70, and/or an amino acid sequence comprising SEQ ID NO:74. In certain embodiments, the polypeptide consists essentially of SEQ ID NO:70, and/or SEQ ID NO:71. In certain embodiments, the polypeptide consists essentially of SEQ ID NO:70, and/or SEQ ID NO:74.

In some ments, a binding agent comprises a polypeptide comprising a sequence selected from the group ting of: SEQ ID N0210, SEQ ID NO:11, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:53, SEQ ID N‘Oz55, SEQ ID NO:57, SEQ ID N0259, SEQ ID NO:68, and SEQ ID NO:69. In some embodiments, a RSPO2-binding agent comprises a polypeptide comprising a sequence selected from the group consisting of: SEQ ID NO:27, SEQ ID N0228, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:70, SEQ ID NO:7l, SEQ ID NO:73, SEQ ID NO:74, and SEQ ID NO:76.

In certain embodiments, a RSPOl—binding agent comprises the heavy chain variable region and light chain variable region of the 89M5 antibody. In certain embodiments, a RSPOl-binding agent comprises the heavy chain and light chain of the 89M5 antibody (with or without the leader sequence). In n embodiments, a RSPOl- binding agent is the 89M5 antibody. In certain embodiments, a RSPOl-binding agent comprises the heavy chain variable region and/or light chain le region of the 89M5 antibody in a humanized form of the antibody. In certain embodiments, the RSPOl- binding agent comprises the heavy chain le region and/or light chain le region of the h89M5-H2L2 antibody. In certain embodiments, a RSPOl-binding agent comprises the heavy chain and light chain of the 89M5 antibody (with or without the leader sequence) in a humanized form of the antibody. In certain embodiments, a RSPOl-binding agent comprises the heavy chain and light chain of the h89M5-H2L2 dy (with or without the leader sequence). In some embodiments, the humanized version of 89M5 is an IgGl antibody. In some embodiments, the humanized version of 89M5 is an IgG2 dy. The hybridoma cell line producing the 89M5 antibody was deposited with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, VA, USA, under the conditions of the Budapest Treaty on June 30, 2011 and assigned ATCC deposit designation number PTA-11970.

In certain embodiments, a RSPOl-binding agent comprises, ts essentially of, or consists of, the dy 89M5. In certain embodiments, a binding agent comprises, ts essentially of, or consists of, the antibody h89M5-H2L2.

In certain ments, a RSPOZ-binding agent comprises the heavy chain variable region and light chain le region of the 130M23 antibody. In certain embodiments, a RSPO2-binding agent comprises the heavy chain and light chain of the 130M23 antibody (with or without the leader sequence). In certain embodiments, a RSPOZ-binding agent is the 130M23 antibody. In certain embodiments, a RSPOZ- binding agent comprises the heavy chain variable region and/or light chain variable region of the 130M23 antibody in a humanized form of the antibody. In n embodiments, the RSPOZ-binding agent comprises the heavy chain variable region and/or light chain variable region of the h130M23-H1L2 antibody. In certain embodiments, the RSPOZ-binding agent comprises the heavy chain variable region and/or light chain variable region of the h130M23-H1L6 antibody. In certain ments, a RSPOZ- binding agent comprises the heavy chain and light chain of the 130M23 antibody (with or without the leader sequence) in a humanized form of the antibody. In certain embodiments, a RSPOZ-binding agent comprises the heavy chain and light chain of the h130M23—H1L2 antibody (with or without the leader sequence). In certain embodiments, a RSPOZ-binding agent comprises the heavy chain and light chain of the h130M23—H1L6 antibody (with or without the leader sequence). In some embodiments, the humanized version of 130M23 is an IgGl antibody. In some embodiments, the humanized version of 130M23 is an IgGZ dy. The hybridoma cell line producing the 130M23 antibody was deposited with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, VA, USA, under the conditions of the Budapest Treaty on August , 2011 and assigned ATCC deposit designation number PTA-12021. {£11.40} In n embodiments, a RSPOZ-binding agent comprises, consists essentially of, or consists of, the antibody 130M23. In certain embodiments, a binding agent comprises, consists essentially of, or consists of, the antibody 3-H1L2. In certain embodiments, a binding agent comprises, ts essentially of, or consists of, the antibody h130M23-H1L6.

Many proteins, including antibodies, contain a signal sequence that directs the transport of the proteins to various locations. Signal sequences (also referred to as signal peptides or leader sequences) are located at the inus of t ptides. They target the polypeptide to the endoplasmic reticulum and the proteins are sorted to their destinations, for example, to the inner space of an organelle, to an interior membrane, to the cell's outer membrane, or to the cell exterior via secretion. Most signal ces are d from the protein by a signal peptidase after the proteins are transported to the endoplasmic reticulum. The cleavage of the signal sequence from the polypeptide usually occurs at a specific site in the amino acid sequence and is ent upon amino acid residues within the signal sequence. Although there is usually one specific cleavage site, more than one ge site may be recognized and/or may be used by a signal peptidase resulting in a non-homogenous N-terminus of the polygjeptide. For example, the use of different cleavage sites within a signal sequence can result in a polypeptide expressed with different N-terminal amino acids. Accordingly, in some embodiments, the polypeptides as described herein may comprise a mixture of polypeptides with different N-termini. In some embodiments, the N-termini differ in length by 1, 2, 3, 4, or 5 amino acids. In some ments, the polypeptide is substantially homogeneous, i.e., the polypeptides have the same N-terminus. In some embodiments, the signal sequence of the polypeptide comprises one or more (e.g., one, two, three, four, five, six, seven, eight, nine, ten, etc.) amino acid substitutions and/or deletions as ed to a “native” or “parenta ” signal sequence. 3n some embodiments, the signal sequence of the polypeptide ses amino acid substitutions and/or deletions that allow one ge site to be dominant, thereby resulting in a substantially homogeneous polypeptide with one N- terminus. In some embodiments, the signal sequence of the polypeptide is replaced with a different signal sequence. In some embodiments, a signal ce of the polypeptide affects the expression level of the polypeptide. In some embodiments, a signal sequence of the polypeptide increases the expression level of the polypeptide. In some embodiments, a signal sequence of the polypeptide decreases the expression level of the polypeptide.

In n ments, a RSPOl-binding agent (e.g., dy) es for c binding to RSPOI with an antibody that comprises a heavy chain variable region comprising SEQ ID NO:10 and a light chain variable region comprising SEQ ID NO:1 1.

In certain embodiments, a RSPOl-binding agent (e.g., antibody) competes for specific binding to RSPOl with an antibody that comprises a heavy chain variable region comprising SEQ ID NO:55 and a light chain variable region comprising SEQ ID NO:59.

In certain embodiments, a RSPOl—binding agent (e.g., dy) competes for specific binding to RSPOl with an antibody that comprises a heavy chain comprising SEQ ID N025 and a light chain sing SEQ ID NO:26. In certain embodiments, a RSPOl- binding agent (e.g., antibody) competes for specific binding to RSPOl with an antibody that comprises a heavy chain comprising SEQ ID NO:68 and a light chain comprising SEQ ID NO:69. In certain embodiments, a RSPOl-binding agent competes with antibody 89M5 or h89M5-H2L2 for c binding to human RSPOl. In some embodiments, a binding agent or antibody competes for specific binding to RSPOl in an in vitro competitive binding assay. In some embodiments, the RSPOl is human RSPOl. In some embodiments, the RSPOI is mouse RSPOI. [0143} In certain embodiments, a RSPOl-binding agent (e.g., an dy) binds the same epitope, or essentially the same epitope, on RSPOl as an antibody of the invention.

In another embodiment, a RSPOl-binding agent is an antibody that binds an epitope on RSPOl that overlaps with the epitope on RSPOl bound by an antibody of the invention.

In certain embodiments, a RSPOl-binding agent (e.g., an antibody) binds the same epitope, or essentially the same epitope, on RSPOl as antibody 89M5 or h89M5—H2L2.

In another embodiment, the RSPOI -binding agent is an antibody that binds an epitope on -5]- RSP‘OI that overlaps with the epitope on RSP01 bound by antibody 89M5 or h89M5- H2L2.

In certain embodiments, the RSPOI-binding agent is an agent that competes for specific binding to RSPOI with an antibody produced by the hybridoma having ATCC deposit designation number PTA-11970 (e.g., in a competitive binding assay).

In n embodiments, a binding agent (e.g., antibody) competes for specific binding to RSP02 with an antibody that comprises a heavy chain variable region comprising SEQ ID N027 and a light chain variable region comprising SEQ ID NO:28.

In certain embodiments, a RSP02-binding agent (e.g., antibody) competes for specific binding to RSP02 with an antibody that ses a heavy chain variable region comprising SEQ ID NO:63 and a light chain variable region comprising SEQ ID NO:67 or SEQ ID NO:76. In certain embodiments, a RSP02-binding agent (e.g., antibody) competes for specific binding to RSP02 with an antibody that comprises a heavy chain sing SEQ ID NO:41 and a light chain comprising SEQ ID NO:42. In certain embodiments, a RSP02-binding agent (e.g., antibody) competes for c binding to RSP02 with an antibody that comprises a heavy chain comprising SEQ ID NO:70 and a light chain comprising SEQ ID NO:71 or SEQ ID NO:74. In certain embodiments, a RSP02-binding agent competes with antibody 130M23, h130M23-H1L2, or h130M23— H1L6 for specific binding to human RSP02. In some ments, a RSP02-binding agent or antibody es for specific g to RSP02 in an in vitro competitive binding assay. In some embodiments, the RSP02 is human RSP02. In some embodiments, the RSP02 is mouse RSP02.

In certain embodiments, a RSP02-binding agent (e.g., an antibody) binds the same epitope, or essentially the same epitope, on RSP02 as an antibody of the invention.

In another embodiment, a RSP02-binding agent is an antibody that binds an epitope on RSP02 that overlaps with the epitope on RSP02 bound by an dy of the invention.

In certain embodiments, a RSP02-binding agent (e.g., an antibody) binds the same epitope, or essentially the same epitope, on RSP02 as antibody 130M23, h130M23- H1L2, or h130M23-H1L6. In r embodiment, the RSP02-binding agent is an antibody that binds an e on RSP02 that overlaps with the epitope on RSP02 bound by antibody 130M23, h130M23-H1L2, or hl30M23-H1L6..

In certain embodiments, the RSP02-binding agent is an agent that competes for specific binding to RSP02 with an antibody produced by the hybridoma having ATCC deposit designation number PTA-12021 (e.g., in a competitive binding assay).

In certain embodiments, the RSPO-binding agent (e.g., an antibody) described herein. binds at least one human RSPO protein and modulates RSPO activity. In some embodiments, the RSPO-binding agent is a RSPO antagonist and ses RSFO activity. In some embodiments, the RSPO-binding agent is a RSPO antagonist and ses B-catenin activity.

In certain embodiments, a RSPOl-binding agent (e.g., an antibody) described herein binds human RSPOl and modulates RSPOl activity. In some embodiments, a binding agent is a RSPOl nist and decreases RSPOl activity. In some embodiments, a RSPOl—binding agent is a RSPOl antagonist and decreases B-catenin activity. [01:30] In certain embodiments, a RSP02-binding agent (e.g., an antibody) described herein binds human RSPOZ and modulates RSP02 ty. In some embodiments, a RSP02-binding agent is a RSP02 antagonist and decreases RSP02 activity. In some embodiments, a RSP02-binding agent is a RSP02 antagonist and decreases B-catenin activity.

In certain embodiments, a binding agent (e.g., an antibody) described herein binds human RSPO3 and modulates RSPO3 activity. In some embodiments, a binding agent is a RSPO3 antagonist and decreases RSPO3 activity. In some embodiments, a RSPO3-binding agent is a RSPO3 antagonist and decreases B-catenin activity.

In n embodiments, the RSPO-binding agent (e.g., an antibody) is an antagonist of at least one human RSPO protein. In some embodiments, the RSPO- binding agent is an antagonist of at least one RSPO and inhibits RSPO activity. In certain embodiments, the RSPO-binding agent inhibits RSPO activity by 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%. In some embodiments, the RSPO-binding agent inhibits activity of one, two, three, or four RSPO proteins. In some ments, the RSPO—binding agent ts activity of human RSPOl, RSP02, RSPO3, and/or RSPO4. In certain ments. a RSPOl-binding agent that inhibits human RSPOl activity is antibody 89M5 or h89M5-H2L2. In certain embodiments, a binding agent that inhibits human RSP02 activity is antibody 130M23, 3—H1L2, or h130M23-H1L6.

In certain embodiments, the RSPO-binding agent (e.g., antibody) is an antagonist of at least one human RSPO protein. In certain embodiments, the RSPO-binding agent ts RSPO signaling by 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%. In some embodiments, the RSPO-binding agent inhibits signaling by one, two, three, or four RSPO proteins. In some embodiments, the RSPO-binding agent inhibits ing of human RSPOl, RSP02, RSPO3, and/or RSPO4. In n embodiments, a RSPOl-_ binding agent that inhibits RSPOl signaling is antibody 89M5 or H2L2. In certain ments, a RSP02-binding agent that inhibits RSP02 signaling is antibody 130M23, h130M23-H1L2, or 3-H1L6.

In certain embodiments, the RSPO—binding agent (e.g., antibody) is an antagonist of B-catenin signaling. In certain embodiments, the RSPO-binding agent inhibits B— catenin signaling by 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%. In certain embodiments, a ESPOl-binding agent that inhibits B—catenin signaling is antibody 89M5 or h89M5-H2L2. In certain embodiments, a RSP02-binding agent that inhibits B-catenin signaling is dy 130M23, hl30M23—H1L2, or hl30M23-H1L6.

In certain embodiments, the RSPO-binding agent (e.g., antibody) inhibits binding of at least one RSFO protein to a receptor. In certain embodiments, the RSPO-binding agent inhibits binding of a human RSPO protein to one or more of its receptors. In some embodiments, the RSPO-binding agent inhibits binding of a RSPO protein to at least one LGR protein. In some embodiments, the RSPO-binding agent ts binding of a RSPO protein to LGR4, LGR5, and/or LGR6. In some embodiments, a RSPOl-binding agent ts binding of RSPOl to LGR4. In some embodiments, a binding agent inhibits binding of RSPOI to LGRS. In some embodiments, a RSPOl-binding agent inhibits binding of RSPOl to LGR6. In some embodiments, a RSP02-binding agent inhibits binding of RSP02 to LGR4. In some embodiments, a RSP02—binding agent inhibits binding of RS902 to LURE. in some embodiments, a RSP02-binding agent inhibits binding oi‘RSPO‘Z to {6. in certain embodiments, the inhibition of binding of a REPS—binding agent to at least one LGR protein, in at ieast about l0‘?/§§, at least about %, at least about 50%, at least about 75%, at least about 90%, or at least about 95%. In n ments, a RSPO-binding agent that inhibits binding of at least one RSPO to at least one LGR n further inhibits B-catenin signaling. In certain embodiments, a RSPOl-binding agent that inhibits binding of human RSPOl to at least one LGR protein is antibody 89M5 or h89M5-H2L2. In n embodiments, a RSP02-binding agent that inhibits binding of human RSP02 to at least one LGR protein is antibody 130M23, hl30M23-H1L2, or hl30M23-H1L6.

In certain embodiments, the RSPO-binding agent (e.g., dy) blocks binding of at least one RSPO to a receptor. In certain embodiments, the inding agent blocks binding of a human RSPO protein to one or more of its receptors. In some embodiments, the RSPO-binding agent blocks binding of a RSPO to at least one LGR protein. In some embodiments, the RSPO-binding agent blocks binding of at least one RSPO protein to LGR4, LGRS, and/or LGR6. In some embodiments, a RSPOl-binding agent blocks binding of RSPOI to LGR4. In some ments, a RSPOl-binding agent blocks binding of RSPOl to LGR5. In some embodiments, a RSPOl-binding agent blocks binding of RSPOl to LGR6. In some embodiments, a RSP02-binding agent blocks binding of RSP02 to LGR4. In some embodiments, a RSP02-binding agent blocks binding of RSP02 to LGR5. In some ments, a RSP02-binding agent blocks binding of RSP02 to LGR6. In certain embodiments, the blocking of binding of a RSPO-binding agent to at least one LGR protein is 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, a RSPO-binding agent that blocks binding of at least one RSPO protein to at least one LGR protein further inhibits B-catenin signaling. In certain embodiments, a RSPOl-binding agent that blocks binding of human RSPOl to at least one LGR protein is antibody 89M5 or H2L2. In certain embodiments, a RSP02- binding agent that blocks binding of human RSP02 to at least one LGR protein is dy 130M23, h130M23-H1L2, 0r hl30M23-H1L6.

In certain embodiments, the RSPO-binding agent (e.g., an antibody) inhibits B- catenin signaling. It is understood that a RSPO-binding agent that inhibits B-catenin ing may, in certain embodiments, inhibit signaling by one or more receptors in the B-catenin signaling pathway but not necessarily t signaling by all receptors. In certain alternative embodiments, B—catenin signaling by all human receptors may be inhibited. In certain embodiments, B-catenin signaling by one or more receptors selected from the group ting of LGR4, LGR5, and LGR6 is inhibited. In certain embodiments, the inhibition of B—catenin signaling by a RSPO-binding agent is a ion in the level of nin signaling of 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 some embodiments, a RSPOl-binding agent that inhibits nin signaling is antibody 89M5 or h89M5-H2L2. In some embodiments, a RSPOZ-binding agent that inhibits B-catenin signaling is antibody 130M23, h130M23-H1L2, or h130M23-H1L6.

In certain embodiments, the RSPO-binding agent (e.g., an dy) inhibits activation of B-catenin. It is understood that a RSPO-binding agent that inhibits activation of B—catenin may, in certain embodiments, inhibit activation of B-catenin by one or more receptors, but not necessarily t activation of B-catenin by all receptors.

In certain alternative embodiments, activation of B-catenin by all human receptors may be inhibited. In certain embodiments, activation of B-catenin by one or more receptors selected from the group consisting of LGR4, LGRS, and LGR6 is inhibited. In certain embodiments, the inhibition of activation of B-catenin by a RSPO-binding agent is a reduction in the level of activation of B-catenin of 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 some embodiments, a binding agent that inhibits activation of B-catenin is antibody 89M5 or h89M5-H2L2. In some embodiments, a RSPOZ-binding agent that inhibits activation of B-catenin is antibody 130M23, hl30M23-H1L2, or h130M23-H1L6.

In vivo and in vitro assays for determining whether a RSPO-binding agent (or candidate RSPO-binding agent) inhibits B—catenin signaling are known in the art. For example, cell-based, luciferase reporter assays utilizing a c reporter vector containing multiple copies of the TCF-binding domain upstream of a firefly rase reporter gene may be used to measure B-catenin ing levels in vitro (Gazit et al., 1999, Oncogene, 18; 5959-66; TOPflash, Millipore, Billerica MA). The level of B» catenin signaling in the ce of one or more Wnts (e.g., Wnt(s) sed by ected cells or provided by Wnt—conditioned media) with or without a RSPO protein or RSPO-conditioned media in the presence of a RSPO-binding agent is compared to the level of signaling without the RSPO-binding agent present. In addition to the TCF/Luc reporter assay} the effect of a RSPO—binding agent (or candidate agent) on B-catenin signaling may be measured in vitro or in vivo by measuring the effect of the agent on the level of expression of B-catenin—regulated genes, such as c-myc (He et al., 1998, Science, 281 :1509-12), cyclin D1 (Tetsu et al., 1999, Nature, 3.98:422-6) and/or fibronectin (Gradl et al. 1999, Mol. Cell Biol., 1.925576-87). In certain embodiments, the effect of a RSPO- binding agent on B-catenin signaling may also be assessed by measuring the effect of the agent on the phosphorylation state of Dishevelled—l , elled-Z, Dishevelled-3, LRPS, LRP6, and/or B-catenin.

In certain embodiments, the RSPO-binding agents have one or more of the following s: inhibit proliferation of tumor cells, inhibit tumor growth, reduce the tumorigenicity of a tumor, reduce the tumorégenicity of a tumor by reducing the frequency of cancer stem cells in the tumor, inhibit tumor growth, trigger cell death of tumor cells, induce cells in a tumor to differentiate, differentiate tumorigenic cells to a non-tumorigenic state, induce expression of differentiation s in the tumor cells, prevent metastasis of tumor cells, or decrease al of tumor cells.

In certain embodiments, the RSPO—binding agents are capable of ting tumor growth. In certain embodiments, the inding agents are capable of inhibiting tumor growth in vivo (e.g., in a xenograft mouse model, and/or in a human having In certain embodiments, the RSPO-binding agents are capable of reducing the tumorigenicity of a tumor. In certain embodiments, the RSPO-binding agent or dy is capable of reducing the tumorigenicity of a tumor comprésing cancer stem cells in an animal model, such as a mouse xenograft model. In certain embodiments, the number or frequency of cancer stem cells in a tumor is reduced by at least about two-fold, about three-fold, about five-fold, about ten-fold, about 50-fold, about lOO-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. Additional examples and guidance regarding the use of limiting dilution assays to ine a reduction in the number or frequency of cancer stem cells in a tumor can be found, e.g., in ational Publication Number WO 2008/042236, US. Patent Publication No. 2008/0064049, and US. Patent Publication No. 2008/0178305.

In certain embodiments, the RSPO-binding agents described herein have a circulating half-life in mice, cynomolgus monkeys, or humans of at least about 5 hours, 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 RSPO-binding agent is an IgG (e.g., IgGl or IgG2) antibody that has a circulating half-life in mice, cynomolgus monkeys, or humans of at least about 5 hours, 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 (or decreasing) the ife of agents such as polypeptides and antibodies are known in the art. For example, known methods of increasing the ating half-life of IgG antibodies include the introduction of mutations in the Fc region which increase the pH-dependent binding of the antibody to the al Fc receptor (FcRn) at pH 6.0 (see, e.g., US. Patent Publication Nos. 2005/0276799, 2007/0148164, and 2007/0122403).

Known methods of increasing the circulating half-life of dy fragments lacking the Fc region include such techniques as PEGylation.

In some embodiments, the RSPO—binding agents are polyclonal antibodies.

Polyclonal antibodies can be ed by any known method. In some embodiments, polyclonal antibodies are raised by immunizing an animal (e.g., a rabbit, rat, mouse, goat, donkey) by multiple subcutaneous or intraperitoneal injections of the relevant antigen (e. g., a purified peptide fragment, full-length recombinant protein, or fusion protein). The antigen can be ally conjugated to a r such as keyhole limpet hemocyanin (KLH) or serum albumin. The antigen (with or t a carrier protein) is diluted in sterile saline and usually combined with an nt (e.g., Complete or Incomplete Freund’s Adjuvant) to form a stable emulsion. After a sufficient period of time, onal antibodies are recovered from blood, ascites, and the like, of the immunized animal. The polyclonal antibodies can be purified from serum or ascites ing to standard methods in the art including, but not limited to, affinity chromatography, ion- exchange chromatography, gel electrophoresis, and dialysis.

In some embodiments, the RSPO-binding agents are monoclonal antibodies.

Monoclonal antibodies can be prepared using oma methods known to one of skill in the art (see e.g., Kohler and Milstein, 1975, Nature, 256:495-497). In some embodiments, using the hybridoma method, a mouse, hamster, or other appropriate host animal, is immunized as described above to elicit from lymphocytes the production of antibodies that will specifically bind the immunizing antigen. In some embodiments, cytes can be immunized in vitro. In some embodiments, the immunizing antigen can be a human protein or a portion thereof In some embodiments, the immunizing antigen can be a mouse protein or a portion thereof.

Following immunization, lymphocytes are isolated and fused with a suitable myeloma cell line using, for example, polyethylene glycol, to form hybridoma cells that can then be selected away from unfused lymphocytes and myeloma cells. Hybridomas that produce monoclonal antibodies directed specifically against a chosen antigen may be identified by a variety of methods including, but not limited to, immunoprecipitation, immunoblotting, and in vitro binding assay (e.g., flow cytometry, FACS, ELISA, and radioimmunoassay). The hybridomas can be propagated either in in vitro culture using standard methods (J.W. Goding, 1996, Monoclonal Antibodies: Principles and ce, 3rd Edition, Academic Press, San Diego, CA) or in vivo as ascites tumors in an animal.

The onal dies can be purified from the culture medium or ascites fluid according to standard methods in the art including, but not limited to, aéfinity chromatography, ion-exchange chromatography, gel electrophoresis, and is.

In certain embodiments, monoclonal antibodies can be made using recombinant DNA techniques as known to one skilled in the art (see e.g., US. Patent No. 4,816,567).

The polynucleotides encoding a monoclonal antibody are isolated from mature B-cells or hybridoma cells, such as by RT-PCR using ucleotide primers that cally amplify the genes encoding the heavy and light chains of the antibody, and their sequence is determined using conventional ques. The isolated cleotides encoding the heavy and light chains are then cloned into suitable expression vectors which produce the monoclonal dies when transfected into host cells such as E. coli, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin proteins. In other embodiments, recombinant onal antibodies, or fragments thereof, can be isolated from phage display libraries expressing CDRs of the desired species (see e.g., McCafferty et a1., 1990, Nature, 2—554; Clackson et a1., 1991, Nature, 352:624-628; and Marks et a1., 1991, J. M0]. Biol, 222:581-597).

The polynucleotide(s) encoding a monoclonal antibody can further be modified in a number of different manners using recombinant DNA technology to generate alternative antibodies. In some embodiments, the constant domains of the light and heavy chains of, for example, a mouse onal antibody can be substituted for those regions of, for example, a human dy to generate a ic antibody, or for a non-immunoglobulin poiypeptide to generate a insion antibody. in some embodiments, the constant regions are indicated or removed to generate the desired antibody fragment. of a monoclonai antibody. Siteudireoted or higin—density nesis of the hie region can be used to optimize specificity, affinity, etc. of a monoctonai antibody. in some ments, the nionooionai antibody against a human RSPO protein is a humanized antibody. Typicaliy, humanized antibodies are human oglobntins in which residues from the CDRS are reptaeed by residues from a (“DR of a nonhuman species (egg mouse, rat, rabbit, hamster, etc.) that have the desired specificity, affinity, and/or binding capability using methods known to one d in the art, in some embodiments, the iii! framework region residues of a human innnnnogiobniin are teniaced with the corresponding residues in an antibody from a nonuhmnan species that has the desired specificity, affinity, and/’or binding capability. in some embodiments, the humanized antibody can be fili'filfif modified by the substitution of additionai residues either in the FV framework region and/or within the replaced nonuhainan residues to refine and ze antibody specificity, affinity, and/oi lity. in. l, the humanized antibody will conipiise snhstantialiy ail of at least one, and typieaiiy two or three, variable domain regions containing all, or substantially aii, of the CDRS that correspond to the nonhuman. iininnnoglobniin s aii, or snhstantiaiiy alt, of the framework regions are those of a human iininunoglobniin consensus sequence. in some embodiments, the humanized antibody can also comprise at least a portion of an immunogiohniin nt region or domain {tic}, typicaiiy that of a human iinmnnogiobniin in certain embodiments, snoh humanized antibodies are used therapeutically e they may reduce antigenicity and RAMA n anti«monse antibody) tesnonses when administered to a human subject. One sitiiied in the an wouid be able to obtain a tinietional humanized antibody with reduced. imntnnogenieity foiiowing known techniques {see e.g., {3.8. i’atent Nos, 5,225,539; 5,585,98g; 5,693,761; and 5,693,?62). {$173} In certain embodiments; the RSt-‘tiiuhinding agent is a human antibody. li—inman antibodies can be directly prepared using various techniques known in the art, in some embodiments, immortalized human B lymphocytes innnuniaed in vitro or isolated from an immunized individnai that produces an antibody directed against a target antigen can be ted (see, e.g., Cole et ai, 1985, onni dies and er Ingram), Alan R. Liss, p. 77; Boemer et a1., 1991, J. l, 147186-95; and US. Patent Nos. ,750,373; 5,567,610 and 5,229,275). In some embodiments, the human antibody can be selected from a phage y, where that phage library expresses human antibodies (Vaughan et a1., 1996, Nature Biotechnology, 14:309-314; Sheets et a1., 1998, PNAS, 95:6157-6162; Hoogenboom and Winter, 1991,.1. M01. Biol., 1; Marks et a1., 1991, .1. M01. Biol, 1). atively, phage display technology can be used to produce human antibodies and antibody fragments in vitro, from immunoglobulin variable domain gene repertoires from nized donors. Techniques for the generation and use of antibody phage libraries are also described in US. Patent Nos. 5,969,108; 6,172,197; ,885,793; 6,521,404; 6,544,731; 6,555,313; 915; 6,593,081; 6,300,064; 068; 6,706,484; and 7,264,963; and Rothe et a1., 2008, .1. M01. Bi0., 376:1182-1200. Affinity maturation strategies including, but not limited to, chain shuffling (Marks et a1., 1992, Bio/Technology, 102779-783) and site—directed mutagenesis, are known in the art and may be ed to generate high affinity human antibodies.

In some embodiments, human antibodies can be made in transgenic mice that contain human immunoglobulin loci. These mice are capable, upon immunization, of producing the full repertoire of human antibodies in the absence of endogenous immunoglobulin production. This ch is described in US. Patent Nos. 5,545,807; ,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016.

This invention also encompasses bispecific antibodies that specifically recognize at least one human RSPO protein. Bispecific antibodies are capable of specifically recognizing and binding at least two different epitopes. The different epitopes can either be within the same molecule (e.g., two epitopes on human RSPOl) or on different molecules (e.g., one epitope on RSPOl and one epitope on RSPO2). In some embodiments, the bispecific dies are monoclonal human or humanized antibodies.

In some embodiments, the antibodies can specifically ize and bind a first antigen target, (e. g., RSPOl) as well as a second antigen target, such as an effector molecule on a leukocyte (e.g., CD2, CD3, CD28, or B7) or 3 Fc receptor (e.g., CD64, CD32, or CD16) so as to focus cellular defense mechanisms to the cell expressing the first antigen target.

In some embodiments, the antibodies can be used to direct cytotoxic agents to cells which express a particular target antigen. These antibodies s an antigen-binding arm and an arm which binds a cytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA. In certain embodiments, the bispecific antibody specifically binds RSPOl, as well as either an additional RSPO protein selected from the group consisting of RSPO2, RSPO3, and RSPO4. In certain embodiments, the bispecific antibody specifically binds RSPO2, as well as either an additional RSPO protein selected from the group ting of RSPOI, RSPO3, and RSPO4.

Techniques for making bispecific antibodies are known by those skilled in the art, see for example, Millstein et al., 1983, Nature, 305:537-539; Brennan et al., 1985, Science, 229281; Suresh et al., 1986, Methods in Enzymol., 121:120; Traunecker et al., 1991, EMBOJ, 10:3655-3659; Shalaby et al., 1992, .1 Exp. Med, 175:217-225; ny et al., 1992, J Immunol., 148:1547-1553; Gruber et al., 1994, J, Immunol., 15225368; US Patent No. 5,731,168; and US. Patent Publication No. 2011/0123532). Bispeciflc antibodies can be intact antibodies or antibody fragments. Antibodies with more than two valencies are also contemplated. For example, trispecific antibodies can be ed (Tutt et al., 1991, J. Immunol., 147:60). Thus, in certain embodiments the antibodies to RSPOl are multispecific.

In certain ments, the antibodies (or other ptides) described herein may be monospecific. For example, in n embodiments, each of the one or more n-binding sites that an antibody contains is capable of binding (or binds) a gous epitope on RSPO ns. In certain embodiments, an antigen-binding site of a monospecific antibody described herein is capable of binding (or binds), for example, RSPOI and RSPO2 (i.e., the same epitope is found on both RSPOI and RSPO2 proteins). {$175} In certain embodiments, the RSPO-binding agent is an antibody nt. dy fragments may have different functions or capabilities than intact antibodies; for example, antibody fragments can have sed tumor penetration. Various techniques are known for the production of antibody fragments including, but not limited to, proteolytic digestion of intact antibodies. In some embodiments, antibody fragments include a F(ab')2 fragment produced by pepsin digestion of an dy molecule. In some embodiments, antibody fragments include a Fab nt generated by reducing the disulfide bridges of an F(ab')2 fragment. In other embodiments, antibody fragments e a Fab fragment generated by the treatment of the antibody molecule with papain and a reducing agent. In certain embodiments, antibody fragments are produced recombinantly. In some embodiments, antibody fragments include FV or single chain FV (scFv) fragments. Fab, Fv, and scFv antibody fragments can be sed in and secreted from E. coli or other host cells, ng for the production of large amounts of these fragments. In some embodiments, antibody fragments are isolated from antibody phage libraries as discussed herein. For example, methods can be used for the construction of Fab expression libraries (Huse et al., 1989, Science, 246:1275-1281) to allow rapid and effective identification of onal Fab fragments with the desired specificity for a RSPO protein or derivatives, nts, analogs or homologs thereof. In some embodiments, antibody fragments are linear antibody fragments. In certain embodiments, antibody fragments are monospecific or bispecific. In certain embodiments, the RSPO- binding agent is a scFv. s techniques can be used for the tion of single- chain antibodies specific to one or more human RSPOs (see, e.g., US. Patent No. 4,946,778). {@1776} it can iiirther be desirable, ally in the case. of antihcdy fragments, in mndify an antibody in crde‘r to increase its serum haifdiie. This can he ed, fer example, by eratinn of a salvage receptor binding epitepe into the antibedy fragment by mntatinn cf the apprepriate region in the antibcdy fragment or by incerpnrating the epitepe inte a peptide tag that is then fused in the antibcdy fragment at. either end er in the middle (e.g., by DNA or peptide synthesis). {@177} iietercccnjngate antihedies are also» within the scope 0f the present invention.

Hetercecniugate antihndies are composed (if two ecvalentiy joined anti hedies. Such antihedies have, far example, been ed to target immune cells to unwanted cells (US. Patent No. 4,676,980). It is also contemplated that the heteroconjugate antibodies can be ed in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins can be constructed using a de exchange on or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methylmercaptobutyrimidate.

For the purposes of the present invention, it should be appreciated that modified antibodies can comprise any type of variable region that es for the association of the antibody with the target (i.e., a human RSPOI or human RSPOZ). In this regard, the variable region may comprise or be derived from any type of mammal that can be induced to mount a humoral response and generate globulins against the desired tumor associated antigen. As such, the variable region of the modified antibodies can be, for example, of human, murine, non—human primate (e.g. cynomolgus monkeys, macaques, etc.) or rabbit . In some embodiments, both the variable and constant regions of the d immunoglobulins are human. In other embodiments, the variable regions of compatible antibodies ly derived from a non-human source) can be engineered or specifically ed to improve the binding properties or reduce the immunogenicity of the molecule. In this respect, variable regions useful in the present invention can be humanized or otherwise altered h the inclusion of imported amino acid sequences.

In certain embodiments, the variable domains in both the heavy and light chains are d by at least partial replacement of one or more CDRs and, if necessary, by partial framework region replacement and sequence modification and/or alteration.

Although the CDRs may be derived from an antibody of the same class or even subclass as the antibody from which the framework regions are derived, it is envisaged that the CDRS will be derived from an antibody of different class and preferably from an antibody from a different species. It may not be necessary to e all of the CDRs with all of the CDRs from the donor variable region to transfer the antigen binding capacity of one variable domain to another. Rather, it may only be necessary to transfer those residues that are necessary to maintain the activity of the antigen-binding site. Given the explanations set forth in U.S. Patent Nos. 5,585,089, 761 and 5,693,762, it will be well within the competence of those skilled in the art, either by carrying out routine experimentation or by trial and error testing to obtain a functional antibody with d immunogenicity.

Alterations to the variable region notwithstanding, those skilled in the art will appreciate that the modified antibodies of this invention will comprise antibodies (e.g., full-length antibodies or immunoreactive fragments f) in which at least a fraction of one or more of the constant region domains has been deleted or otherwise altered so as to provide d biochemical characteristics such as increased tumor localization or increased serum half-life when compared with an antibody of approximately the same immunogenicity comprising a native or unaltered constant region. In some embodiments, the constant region of the ed antibodies will comprise a human constant region.

Modifications to the nt region compatible with this invention comprise additions, deletions or tutions of one or more amino acids in one or more domains. The d antibodies disclosed herein may comprise alterations or modifications to one or more of the three heavy chain constant domains (CH1, CH2 or CH3) and/or to the light chain constant domain (CL). In some embodiments, one or more domains are partially or entirely deleted from the constant regions of the modified antibodies. In some embodiments, the modified antibodies will se domain deleted constructs or variants wherein the entire CH2 domain has been removed (ACH2 constructs). In some embodiments, the omitted constant region domain is replaced by a short amino acid spacer (e.g., 10 amino acid residues) that provides some of the molecular flexibility typically imparted by the absent nt region.

In some embodiments, the modified antibodies are engineered to fuse the CH3 domain directly to the hinge region of the antibody. In other ments, a peptide spacer is inserted between the hinge region and the modified CH2 and/or CH3 domains.

For example, constructs may be expressed wherein the CH2 domain has been deleted and the remaining CH3 domain (modified or unmodified) is joined to the hinge region with a -20 amino acid spacer. Such a spacer may be added to ensure that the regulatory elements of the constant domain remain free and accessible or that the hinge region remains flexible. However, it should be noted that amino acid spacers may, in some cases, prove to be immunogenic and elicit an unwanted immune response against the construct. Accordingly, in certain embodiments, any spacer added to the construct wéll be relatively non-immunogenic so as to maintain the desired biological qualities of the d antibodies.

In some embodiments, the modified antibodies may have only a partial deletion of a constant domain or substitution of a few or even a single amino acid. For example, the mutation of a single amino acid in selected areas of the CH2 domain may be enough to ntially reduce Fc g and y increase cancer cell localization and/or tumor penetration. Similarly, it may be desirable to simply delete the part of one or more constant region domains that control a specific effector function (e.g. complement Clq binding) to be modulated. Such partial deletions of the constant regions may improve ed characteristics of the antibody (serum half-life) while leaving other desirable functions associated with the subject nt region domain intact. Moreover, as alluded to above, the nt regions of the sed dies may be modified through the mutation or substitution of one or more amino acids that enhances the profile of the resulting construct. In this t it may be possible to disrupt the activity provided by a conserved binding site (e.g., Fc binding) while substantially maintaining the configuration and immunogenic profile of the d antibody. In certain embodiments, the d antibodies comprise the addition of one or more amino acids to the constant region to e desirable characteristics such as decreasing or increasing effector fianction or provide for more xin or carbohydrate attachment sites.

It is known in the art that the constant region mediates several effector functions.

For example, binding of the C1 component of complement to the Fc region of IgG or IgM antibodies (bound to antigen) activates the complement system. Activation of complement is ant in the opsonization and lysis of cell pathogens. The activation of complement also stimulates the atory response and can also be involved in mune hypersensitivity. In addition, the Fc region. of an antibody can bind a cell expressing a Fc receptor (FcR). There are a number of Fc receptors which are specific for different s of antibody, including IgG (gamma receptors), lgE (epsilon receptors), IgA (alpha receptors) and IgM (mu receptors). g of antibody to Fc receptors on cell surfaces triggers a number of important and diverse biological responses including engulfment and destruction of antibody-coated particles, clearance of immune complexes, lysis of antibody-coated target cells by killer cells (called antibody-dependent cell cytotoxicity or ADCC), release of inflammatory ors, placental transfer, and control of immunoglobulin production.

In certain embodiments, the RSPO-binding antibodies provide for altered effector functions that, in turn, affect the biological profile of the administered antibody. For example, in some embodiments, the deletion or vation (through ‘point mutations or other means) of a constant region domain may reduce Fc receptor g of the circulating modified antibody (e.g., anti—RSPOI antibody) thereby increasing cancer cell localization and/or tumor penetration. In other embodiments, the constant region modifications increase or reduce the serum half-life of the antibody. In some embodiments, the constant region is modified to eliminate disulfide linkages or oligosaccharide moieties. Modifications to the constant region in accordance with this invention may easily be made using well known biochemical or molecular engineering techniques well within the purview ofthe skilled artisan.

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

The present invention further embraces variants and equivalents which are substantially homologous to the chimeric, zed, and human antibodies, or antibody fragments f, set forth . These can contain, for example, conservative substitution ons, i.e. the substitution of one or more amino acids by similar amino acids. For example, conservative substitution refers to the substitution of an amino acid with another within the same general class such as, for example, one acidic amino acid with r acidic amino acid, one basic amino acid with another basic amino acid or one neutral amino acid by another neutral amino acid. What is intended by a conservative amino acid substitution is well known in the art and described herein.

Thus, the present invention es methods for producing an antibody that binds at least one RSPO protein. In some embodiments, the method for producing an antibody that binds at least one RSPO protein comprises using hybridoma techniques. In some embodiments, a method for producing an antibody that binds human RSPOl is provided.

In some embodiments, the method comprises using amino acids 31-263 of human RSPOl. In some embodiments, the method comprises using amino acids 31-263 of SEQ ID N021. In some embodiments, a method for producing an antibody that binds human RSP02 is provided. in some embodiments, the method comprises using amino acids 22- 205 of human RSP02. In some embodiments, the method comprises using amino acids 22-205 of SEQ ID N022. In some embodiments, a method for producing an antibody that binds human RSPO3 is provided. In some embodiments, the method comprises using amino acids 22-272 of human RSPO3. In some embodiments, the method comprises using amino acids 22-272 of SEQ ID NO:3. In some embodiments, the method of generating an antibody that binds at least one human RSPO protein comprises ing a human phage y. The present invention further provides methods of identifying an antibody that binds at least one RSPO protein. In some embodiments, the antibody is identified by screening by FACS for g to a RSPO protein or a portion thereof. In some embodiments, the antibody is identified by screening using ELISA for binding to a RSPO protein. In some embodiments, the antibody is identified by screening by FACS for blocking of binding of a RSPO protein to a human LGR protein. In some embodiments, the antibody is fied by screening for inhibition or blocking of B- catenin signaling. {$188} In some embodiments, a method of generating an antibody to human RSPOl protein comprises immunizing a mammal with a polypeptide comprising amino acids 31- 263 of human RSPOl. In some embodiments, a method of generating an dy to human RSPOl n comprises zing a mammal with a ptide comprising at least a portion of amino acids 21-263 of human RSPOl. In some embodiments, the method further comprises isolating antibodies or dy-producing cells from the mammal. In some embodiments, a method of generating a monoclonal antibody which binds RSPOl protein comprises: (a) immunizing a mammal With a polypeptide comprising at least a portion of amino acids 21-263 of human RSPOl; (b) isolating antibody producing cells from the immunized mammal; (c) fusing the antibody-producing cells with cells of a myeloma cell line to form hybridoma cells. In some embodiments, the method further comprises (d) selecting a hybridoma cell expressing an antibody that binds RSPOl n. In some embodiments, the at least a portion of amino acids 21-263 of human RSPOl is selected from the group consisting of SEQ ID N0s:5-9. In some embodiments, the at least a portion of amino acids 21-263 of human RSPOl is SEQ ID N029. In some embodiments, the at least a n of amino acids 21-263 of human RSPOl is SEQ ID N0:6 or SEQ ID N0:7. In some embodiments, the at least a portion of amino acids 21—263 of human RSPOl is SEQ ID N0:6 and SEQ ID N0:7. In certain embodiments, the mammal is a mouse. In some embodiments, the antibody is selected using a polypeptide comprising at least a portion of amino acid 21-263 of human RSPOl.

In certain embodiments, the ptide used for selection comprising at least a portion of amino acids 21-263 of human RSPOl is selected from the group consisting of SEQ ID NOS:5-9. In some embodiments, the antibody binds RSPOl and at least one other RSPO protein. In certain embodiments, the at least one other RSPO protein is selected from the group consisting of RSPOZ, RSP03 and RSP04. In certain embodiments, the antibody binds RSPOl and RSPOZ. In n embodiments, the antibody binds RSPOI and RSP03. In certain embodiments, the antibody binds RSPOl and RSP04. In certain embodiments, the antibody binds RSPOI, RSP02, and RSP03. In certain embodiments, the antibody binds RSPOl, RSP02, and RSPO4. In certain embodiments, the antibody binds RSPOl, RSPO3, and RSPO4. In some embodiments, the antibody binds both human RSPOl and mouse RSPOl.

In some embodiments, a method of generating an antibody to human RSP02 protein ses immunizing a mammal with a polypeptide comprising amino acids 22- 205 of human RSPO2. In some embodiments, a method of generating an antibody to human RSP02 protein comprises zing a mammal with a polypeptide comprising at least a portion of amino acids 22-243 of human RSPO2. In some embodiments, the method further comprises isolating antibodies or antibody-producing cells from the mammal. In some embodiments, a method of generating a monoclonal antibody which binds RSP02 protein ses: (a) zing a mammal with a polypeptide comprising at least a portion of amino acids 22-243 of human RSP02; (b) isolating dy producing cells from the zed mammal; (c) fusing the antibody-producing cells with cells of a myeloma cell line to form hybridoma cells. In some embodiments, the method further comprises (d) selecting a hybridoma cell expressing an antibody that binds RSP02 protein. In some ments, the at least a portion of amino acids 22-243 of human RSP02 is selected from the group consisting of SEQ ID NOsz44-47. In some embodiments, the at least a portion of amino acids 22-243 of human RSP02 is SEQ ID NO:44. In some embodiments, the at least a portion of amino acids 22-243 of human RSP02 is SEQ ID NO:45 or SEQ ID NO:46. In some embodiments, the at least a portion of amino acids 22—243 of human RSP02 is SEQ ID NO:45 and SEQ ID NO:46. In certain embodiments, the mammal is a mouse. In some embodiments, the antibody is selected using a polypeptide comprising at least a portion of amino acid 22-243 of human RSPO2. In certain embodiments, the polypeptide used for selection comprising at least a portion of amino acids 22-243 of human RSP02 is selected from the group consisting of SEQ ID NOs244-47. In some embodiments, the antibody binds RS i302 and at least one other RSPO protein. In certain embodiments, the at least one other RSPO protein is selected from the group consisting of RSPOI, RSPO3 and RSPO4. In certain embodiments, the antibody binds RSP02 and RSPOl. In certain embodiments, the antibody binds RSP02 and RSPO3. In certain embodiments, the antibody binds RSP02 and RSPO4. In n embodiments, the antibody binds RSP02, RSPOl, and RSPO3.

In certain embodiments, the dy binds RSP02, RSPO3, and RSPO4. In certain embodiments, the antibody binds RSP02, RSPOl, and RSPO4. In some ments, the antibody binds both human RSP02 and mouse RSP02.

In some embodiments, a method of generating an antibody to human RSPO3 protein comprises immunizing a mammal with a polypeptide comprising amino acids 22- 272 of human RSPO3. In some embodiments, a method of generating an antibody to human RSPO3 protein comprises immunizing a mammal with a polypeptide comprising at least a n of amino acids 22-272 of human RSPO3. In some embodiments, the method further ses isolating antibodies or antibody-producing cells from the mammal. In some embodiments, a method of generating a monoclonal antibody which binds RSPO3 protein comprises: (a) immunizing a mammal with a ptide comprising at least a portion of amino acids 22-272 of human RSPO3; (b) isolating antibody producing cells from the immunized mammal; (c) fasing the antibody-producing cells with cells of a myeloma cell line to form hybridoma cells. In some embodiments, the method further comprises (d) selecting a hybridoma cell expressing an antibody that binds RSPO3 protein. In some embodiments, the at least a portion of amino acids 22-272 of human RSPO3 is selected from the group consisting of SEQ ID NOsz48-51. In some embodiments, the at least a portion of amino acids 22-272 of human RSPO3 is SEQ ID NO:48. In some embodiments, the at least a portion of amino acids 22-272 of human RSPO3 is SEQ ID NO:49 or SEQ ID NO:50. In some embodiments, the at least a portion of amino acids 22-272 of human RSPO3 is SEQ ID NO:49 and SEQ ID NO:50. In n ments, the mammal is a mouse. In some embodiments, the antibody is selected using a polypeptide comprising at least a portion of amino acid 22-272 of human RSPO3. In certain ments, the polypeptide used for ion comprising at least a portion of amino acids 22-272 of human RSPO3 is selected from the group consisting of SEQ ID NOs:48-51. In some embodiments, the antibody binds RSPO3 and at least one other RSPO protein. In certain embodiments, the at least one other RSPO protein is selected from the group cOnsisting of RSP02, RSPO4 and RSPOl. In certain embodiments, the antibody binds RSPO3 and RSPOl. In certain embodiments, the antibody binds RSPO3 and RSP02. In certain embodiments, the antibody binds RSPO3 and RSPO4. In certain ments, the antibody binds RSPO3, RSPOI, and RSP02.

In certain embodiments, the antibody binds RSPO3, RSPOl, and RSPO4. In certain embodiments, the antibody binds RSPO3, RSPOZ, and RSPO4. In some embodiments, the antibody binds both human RSPO3 and mouse RSPO3.

In some embodiments, the antibody generated by the methods described herein is a RSPO antagonist. In some embodiments, the antibody generated by the methods described herein inhibits B-catenin signaling.

In some embodiments, a method of producing an antibody to at least one human RSPO protein comprises identifying an antibody using a ne-bound heterodimeric molecule comprising a single antigen-binding site. In some miting embodiments, the antibody is identified using s and polypeptides described in International Publication W0 201 l/100566, which is incorporated by reference herein in its entirety.

In some embodiments, a method of producing an antibody to at least one human RSPO protein comprises screening an antibody-expressing library for antibodies that bind a human RSPO protein. In some embodiments, the dy-expressing y is a phage library. In some embodiments, the screening comprises panning. In some embodiments, the antibody-expressing library (e.g., phage library) is screened using at least a portion of amino acids 21-263 of human RSPO]. In some embodiments, antibodies identified in the first screening, are screened again using a different RSPO protein thereby identifying an antibody that binds RSPOl and a second RSPO protein. In certain embodiments, the polypeptide used for screening comprises at least a portion of amino acids 21-263 of human RSPOI selected from the group consisting of SEQ ID NOsz5-9. In some ments, the antibody identified in the screening binds RSPOI and at least one other RSPO protein. In certain embodiments, the at least one other RSPO protein is selected from the group consisting of RSPOZ, RSPO3 and RSPO4. In certain embodiments, the antibody identified in the screening binds RSPOl and RSPOZ. In certain embodiments, the dy identified in the screening binds RSPOI and RSPO3. In certain embodiments, the antibody identified in the screening binds RSPOl and RSPO4. In some embodiments, the antibody identified in the screening binds both human RSPOl and mouse RSPOl. In some embodiments, the dy identified in the screening is a RSPOl nist. In some embodiments, the antibody identified if: the ing inhibits B-catenin signaling d by RSPOl. In some embodiments, the dy— expressing library (e.g., phage library) is screened using at least a portion of amino acids 22-205 of human RSPOZ. In some ments, antibodies identified in the first screening, are screened again using a different RSPO n thereby identifying an antibody that binds RSP02 and a second RSPO protein. In certain ments, the polypeptide used for screening comprises at least a portion of amino acids 22-205 of human RSP02 selected from the group consisting of SEQ ID NOsz44-47. In some embodiments, the antibody identified in the screening binds RSP02 and at least one other RSPO protein. In certain embodiments, the at least one other RSPO protein. is selected from the group consisting of RSPOI, RSPO3 and RSPO4. In certain embodiments, the dy identified in the screening binds RSP02 and RSPO3. In certain ments, the antibody identified in the screening binds RSP02 and RSPO4. In certain embodiments, the dy identified in the screening binds RSP02 and RSPOl. In some embodiments, the dy identified in the screening binds both human RSP02 and mouse RSP02. In some embodiments, the antibody identified in the ing is a RSP02 antagonist. In some embodiments, the antibody identified in the screening inhibits B-catenin signaling induced by RSP02.

In certain embodiments, the antibodies described herein are isolated. In certain embodiments, the antibodies described herein are substantially pure.

In some embodiments of the present: ion, the RSPO-binding agents are ptides. The polypeptides can be recombinant polypeptides, natural polypeptides, or synthetic polypeptides comprising an antibody, or fragment thereof, that bind at least one human RSPO protein. It will be recognized in the art that some amino acid sequences of the invention can be varied without significant effect of the ure or function of the protein. Thus, the invention further includes variations of the polypeptides which show substantial activity or which include regions of an antibody, or fragment thereof, against a human RSPO protein. In some embodiments, amino acid sequence variations of inding polypeptides include deletions, insertions, inversions, s, and/or other types of substitutions.

The polypeptides, analogs and variants thereof, can be further modified to contain additional chemical moieties not normally part of the polypeptide. The derivatized moieties can improve the solubility, the biological half-life, and/or absorption of the polypeptide. The moieties can also reduce or eliminate any undesirable side effects of the ptides and variants. An ew for chemical moieties can be found in Remington: The Science and Practice ofPharmacy, 215’ Edition, 2005, University of the Sciences, Philadelphia, PA.

The isolated polypeptides described herein can be produced by any suitable method known in the art. Such s range from direct protein synthesis methods to constructing a DNA sequence encoding polypeptide ces and expressing those sequences in a suitable host. In some embodiments, a DNA sequence is constructed using recombinant logy by isolating or synthesizing a DNA sequence encoding a Wild- type protein of st. Optionally, the sequence can be mutagenized by site-specific nesis to provide functional analogs thereof. See, e.g., Zoeller et al., 1984, PNAS, 81 :5662—5066 and US. Patent No. 4,588,585.

In some embodiments, a DNA sequence encoding a polypeptide of interest may be constructed by chemical sis using an oligonucleotide synthesizer.

Oligonucleotides can be ed based on the amino acid sequence of the desired polypeptide and selecting those codons that are favored in the host cell in which the recombinant polypeptide of interest will be produced. Standard methods can be applied to synthesize a polynucleotide ce encoding an isolated polypeptide of interest. For e, a complete amino acid sequence can be used to construct a ranslated gene. Further, a DNA oligomer containing a nucleotide sequence coding for the particular isolated polypeptide can be synthesized. For example, several small ucleotides coding for portions of the desired polypeptide can be synthesized and then ligated. The individual oligonucleotides typically contain 5' or 3' overhangs for complementary assembly.

Once led (by synthesis, site-directed mutagenesis, or another method), the cleotide sequences encoding a particular polypeptide of interest can be ed into an expression vector and operatively linked to an expression control sequence appropriate for expression of the protein in a desired host. Proper assembly can be confirmed by nucleotide sequencing, restriction enzyme mapping, and/or sion of a 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, the gene must be operatively linked to transcriptional and transiational expression control sequences that are fanctional in the chosen expression host.

In certain embodiments, recombinant expression vectors are used to amplify and express DNA encoding antibodies, or fragments thereof, t a human RSPO protein.

For example, recombinant expression vectors can be replicable DNA constructs which have synthetic or erived DNA fragments encoding a ptide chain of a inding agent, an anti-RSPO antibody, or fragment thereof, ively linked to suitable transcriptional and/or ational regulatory elements derived from mammalian, ial, viral or insect genes. A transcriptional unit generally comprises an assembly of (1) a genetic element or elements having a regulatory role in gene expression, for example, transcriptional promoters or enhancers, (2) a ural or coding sequence which is transcribed into mRNA and translated into protein, and (3) appropriate transcription and translation initiation and termination sequences. Regulatory elements can include an operator sequence to l transcription. The ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants can additionally be incorporated. DNA regions are tively linked” when they are functionally related to each other. For example, DNA for a signal peptide (secretory ) is operatively linked to DNA for a polypeptide if it is expressed as a precursor which participates in the secretion of the polypeptide; a promoter is operatively linked to a coding sequence if it controls the transcréption of the ce; or a ribosome binding site is operatively linked to a coding sequence if it is positioned so as to permit translation. In some embodiments, structural elements intended for use in yeast expression systems include a leader sequence enabling extracellular secretion of translated protein by a host cell. In other embodiments, where recombinant protein is expressed Without a leader or transport sequence, it can include an N-terminal methionine residue. This residue can optionally be uently cleaved from the expressed recombinant protein to provide a final product.

The choice of an expression control sequence and an expression vector depends upon the choice of host. A wide variety of expression host/vector ations can be employed. Useful expression vectors for eukaryotic hosts include, for example, vectors comprising expression control sequences from SV40, bovine papilloma virus, adenovirus, and cytomegalovirus. Useful expression vectors for ial hosts include known ial plasmids, such as plasmids from E. coli, including pCRl, pBR322, pMB9 and their derivatives, and wider host range plasmids, such as M13 and other filamentous single-stranded DNA phages.

Suitable host cells for expression of a RSPO-binding polypeptide or dy (or a RSPO protein to use as an antigen) include prokaryotes, yeast cells, insect cells, or higher eukaryotic cells under the control of appropriate promoters. Prokaryotes include gram- negative or positive organisms, for e E. coli or Bacillus. Higher eukaryotic cells include established cell lines of mammalian origin as described below. Cell-free translation systems may also be employed. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts are described by Pouwels et a1. (1985, Cloning Vectors: A Laboratory Manual, Elsevier, New York, NY).

Additional information regarding methods of protein production, ing antibody production, can be found, e.g., in US. Patent Publication No. 2008/0187954, US. Patent Nos. 746 and 6,660,501, and International Patent Publication No. WO 04009823.

Various mammalian or insect cell culture systems are used to express recombinant polypeptides. Expression of recombinant proteins in mammalian cells can be preferred because such proteins are generally correctly folded, appropriately modified, and tely functional. Examples of suitable mammalian host cell lines include COS-7 (monkey kidney-derived), L-929 (murine fibroblast-derived), C127 (murine mammary tumor-derived), 3T3 (murine fibroblast-derived), CHO (Chinese hamster ovary—derived), HeLa (human cervical cancer-derived), BHK (hamster kidney fibroblast-derived), and HEK-293 (human embryonic kidney-derived) cell lines and variants thereof. Mammalian expression s can comprise anscribed elements such as an origin of replication, a suitable er and enhancer linked to the gene to be expressed, and other ' or 3' g non-transcribed ces, and 5' or 3' non-translated sequences, such as necessary me binding sites, a polyadenylation site, splice donor- and acceptor sites, and riptional termination sequences. Baculovirus systems for production of heterologous proteins in insect cells are well-known to those of skill in the art (see, e.g., Luckow and Summers, 1988, Bio/Technology, 6:47).

Thus, the present invention provides cells comprising the RSPO-binding agents described herein. In some embodiments, the cells produce the RSPO-binding agents described herein. In certain ments, the cells produce an antibody. In n embodiments, the cells produce dy 89M5. In certain embodiments, the cells produce dy h89M5-H2L2. In n embodiments, the cells produce antibody 130M23. In certain embodiments, the cells produce dy h130M23-H1L2. In certain embodiments, the cells produce antibody h130M23-H1L6. In some embodiments, the cell is a hybridoma cell line having ATCC deposit number PTA-11970. In some embodiments, the cell is a hybridoma cell line having ATCC deposit number PTA-12021.

The proteins produced by a ormed host can be purified according to any suitable method. rd methods include chromatography (e. g., ion exchange, affinity, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for protein purification. Affinity tags such as hexa—histidine, maltose binding domain, influenza coat ce, and glutathione-S-transferase can be attached to the protein to allow easy purification by passage over an appropriate affinity column. Isolated proteins can also be physically characterized using such techniques as proteolysis, mass spectrometry (MS), nuclear magnetic resonance (NMR), high performance liquid chromatography (HPLC), and x-ray crystallography.

In some embodiments, supematants from sion systems which secrete recombinant protein into culture media can be first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. Following the concentration step, the concentrate can be applied to a suitable purification . In some ments, an anion exchange resin can be employed, for example, a matrix or substrate having t diethylaminoethyl (DEAE) groups. The matrices can be acrylamide, agarose, dextran, cellulose, or other types commonly employed in n purification. In some embodiments, a cation exchange step can be employed. le cation exchangers include various insoluble matrices comprising sulfopropyl or carboxymethyl groups. In some embodiments, a hydroxyapatite media can be employed, ing but not d to, ceramic hydroxyapatite (CHT). In certain embodiments, one or more reverse-phase HPLC steps employing hydrophobic RP-HPLC media, e.g., silica gel having pendant methyl or other aliphatic groups, can be employed to further purify a RSPO-binding agent. Some or all. of the foregoing purification steps, in various combinations, can also be employed to provide a homogeneous recombinant n.

In some embodiments, recombinant protein produced. in bacterial culture can be isolated, for e, by initial extraction from cell pellets, followed by one or more —76- tration, salting—out, aqueous ion exchange, or size exclusion chromatography steps.

HPLC can be employed for final purification steps. Microbial cells employed in expression of a recombinant protein can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents.

Methods known in the art for purifying antibodies and other proteins also include, for example, those described in US. Patent Publication No. 2008/0312425, 2008/0177048, and 2009/0187005.

In certain embodiments, the RSPO-binding agent is a polypeptide that is not an antibody. A variety of methods for identifying and producing non-antibody ptides that bind with high affinity to a n target are known in the art. See, e.g., Skerra, 2007, Curr. Opin. Biotechnol, 18:295-304; Hosse et al., 2006, Protein Science, 15:14-27; Gill et al., 2006, Curr. Opin. Biotechnol., 172653-658; Nygren, 2008, FEBS 1, 2752668- 76; and Skerra, 2008, FEBS J, 275:2677-83. In certain embodiments, phage display technology may be used to produce and/or identify a RSPO—binding ptide. In certain embodiments, the polypeptide comprises a n scaffold of a type selected from the group consisting of protein A, protein G, a lipocalin, a fibronectin domain, an n consensus repeat domain, and doxin.

In n embodiments, the RSPO-binding agents or antibodies can be used in any one of a number of conjugated (i.e. an immunoconjugate or radioconjugate) or non- conjugated forms. In certain embodiments, the antibodies can be used in a non- conjugated form to harness the subject’s natural e mechanisms ing complement-dependent cytotoxicity and antibody dependent cellular toxicity to eliminate the malignant or cancer cells.

In some ments, the RSPO-binding agent (e.g., an antibody or polypeptide) is conjugated to a cytotoxic agent. In some ments, the cytotoxic agent is a chemotherapeutic agent including, but not limited to, methotrexate, adriamicin, doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other alating . In some embodiments, the cytotoxic agent is an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof, including, but not limited to, diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), Momordica charantia tor, , crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the hecenes. In some embodiments, the cytotoxic agent is a radioisotope to produce a radioconjugate or a radioconjugated antibody. A variety of radionuclides are available for the production of radioconjugated antibodies including, but not limited to, 90Y, '251, 131I, 123I, 111In, 13 1In, 105Rh, 153 Sm, 67Cu, 67Ga, 166Ho, 177Lu, 186Re, [88Re and 212Bi. Conjugates of an antibody and one or more small molecule toxins, such as a calicheamicin, maytansinoids, a trichothene, and CC1065, and the derivatives of these toxins that have toxin activity, can also be used. ates of an antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N—succinimidyl(2-pyridyidithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of sters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl te), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis- diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as e 2,6-diisocyanate), and bis-active fluorine compounds (such as l,5-difluoro-2,4-dinitroEenzene).

III. cleotides In certain embodiments, the invention encompasses polynucleotides comprising polynucleotides that encode a polypeptide that cally binds at least one human RSPO or a fragment of such a polypeptide. The term ucleotides that encode a polypeptide” encompasses a polynucleotide which includes only coding sequences for the ptide as well as a polynucleotide which includes additional coding and/or non- coding sequences. For example, the invention provides a polynucleotide comprising a polynucleotide sequence that encodes an antibody to a human RSPO protein or encodes a fragment of such an antibody. The polynucleotides of the invention can be in the form of RNA or in the form of DNA. DNA includes cDNA, genomic DNA, and synthetic DNA; and can be double-stranded or -stranded, and if single stranded can be the coding strand or non-coding (anti—sense) strand.

In certain embodiments, the polynucleotide comprises a polynucleotide encoding a polypeptide sing a sequence selected from the group consisting of SEQ ID NO:10, SEQ ID NO:11, SEQ ID N021, SEQ ID NO:22, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:68, and SEQ ID NO:69. In certain ments, the polynucleotide comprises a polynucleotide encoding a polypeptide comprising a sequence selected from the group consisting of SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:4l, SEQ ID NO:42, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:74, and SEQ ID NO:76. In some embodiments, the polynucleotide comprises a polynucleotide sequence ed from the group consisting of SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56 and SEQ ID NO:58. In some embodiments, the polynucleotide ses a polynucleotide sequence selected from the group consisting of SEQ ID N0235, SEQ ID NO:36, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:72, and SEQ ID NO:75.

In some embodiments, a d comprises a polynucleotide comprising SEQ ID NO:52. In some embodiments, a plasmid comprises a polynucleotide comprising polynucleotide ce SEQ ID NO:56. In some embodiments, a plasmid ses a polynucleotide comprising polynucleotide sequence SEQ ID NO:60. In some embodiments, a plasmid comprises a polynucleotide comprising polynucleotide ce SEQ ID NO:64. In some embodiments, a plasmid ses a polynucleotide comprising cleotide sequence SEQ ID NO:72. In some ments, a plasmid comprises a polynucleotide that encodes an amino acid sequence comprising SEQ ID NO:68 and/or SEQ ID NO:69. In some embodiments, a plasmid comprises a polynucleotide that encodes an amino acid sequence comprising SEQ ID NO:70 and/or SEQ ID NO:71. In some embodiments, a plasmid comprises a polynucleotide that encodes an amino acid sequence comprising SEQ ID NO:70 and/or SEQ ID NO:74.

In certain embodiments, the polynucleotide comprises a polynucleotide having a nucleotide sequence at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, and in some embodiments, at least 96%, 97%, 98% or 99% identical to a polynucleotide comprising a sequence selected from the group consisting of SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, and SEQ ID NO:58. In certain embodiments, the polynucleotide comprises a polynucleotide having a nucleotide sequence at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, and in some embodiments, at least 96%, 97%, 98% or 99% identical to a polynucleotide comprising a sequence selected from the group consisting of SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:72, and SEQ ID NO:75. Also provided is a polynucleotide that comprises a polynucleotide that hybridizes to SEQ ID NO:19, SEQ ID NO:20, SEQ ID N023, SEQ ID N0224, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, and SEQ ID NO:58, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:72, or SEQ ID NO:75. In certain ments, the hybridization is under conditions of high stringency.

In some embodiments, an dy is encoded by a polynucleotide comprising SEQ ID N023 and SEQ ID NO:24. In some embodiments, an antibody is encoded by a polynucleotide comprising SEQ ID NO:52 and SEQ ID NO:56. In some embodiments, an antibody is encoded by a polynucleotide comprising SEQ ID NO:39 and SEQ ID NO:40. In some embodiments, an antibody is encoded by a cleotide comprising SEQ ID NO:6O and SEQ ID NO:64. In some embodiments, an antibody is encoded by a polynucleotide comprising SEQ ID NO:60 and SEQ ID NO:72.

In certain embodiments, the polynucleotides comprise the coding sequence for the mature polypeptide fused in the same reading frame to a polynucleotide which aids, for example, in expression and secretion of a polypeptide from a host cell (e.g., a leader sequence or signal sequence which functions as a secretory sequence for lling transport of a polypeptide from the cell). The polypeptide having a leader sequence is a preprotein and can have the leader sequence cleaved by the host cell to form the mature form of the polypeptide. The polynucleotides can also encode for a proprotein which is the mature protein plus additional 5' amino acid es. A mature protein having a prosequence is a proprotein and is an ve form of the protein. Once the prosequence is cleaved an active mature protein s.

In certain embodiments, the polynucleotides comprise the coding sequence for the mature polypeptide fused in the same reading frame to a marker sequence that allows, for example, for ation of the encoded polypeptide. For example, the marker sequence can be a istidine tag supplied by a pQE-9 vector to provide for purification of the mature ptide fused to the marker in the case of a bacterial host, or the marker sequence can be a hemagglutinin (HA) tag derived from the influenza lutinin n when a mammalian host (e.g., COS-7 cells) is used. In some embodiments, the marker sequence is a FLAG-tag, a peptide of sequence DYKDDDDK (SEQ ID NO:18) which can be used in ction with other y tags.

The present ion further relates to variants of the hereinabove described polynucleotides encoding, for example, fragments, analogs, and/or derivatives.

In n embodiments, the present invention provides polynucleotides comprising polynucleotides having a nucleotide sequence at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, and in some embodiments, at least about 96%, 97%, 98% or 99% identical to a polynucleotide encoding a polypeptide comprising a RSPO-binding agent (e. g., an antibody), or fragment thereof, described herein.

As used herein, the phrase a polynucleotide having a nucleotide sequence at least, for example, 95% “identical” to a reference nucleotide sequence is intended to mean that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence can include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a cleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence can be deleted or substituted with r nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence can be inserted into the nce sequence. These mutations of the reference sequence can occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.

The polynucleotide variants can contain alterations in the coding regions, non- coding regions, or both. In some embodiments, the polynucleotide variants n tions which produce silent substitutions, additions, or deletions, but do not alter the ties or activities of the encoded polypeptide. In some ments, nucleotide ts are produced by silent substitutions due to the racy of the genetic code. In some embodiments, nucleotide variants comprise nucleotide sequences which result in expression differences (e.g., increased or decreased expression), even though the amino acid sequence is not changed. Polynucleotide variants can be produced for a variety of reasons, for example, to optimize codon expression for a particular host (i.e., change codons in the human mRNA to those preferred by a bacterial host such as E. coli).

In certain embodiments, the polynucleotides are isolated. In certain ments, the polynucleotides are substantially pure.

Vectors and cells comprising the polynucleotides described herein are also provided. In some embodiments, an expression vector comprises a polynucleotide molecule. In some ments, a host cell comprises an expression vector comprising the polynucleotide molecule. In some embodiments, a host cell comprises a polynucleotide molecule.

IV. s of use and pharmaceutical itions [0225} The RSPO-binding agents (including polypeptides and dies) of the invention are useful in a variety of applications ing, but not limited to, therapeutic treatment methods, such as the treatment of cancer. In certain embodiments, the agents are useful for inhibiting nin ing, inhibiting tumor growth, inducing differentiation, reducing tumor volume, reducing the frequency of Cancer stem cells in a tumor, and/or reducing the tumorigenicity of a tumor. The methods of use may be in vitro, ex vivo, or in vivo methods. In certain ments, a inding agent or polypeptide or antibody is an antagonist of human RSPOl. In certain embodiments, a RSPO-binding agent or polypeptide or antibody is an antagonist of human RSPOZ. In certain embodiments, a RSPO—binding agent or polypeptide or antibody is an antagonist of human RSPO3.

In certain embodiments, the RSPO-binding agents are used in the treatment of a disease associated with activation of B-catenin, increased B-catenin signaling, and/or nt nin signaling. In certain embodiments, the disease is a disease dependent upon B-catenin signaling. In certain embodiments, the disease is a disease ent upon B-catenin activation. In certain embodiments, the RSPO-binding agents are used in the ent of disorders characterized by increased levels of stem cells and/or progenitor cells. In some embodiments, the methods comprise administering a therapeutically effective amount of a RSPOl—binding agent (e.g., antibody) to a subject.

In some embodiments, the methods comprise administering a therapeutically effective amount of a RSPOZ-binding agent (e.g., antibody) to a subject. In some embodiments, the methods comprise administering a therapeutically effective amount of a RSPO3- binding agent (e.g., antibody) to a subject. In some embodiments, the subject is human.

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

In some embodiments, the method of inhibiting growth of a tumor ses contacting the tumor or tumor cells with a RSPO-binding agent (e.g., antibody) in vivo.

In certain embodiments, contacting a tumor or tumor cell with a inding agent is undertaken in an animal model. For example, a RSPO-binding agent may be administered to immunocompromised mice (e.g. NOD/SCID mice) which have xenografts. In some embodiments, cancer cells or cancer stem cells are ed from a t sample such as, for example, a tissue biopsy, pleural effusion, or blood sample and injected into immunocompromised mice that are then administered a RSPO-binding agent to inhibit tumor cell growth. In some embodiments, a RSPOl-binding agent is administered to the animal. In some embodiments, a RSPO2-binding agent is administered to the . In some embodiments, a RSPO3-binding agent is administered to the . In some embodiments, the RSPO-binding agent is stered at the same time or shortly after introduction of tumorigenic cells into the animal to prevent tumor growth entative model”). In some embodiments, the RSPO-binding agent is administered as a therapeutic after tumors have grown to a specified size (“therapeutic model”). In some embodiments, the RSPO-binding agent is an antibody. In some embodiments, the RSPO-binding agent is an anti-RSPOl antibody.

In some embodiments, the anti-RSPOI dy is antibody 89M5. In some embodiments, the anti-RSPOI antibody is antibody h89M5-H2L2. In some embodiments, the inding agent is an anti-RSPOZ antibody. In some embodiments, the anti-RSP02 antibody is antibody 130M23. In some embodiments, the SPOZ antibody is antibody h130M23-H1L2. In some embodiments, the anti- RSP02 antibody is antibody h130M23-H1L6. In some embodiments, the RSPO—binding agent is an anti-RSPO3 antibody.

In certain embodiments, the method of inhibiting growth of a tumor comprises administering to a subject a eutically effective amount of a RSPO-binding agent which comprises a heavy chain CDR1 comprising TGYTMH (SEQ ID NO:12), a heavy chain CDR2 comprising GINPNNGGTTYNQNFKG (SEQ ID , and a heavy chain CDR3 comprising KEFSDGYYFFAY (SEQ ID NO:14), and/or a light chain CDR1 comprising KASQDVIFAVA (SEQ ID NO:15), a light chain CDR2 comprising WASTRHT (SEQ ID NO:16), and a light chain CDR3 comprising QQHYSTPW (SEQ ID NO:17). In certain embodiments, the method of inhibiting growth of a tumor comprises administering to a subject a eutically effective amount of a RSPO- binding agent which comprises a heavy chain CDR1 comprising SSYAMS (SEQ ID N029), a heavy chain CDR2 comprising SISSGGSTYYPDSVKG (SEQ ID N030), and a heavy chain CDR3 comprising VYNGDYEDAMDY (SEQ ID NO:31), and/or a light chain CDR1 comprising KASQDVSSAVA (SEQ ID NO:32), a light chain CDR2 comprising WASTRHT (SEQ ID NO:33), and a light chain CDR3 sing QQHYSTP (SEQ ID NO:34).

In certain embodiments, the method of inhibiting growth of a tumor comprises administering to a t a therapeutically effective amount of a RSPO-binding agent. In certain embodiments, the subject is a human. In certain embodiments, the t has a tumor or has had a tumor which was removed. In some ments, the subject has a tumor with an elevated expression level of at least one RSPO protein (e.g., RSPOl, RSP02, or RSPO3). In some ments, the RSPO-binding agent is a RSPOl-binding agent. In some ments, the RSPOl-binding agent is an antibody. In some embodiments, the RSPOl-binding agent is antibody 89M5. In some embodiments, the anti-RSPOI antibody is antibody h89M5-H2L2. In some embodiments, the RSPO- binding agent is a RSP02-binding agent. In some embodiments, the RSP02-binding agent is an antibody. In some embodiments, the RSP02-binding agent is antibody 130M23. In some embodiments, the anti—RSP02 antibody is antibody h130M23-H1L2.

In some embodiments, the RSPO-binding agent is a RSPO3-binding agent. In some embodiments, the RSPO3-binding agent is an antibody.

Ir: certain embodiments, the tumor is a tumor in which B-catenin signaling is active. In some embodiments, the tumor is a tumor in which B—catenin ing is aberrant. In certain ments, the tumor comprises an vating mutation (e.g., a truncating mutation) in the APC tumor suppressor gene. In certain embodiments, the tumor does not comprise an inactivating mutation in the APC tumor suppressor gene. In some embodiments, the tumor comprises a wild-type APC gene. In some embodiments, the tumor does not comprise an activating mutation in the B-catenin gene. In certain embodiments, a cancer for which a subject is being treated involves such a tumor.

In certain embodiments, the tumor expresses RSPOl to which a RSPOl-binding agent or antibody binds. In n embodiments, the tumor has elevated sion levels of RSPOl or over—expresses RSPOl. In some embodiments, the tumor has a high sion level of RSPOl. In general, the phrase “a tumor has elevated expression levels of” a protein (or similar phrases) refers to expression levels of a protein in a tumor as compared to expression levels of the same protein in normal tissue of the same tissue type. However, in some embodiments, the expression levels of a protein in a tumor are “elevated” or “high” as compared to the average expression level of the protein within a group of tissue types. In some embodiments, the expression levels of a protein in a tumor are ted” or “high” as compared to the expression level of the protein in other tumors of the same tissue type or a different tissue type. In certain embodiments, the tumor expresses RSP02 to which a RSP02-binding agent or antibody binds. In certain embodiments, the tumor has elevated expression levels of RSP02 or over-expresses RSP02. In some embodiments, the tumor has a high expression level of RSP02. In certain embodiments, the tumor ses RSPO3 to which a RSPO3-binding agent or dy binds. In certain ments, the tumor has elevated expression levels of RSPO3 or over-expresses RSPO3. In some embodiments, the tumor has a high expression level of RSPO3. In certain embodiments, the tumor expresses RSPO4 to which a RSPO4-binding agent or antibody binds. In certain embodiments, the tumor has elevated sion levels of RSPO4 or over-expresses RSPO4. In some embodiments, the tumor has a high expression level of RSPO4. In some embodiments, the tumor expresses elevated levels of RSPOI, RSP02. RSPO3, and/or RSPO4 as compared to RSPO levels expressed in normal tissue. In some embodiments, the normal tissue is tissue of the same tissue type as the tumor.

In addition, the invention provides a method of ting growth of a tumor in a subject, comprising administering a therapeutically effective amount of a RSPO-binding agent to the subject. In n embodiments, the tumor comprises cancer stem cells. In certain embodiments, the frequency of cancer stem cells in the tumor is reduced by administration of the RSPO-binding agent. The invention also provides a method of reducing the frequency of cancer stem cells in a tumor, comprising contacting the tumor with an effective amount of a RSPO-binding agent (e.g., an anti-RSFO antibody). In some embodiments, a method of reducing the frequency of cancer stem cells in a tumor in a subject, comprising stering to the subject a therapeutically effective amount of a ~ inding agent (e.g., an SPO antibody) is provided. In some embodiments, the RSPO-binding agent is an antibody. In some embodiments, the RSPO-binding agent is an anti-RSPOI antibody. In some embodiments, the anti-RSPOl antibody is 89Mfa. In some embodiments, the anti—RSPOl antibody is dy h89M5-H2L2. In some embodiments, the RSPO-binding agent is an anti—RSP02 antibody. In some embodiments, the anti-RSP02 antibody is 130M23. In some embodiments, the anti- RSPOZ antibody is dy hl30M23-H1L2. In some embodiments, the anti-RSP02 antibody is antibody h130M23—H1L6. In some embodiments, the RSPO-binding agent is an anti-RSPO3 antibody.

In some embodiments, the tumor is a solid tumor. In certain embodiments, the tumor is a tumor selected from the group consisting of colorectal tumor, atic 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 an ovarian tumor. In some embodiments, the tumor is a lung tumor. In n ments, the tumor is a pancreatic tumor. In some embodiments, the tumor is a colorectal tumor that comprises an inactivating mutation in the APC gene.

In some embodiments, the tumor is a colorectal tumor that does not se an inactivating mutation in the APC gene. In some embodiments, the tumor is an ovarian tumor with an elevated expression level of RSPOl. In some embodiments, the tumor is a pancreatic tumor with an elevated expression level of RSPOZ. In some ments, the tumor is a colon tumor with an elevated expression level of RSPOZ. In some embodiments, the tumor is a lung tumor with an elevated expression level of ESPOZ. In -86— some embodiments, the tumor is a melanoma tumor with an ed expression level of RSPOZ. In some embodiments, the tumor is a breast tumor with an ed expression level of RSPOZ. In some embodiments, the tumor is a lung tumor with an elevated expression level of RSPO3. In some embodiments, the tumor is an ovarian tumor with an elevated expression level of RSPO3. In some embodiments, the tumor is a breast tumor with an elevated expression level of RSPO3. In some embodiments, the tumor is a colon tumor with an elevated expression level ofRSPO3. In some embodiments, the tumor is a breast tumor with an elevated expression level of RSPO4. In some embodiments, the tumor is a lung tumor with an elevated expression level of RSPO4. In some embodiments, the tumor is an ovarian tumor with an elevated expression level of RSPO4.

In some embodiments, the tumor is an ovarian tumor with a high expression level of RSPOl. In some embodiments, the tumor is a pancreatic tumor with a high expression level of RSPOZ. In some embodiments, the tumor is a colon tumor with a high sion level of RSPOZ. In some embodiments, the tumor is a lung tumor with a high expression level of RSPOZ. In some embodiments, the tumor is a melanoma tumor with a high expression level of RSPOZ. In some embodiments, the tumor is a breast tumor with a high expression level of RSPOZ. In some embodiments, the tumor is a lung tumor with a high expression level of RSPO3. In some embodiments, the tumor is an ovarian tumor with a high expression level of RSPO3. In some embodiments, the tumor is a breast tumor with a higl’: expression level of RSPO3. In some embodiments, the tumor is a colon tumor with a high expression level of RSPO3. In some embodiments, the tumor is a breast tumor with a high expression level of RSPO4. In some embodiments, the tumor is a lung tumor with a high expression level of RSPO4. In some embodiments, the tumor is an ovarian tumor with a high expression level of RSPO4.

The present invention further provides methods for treating cancer comprising stering a therapeutically effective amount of a inding agent to a subject. In certain embodiments, the cancer is characterized by cells sing elevated levels of at least one RSPO protein as compared to expression levels of the same RSPO protein in normal . In certain embodiments, the cancer is characterized by cells over— expressing RSPOl. In certain embodiments, the cancer is terized by cells over- expressing RSPOZ. In certain embodiments, the cancer is characterized by cells over- expressing RSPO3. In certain embodiments, the cancer oVer—expresses at least one RSPO protein selected from the group ting ofRSPOI , RSPOZ, RSPO3, and/or RSPO4. In certain embodiments, the cancer is characterized by cells expressing B-catenin, wherein the RSPO-binding agent (e.g., an antibody) interferes with nduced B-catenin signaling and/or activation. In some embodiments, the RSPO-binding agent binds RSPOl, and inhibits or reduces growth of the cancer. In some embodiments, the RSPO- binding agent binds RSPO2, and inhibits or reduces growth of the cancer. In some embodiments, the RSPO-binding agent binds RSPO3, and ts or reduces growth of the cancer. In some embodiments, the RSPO-binding agent binds RSPOI, eres with RSPOl/LGR interactions, and inhibits or reduces growth of the cancer. In some embodiments, the RSPO-binding agent binds RSPOZ, interferes with RSPOZ/LGR ctions, and inhibits or reduces growth of the cancer. In some embodiments, the RSPO—binding agent binds RSPO3, interferes with RSPO3/LGR ctions, and inhibits or reduces growth of the cancer. In some embodiments, the RSPO-binding agent binds RSPOl, inhibits B—catenin activation, and ts or reduces growth of the cancer. In some embodiments, the RSPO-binding agent binds RSPOZ, inhibits B-catenin activation, and inhibits or reduces growth of the cancer. In some embodiments, the RSPO-binding agent binds RSPO3, inhibits B-catenin activation, and inhibits or reduces growth of the cancer. In some embodiments, the RSPO-binding agent binds RSPOI, and reduces the frequency of cancer stem cells in the cancer. In some embodiments, the RSPO-binding agent binds RSPOZ, and reduces the frequency of cancer stem cells in the . In some embodiments, the RSPO-binding agent binds RSPO3, and reduces the frequency of cancer stem cells in the cancer. In some embodiments, the inding agent is an antibody. In some embodiments, the RSPO-binding agent is an anti-RSPOI antibody. In some ments, the anti-RSPOI antibody is antibody 89M5. In some embodiments, the anti-RSPOI antibody is antibody h89M5—H2L2. In some embodiments, the RSPO— binding agent is an anti-RSP02 antibody. In some embodiments, the anti-RSPO2 dy is antibody 130M23. In some embodiments, the anti-RSP02 antibody is dy h130M23-H1L2. In some embodiments, the anti-RSPO2 antibody is antibody h130M23-H1L6. In some embodiments, the RSPO-binding agent is an anti—RSPO3 antibody.

The present invention provides for methods of treating cancer sing administering a therapeutically effective amount of a RSPO-binding agent to a subject (e.g., a subject in need of treatment). In certain embodiments, the subject is a human. In n embodiments, the subject has a cancerous tumor. In certain embodiments, the t has had a tumor removed. In some embodiments, a method of treating cancer comprises administering a therapeutically effective amount of a RSPO-binding agent to a subject, wherein the subject has a tumor that has elevated expression of at least one RSPO protein. In some embodiments, the subject has an ovarian tumor that has elevated expression of RSPOl and is administered a RSPOl-binding agent. In some embodiments, the subject has an ovarian tumor that has elevated expression of RSPOI and is administered an SPOI antibody. In some embodiments, the subject has an ovarian tumor that has elevated expression of RSPOI and is administered antibody 89M5.

In some embodiments, the subject has an ovarian tumor that has elevated sion of RSPOl and is administered antibody H2L2. In some embodiments, the subject has an ovarian tumor that has elevated expression of RSP02 and is administered a RSP02-binding agent. In some embodiments, the subject has an ovarian tumor that has elevated expression of RSP02 and is administered an anti—RSP02 antibody. In some embodiments, the subject has an ovarian tumor that has elevated sion of RSP02 and is administered antibody 130M23. In some embodiments, the subject has an ovarian tumor that has elevated expression of RSP02 and is administered antibody 3- H1L2. In some embodiments, the subject has a pancreatic tumor that has elevated expression of RSP02 and is administered dy 130M23. In some embodiments, the subject has a atic tumor that has ed expression of RSP02 and is administered antibody hl30M23-H1L2. In some embodiments, the subject has a pancreatic tumor that has elevated expression of RSP02 and is administered antibody h130M23—H1L6. In some embodiments, the subject has a colon tumor that has elevated expression of RSP02 and is administered antibody . In some embodiments, the subject has a colon tumor that has ed expression of RSP02 and is administered antibody h13OM23— H1L2. In some embodiments, the subject has a colon tumor that has elevated sion of RSP02 and is administered antibody 3-H1L6. In some embodiments, the subject has a lung tumor that has elevated expression of RSPO3 and is administered an anti-RSPO3 antibody. {$237} In certain ments, 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 ovarian cancer. In certain ments, the cancer is colorectal cancer. In certain embodiments, the cancer is breast cancer. In certair: embodiments, the cancer is prostate cancer. In certain embodiments, the cancer is lung cancer.

In addition, the invention provides a method of reducing the tumorigenicity of a tumor in a t, comprising stering to a subject a therapeutically effective amount of a RSPO-binding agent. In certain embodiments, the tumor ses cancer stem cells. In some embodiments, the tumorigenicity of a tumor is reduced by reducing the frequency of cancer stem cells in the tumor. In some embodiments, the methods comprise using the binding agents, RSPO2-binding , or binding agents described herein. In certain embodiments, the frequency of cancer stem cells in the tumor is reduced by administration of a RSPO—binding agent.

In certain embodiments, the methods further comprise a step of determining the level of at least one RSPO protein expression in the tumor or cancer. In some embodiments, the step of determining the level of RSPO expression in the tumor or cancer comprises determining the level of expression of RSPOI, RSPOZ, RSPO3, and/or RSPO4. In some embodiments, the level of sion of RSPOI, RSPOZ, RSPO3, and/or RSPO4 in a tumor or cancer is compared to the level of expression of RSPOI, RSPOZ, RSPO3, and/or RSPO4 in normal tissue. In some embodiments, the level of expression of RSPOI, RSPO2, RSPO3, and/or RSPO4 in a tumor or cancer is compared to pre-determined level of sion of RSPOl, RSPOZ, RSPO3, and/or RSPO4 in normal tissue. In certain embodiments, the methods further comprise a step of determining if the tumor or cancer has an inactivating mutation in the APC gene. In some embodiments, the methods further comprise a step of ining if the tumor or caricer has an activating mutation in the B-catenin gene. In some embodiments, determining the level of RSPO expression is done prior to treatment. In some ments, the subject is administered a RSPO-binding agent or antibody describe herein if the tumor or cancer has an elevated level of RSPO expression as compared to the expression of the same RSPO protein in normal tissue. For example, in some embodiments, the subject is administered a RSPOl-binding agent (e.g., anti-RSPOI antibody) if the tumor or cancer has an elevated level of RSPOl expression as compared to the level of RSPOl expression in normal tissue. In some embodiments, the subject is administered a RSP02-binding agent (e.g., anti-RSP02 antibody) if the tumor or cancer has an elevated level of RSP02 expression as compared to the level of RSP02 sion in normal . In some ments, the subject is administered a RSPO3-binding agent (e.g., anti-RSPO3 antibody) if the tumor or cancer has an elevated level of RSPO3 sion as compared to the level of RSPO3 expression in normal tissue. If a tumor has elevated expression levels of more than one RSPO protein, the subject is first administered a RSPO-binding agent or antibody to the RSPO protein that is the most over-expressed as compared to normal tissue. In some embodiments, the subject is administered a RSPO-binding agent or antibody describe herein if the tumor or cancer has a mutation in the APC gene.

In addition, the present invention provides methods of identifying a human subject for treatment with an RSPO-binding agent, comprising determining if the subject has a tumor that has an elevated level of RSPO expression as compared to expression of the same RSPO protein in normal tissue. In some embodiments, if the tumor has an ed level of RSPO expression the subject is selected for treatment with an antibody that cally binds a RSPO protein. In some embodiments, if selected for treatment, the subject is administered a RSPO-binding agent or antibody describe herein. In some ments, if the tumor has an elevated level of more than one RSPO protein, the subject is administered a inding agent that binds the RSPO protein with the highest level of expression. In certain embodiments, the subject has had a tumor removed. For example, in some ments, the sion level of RSPOl, RSP02, RSPO3, and/or RSPO4 in a tumor is determined, if the tumor has an elevated level of RSPOl expression as compared to the level of RSPOl in normal tissue, the t is selected for treatment with an antibody that specifically binds RSPOl. If selected for treatment, the subject is administered an anti-RSPOl antibody describe herein. In some embodiments, the RSPOl-binding agent is antibody 89M5. In some embodiments, the RSPOl-binding agent is antibody h89M5-H2L2. In certain ments, the t has had a tumor removed. In some embodiments, the expression level of RSPOl, RSP02, RSPO3, and/or RSPO4 in a tumor is determined, if the tumor has an elevated level of RSP02 expression as compared to the level of RSP02 in normal tissue, the subject is selected for treatment with an antibody that specifically binds RSP02. If selected for -9]- ent, the subject is administered an anti-RSP02 antibody describe herein. In some embodiments, the anti-RSPO2 antibody is antibody l3OM23. In some embodiments, the anti-RSP02 antibody is antibody h130M23-H1L2. In some embodiments, the anti— RSP02 antibody is antibody h130M23-H1L6. In certain embodiments, the subject has had a tumor removed.

The t invention provides methods of selecting a human subject for treatment with a RSPO-binding agent, comprising determining if the subject has a tumor that has an elevated expression level of at least one RSPO protein, wherein if the tumor has an elevated expression level of at least one RSPO protein, the subject is selected for treatment with an dy that specifically binds the RSPO protein with the elevated sion level. The present invention provides methods of selecting a human subject for treatment with a RSPO-binding agent, comprising determining if the subject has a tumor that has a high expression level of at least one RSPO protein, wherein if the tumor has a high expression level of at least one RSPO protein, the subject is selected for treatment with an antibody that specifically binds the RSPO protein with the high expression level. In some embodiments, the ted” or “high” sion level is in comparison to the expression level of the same RSPO protein in normal tissue of the same tissue type. In some embodiments, the “elevated” or “high” expression level is in comparison to the expression level of the same RSPO protein in other tumors of the same tumor type. In some embodiments, if selected for treatment, the subject is administered a inding agent or antibody describe herein. In certain embodiments, the t has had a tumor removed. In some embodiments, the RSPO-binding agent is a RSPOl- binding agent. In some embodiments, the RS;Ol-binding agent is antibody 89M5. In some embodiments, the anti-RSPOI antibody is antibody h89M5-H2L2. In some embodiments, the RSPO-binding agent is a RSPO2-binding agent. In some ments, the RSPO2-binding agent is antibody . In some embodiments, the anti-RSPO2 antibody is antibody h130M23-H1L2. In some embodiments, the anti- RSP02 antibody is antibody h130M23-H1L6. In some embodiments, the RSPO-binding agent is a RSPO3—binding agent.

The present invention also provides s of treating cancer in a human subject, comprising: (a) ing a t for treatment based, at least in part, on the subject having a cancer that has an elevated or high expression level of RSPOI, and (b) administering to the subject a therapeutically effective amount of a RSPOl-binding agent bed herein. In some embodiments, the RSPOl-binding agent is antibody 89M5. In some embodiments, the RSPOl-binding agent is dy h89M5-H2L2.

The present invention also provides methods of treating cancer in a human t, comprising: (a) selecting a subject for treatment based, at least in part, on the subject having a cancer that has an elevated or high expression level of RSPOZ, and (in) administering to the subject a therapeutically effective amount of a RSPOZ-binding agent described herein. In some embodiments, the RSPOZ-binding agent is antibody 130M23.

In some embodiments, the RSPOZ-binding agent is antibody h130M23-H1L2. In some embodiments, the RSPOZ—binding agent is antibody hl 30M23-H1 L6.

The t invention also provides methods of treating cancer in a human subject, comprising: (a) selecting a subject for treatment based, at least in part, on the subject having a cancer that has an elevated or high expression level of RSPO3, and (b) stering to the subject a therapeutically effective amount of a RSPO3-binding agent described herein.

Methods for determining the level of RSPO expression in a cell, tumor or cancer are known by those of skill in the art. These methods include, but are not limited to, PCR-based assays, microarray analyses and nucleotide cing (e.g., NextGen sequencing) for nucleic acid sion. Other methods include, but are not limited, Western blot analysis, protein arrays, , and FACS for protein expression.

Methods for determining whether a tumor or cancer has an elevated or high level of RSPO expression can use a variety of samples. In some embodiments, the sample is taken from a subject having a tumor or cancer. In some embodiments, the sample is a fresh tumor/cancer sample. In some embodiments, the sample is a frozen tumor/cancer sample. In some embodiments, the sample is a formalin—fixed n-embedded sample.

In some embodiments, the sample is processed to a cell lysate. In some embodiments, the sample is processed to DNA or RNA.

Methods of treating a disease or disorder in a subject, wherein the disease or disorder is associated with aberrant (e.g., increased levels) B-catenin signaling are r provided. Methods of treating a disease or disorder in a t, n the e or er is characterized by an increased level of stem cells and/or progenitor cells are further provided. In some embodiments, the treatment methods comprise administering a therapeutically effective amount of a RSPO-bindiEg agent, ptide, or antibody to the subject. In some embodiments, the RSPO-binding agent is a RSPOl-binding agent. In some embodiments, the RSPOl-binding agent is an antibody. In some embodiments, the RSPOl-binding agent is antibody 89M5. In some embodiments, the RSPOl-binding agent is antibody H2L2. In some embodiments, the inding agent is a RSPO2—binding agent. In some embodiments, the RSPOZ—binding agent is an dy.

In some embodiments, the RSPOZ-binding agent is antibody . In some embodiments, the RSPOZ-binding agent is dy h130M23-H1L2. In some embodiments, the RSPOZ-binding agent is antibody h130M23-H1L6. In some embodiments, the RSPO-binding agent is a RSPO3-binding agent. In some embodiments, the RSPO3—binding agent is an dy.

The invention also provides a method of inhibiting B-catenin signaling in a cell comprising contacting the cell with an effective amount of a RSPO-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 RSPO-binding agent comprises administering a therapeutically effective amount of the inding agent to the subject. In some embodiments, the method is an in vitro or ex vivo method. In certain embodiments, the RSPO-binding agent inhibits B-catenin signaling. In some embodiments, the RSFO-binding agent inhibits activation of B-catenin. In certain embodiments, the RSPO-binding agent interferes with a RSPO/LGR interaction. In certain embodiments, the LGR is LGR4, LGRS, and/or LGR6. In certain embodiments, the LGR is LGR4. In certain embodiments, the LGR is LGRS. In certain embodiments, the LGR is LGR6. In some embodiments, the RSPO-binding agent is a RSPOl-binding agent.~ In some embodiments, the RSPOl—binding agent is an antibody. In some embodiments, the RSPOl-binding agent is antibody 89M5. In some embodiments, the RSPOl-binding agent is antibody h89M5-H2L2. In some embodiments, the RSPO- binding agent is a RSPOZ-binding agent. In some embodiments, the RSPO2-binding agent is an antibody. In some embodiments, the RSPOZ-binding agent is antibody 130M23. In some embodiments, the binding agent is antibody 3-H1L2.

In some embodiments, the RSPOZ-binding agent is antibody h130M23-H1L6. In some ments, the inding agent is a RSPO3-binding agent. In some ments, the RSPO3 -binding agent is an antibody.

The use of the RSPO-binding agents, polypeptides, or antibodies described herein to induce the differentiation of cells, including, but not limited to tumor cells, is also provided. In some embodiments, methods of inducing cells to entiate comprise contacting the cells with an effective amount of a RSPO-binding agent (e.g., an anti- RSPO antibody) bed herein. In certain embodiments, methods of ng cells in a tumor in a subject to differentiate comprise administering a therapeutically effective amount of a RSPO-binding agent, polypeptide, or antibody to the subject. In some embodiments, methods for inducing differentiation markers on tumor cells comprise administering a therapeutically effective amount of a RSPO-binding agent, polypeptide, or antibody. In some embodiments, the tumor is a solid tumor. In some embodiments, the tumor is ed from the group consisting of colorectal tumor, pancreatic tumor, lung tumor, ovarian tumor, liver tumor, breast tumor, kidney tumor, prostate tumor, gastrointestinal tumor, ma, cervical tumor, r tumor, glioblastoma, and head and neck tumor. In certain embodiments, the tumor is an n tumor. In certain other embodiments, the tumor is a colon tumor. In some embodiments, the tumor is a lung tumor. In certain embodiments, the method is an in viva method. In certain embodiments, the method is an in vitro method. in some embodiments, the RSPO— binding agent is a RSPOl—binding agent. In some embodiments, the RSPOl-binding agent is an antibody. In some embodiments, the RSPOl—binding agent is antibody 89M5.

In some embodiments, the RSPO-binding agent is a RSPOZ-binding agent. In some embodiments, the RSPOZ-binding agent is an antibody. In some embodiments, the RSPOZ—binding agent is antibody . In some embodiments, the RSPO-binding agent is a RSPO3-binding agent. In some embodiments, the RSPO3-binding agent is an antibody.

The invention further provides methods of differentiating tumorigenic cells into non-tumorigenic cells comprising contacting the tumorigenic cells with a RSPO-binding agent. In some embodiments, the method comprises administering the RSPO-binding agent to a subject that has a tumor comprising tumorigenic cells or that has had such a tumor removed. In certain embodiments, the tumorigenic cells are ovarian tumor cells.

In n embodiments, the tumorigenic cells are colon tumor cells. In some ments, the tumorigenic cells are lung tumor cells. In some embodiments, the RSPO-binding agent is a binding agent. In some embodiments, the RSPOl- binding agent is an antibody. In some embodiments, the RSPOl-binding agent is antibody 89M5. In some ments, the RSPO-binding agent is a RSPO2-binding agent. In some embodiments, the RSPOZ-binding agent is an antibody. In some ments, the RSPO2-binding agent is antibody 130M23. In some ments, the RSPO-binding agent is a RSPO3-binding agent. In some ments, the RSPO3— binding agent is an antibody.

In certain ments, the disease treated with the RSPO-binding agents described herein is not a cancer. For e, the disease may be a metabolic disorder such as obesity or diabetes (e.g., type II diabetes) (Jin T., 2008, Diabetologz‘a, 51:1771- 80). Alternatively, the disease may be a bone disorder such as osteoporosis, osteoarthritis, or rheumatoid arthritis (Corr M., 2008, Nat. Clin. Pract. Rheumatol., 4:550- 6; Day et al., 2008, Bone Joint Surg. Am, 90 Suppl 1:19-24). The disease may also be a kidney disorder, such as a polycystic kidney disease (Harris et al., 2009, Ann. Rev. Med, 60:321-337; Schmidt-Ott et al., 2008, Kidney Int, 74:1004-8; Benzing et al., 2007, J. Am.

Soc. Nephrol., 18:1389-98). Alternatively, eye disorders including, but not limited to, macular degeneration and familial exudative Vitreoretinopathy may be treated (Lad et al., 2009, Stem Cells Dev., 18:7-16). Cardiovascular disorders, ing myocardial infarction, sclerosis, and valve disorders, may also be treated (Al-Aly Z., 2008, Transl. Res, 151 :233-9; Kobayashi et al., 2009, Nat. Cell Biol, 11:46-55; van Gijn et al., 2002, Cardiovasc. Res., 55:16-24; Christman et al., 2008, Am. J. Physiol. Heart Circ.

Physiol, 2942H2864-70). In some ments, the disease is a ary disorder such as idiopathic pulmonary arterial hypertension or pulmonary fibrosis (Laumanns et al., 2008, Am. J. Respir. Cell Mol. Biol, 2009, 402683-691; Konigshoff et al., 2008, PLoS ONE, 2). In some embodiments, the disease treated with the RSPO-binding agent is a liver disease, such as cirrhosis or liver fibrosis (Cheng et al., 2008, Am. J. Physiol.

Gastrointest. Liver Physiol, 2942G39-49).

The present invention further provides pharmaceutical compositions comprising the RSPO-binding agents described herein. In certain embodiments, the pharmaceutical compositions further comprise a pharmaceutically acceptable vehicle. These pharmaceutical compositions find use in ting tumor growth and treating cancer in a subject (e.g., a human patient). ~96- In certain embodiments, formulations are prepared for storage and use by combining a purified antibody or agent of the present invention with a ceutically acceptable vehicle (e.g., a carrier or excipient). Suitable ceutically acceptable vehicles include, but are not limited to, nontoxic buffers such as phosphate, citrate, and other organic acids; salts such as sodium chloride; antioxidants including ascorbic acid and nine; preservatives such as octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens, such as methyl or propyl paraben, ol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol; low molecular weight polypeptides (e.g., less than about 10 amino acid residues); proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic rs such as nylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; carbohydrates such as ccharides, disaccharides, glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes such as Zn-protein complexes; and non-ionic surfactants such as TWEEN or polyethylene glycol (PEG). (Remington: The Science and Practice of Pharmacy, 21st Edition, 2005, University of the Sciences in Philadelphia, PA). [0254; The pharmaceutical compositions of the present invention can be administered in any number of ways for either local or systemic treatment. Administration can be topical by epidermal or transderrnal s, ointments, lotions, creams, gels, drops, itories, sprays, liquids and powders; ary by inhalation or insufflation of powders or aerosols, including by nebulizer, intratracheal, and intranasal; oral; or parenteral including intravenous, intraarterial, intratumoral, subcutaneous, intraperitoneal, uscular (e.g., injection or infusion), or intracranial (e.g., intrathecal or intraventricular).

The therapeutic formulation can be in unit dosage form. Such formulations include tablets, pills, capsules, powders, granules, solutions or suspensions in water or non-aqueous media, or suppositories. In solid compositions such as tablets the pal active ient is mixed with a pharmaceutical carrier, tional tableting ingredients include corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium te, dicalcium phosphate or gums, and diluents (e.g., water). These can be used to form a solid preformulation composition containing a homogeneous mixture of a compound of the present invention, or a non~toxic pharmaceutically acceptable salt thereof. The solid preformulation composition is then subdivided into unit dosage forms of a type described above. The tablets, pills, etc. of the formulation or composition can be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action. For example, the tablet or pill can comprise an inner composition covered by an outer component. Furthermore, the two components can be separated by an enteric layer that serves to resist disintegration and permits the inner component to pass intact through the stomach or to be d in release. A y of materials can be used for such enteric layers or coatings, such materials include a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol and cellulose acetate.

The RSPO-binding agents or antibodies described herein can also be entrapped in microcapsules. Such microcapsules are prepared, for example, by coacervation techniques or by interfacial polymerization, for e, hydroxymethylcellulose or gelatin-microcapsules and methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions as described in ton: The Science and Practice ofPharmacy, 21st n, 2005, University of the Sciences in Philadelphia, PA. [0257} In n embodiments, pharmaceutical formulations include a RSPO-binding agent (e.g., an antibody) of the present ion complexed with liposomes. Methods to produce liposomes are known to those of skill in the art. For example, some mes can be generated by reverse phase ation with, a lipid ition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG- PE). Liposomes can be extruded through filters of defined pore size to yield liposomes with the desired diameter.

In certain embodiments, sustained-release preparations can be produced. le examples of sustained-release preparations include semi-permeable matr‘éces of solid hobic rs containing a RSPO-binding agent (e.g., an antibody), where the 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- -98— methacrylate) or poly(vinyl l), polylactides, copolymers of L-glutamic acid and 7 ethyl-L-glutamate, gradable ethylene-Vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOTTM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), sucroseacetate isobutyrate, and poly-D-(-)hydroxybutyric acid.

In certain embodiments, in addition to stering a RSPO-binding agent (e.g., an antibody), the method or treatment further comprises stering at least one additional therapeutic agent. An onal therapeutic agent can be administered prior to, concurrently with, and/or uently to, administration of the RSPO-binding agent.

Pharmaceutical itions comprising a RSPO-binding agent and the additional therapeutic agent(s) are also provided. In some embodiments, the at least one onal therapeutic agent comprises 1, 2, 3, or more additional therapeutic agents.

Combination y with two or more therapeutic agents often uses agents that work by different mechanisms of action, although this is not required. Combination therapy using agents with different mechanisms of action may result in additive or synergetic effects. Combination therapy may allow for a lower dose of each agent than is used in monotherapy, thereby reducing toxic side effects and/or increasing the therapeutic index of the agent(s). Combination therapy may decrease the likelihood that resistant cancer cells will develop. In some embodiments, combination therapy comprises a therapeutic agent that affects (e.g., inhibits or kills) non-tumorégenic cells and a therapeutic agent that affects (e.g., ts or kills) tumorigenic CSCs.

In some embodiments, the combination of a RSPO-binding agent and at least one additional therapeutic agent results in additive or istic results. In some embodiments, the combination therapy results in an increase in the therapeutic index of the RSPO-binding agent. In some embodiments, the ation therapy results in an increase in the therapeutic index of the additional agent(s). In some embodiments, the combination therapy results in a se in the toxicity and/or side effects of the RSPO- binding agent. In some embodiments, the ation therapy results in a decrease in the toxicity and/or side effects of the additional agent(s).

Useful s of therapeutic agents include, for example, antitubulin agents, auristatins, DNA minor groove binders, DNA replication inhibitors, alkylating agents (e.g., platinum complexes such as cisplatin, mono(platinum), bis(platinum) and tri- nuclear platinum xes and carboplatin), anthracyclines, antibiotics, antifolates, antimetabolites, herapy sensitizers, duocarmycins, etoposides, fluorinated pyrémidines, ionophores, lexitropsins, nitrosoureas, ols, purine antimetabolites, puromycins, radiation sensitizers, steroids, taxanes, topoisomerase inhibitors, Vinca alkaloids, or the like. In certain embodiments, the second therapeutic agent is an alkylating agent, an antimetabolite, an antimitotic, a topoisomerase inhibitor, or an enesis inhibitor. In some embodiments, the second therapeutic agent is a platinum complex such as carboplatin or cisplatin. In some embodiments, the additional therapeutic agent is a platinum complex in ation with a taxane.

Therapeutic agents that may be administered in ation with the RSPO— binding agents include chemotherapeutic agents. Thus, in some embodiments, the method or treatment involves the administration of a RSPOl-binding agent or antibody of the present invention in combination with a chemotherapeutic agent or cocktail of multiple different chemotherapeutic agents. In some embodiments, the neethod or treatment involves the administration of a binding agent or antibody of the present invention in combination with a chemotherapeutic agent or cocktail of multiple different chemotherapeutic agents. In some embodiments, the method or teeatment involves the administration of a RSPO3-binding agent or antibody of the present invention in combination with a chemotherapeutic agent or cocktail of multiple different chemotherapeutic agents. Treatment with a RSPO-binding agent (e.g, an antibody) can occur prior to, concurrently with, or subsequent to administration of chemotherapies.

Combined administration can e co-administration, either in a single pharmaceutical formulation or using separate formulations, or consecutive stration in either order but generally within a time period such that all active agents can exert their biological activities simultaneously. Preparation and dosing schedules for such herapeutic agents can be used according to manufacturers' instructions or as determined empirically by the skilled practitioner. Preparation and dosing schedules for such chemotherapy are also described in The Chemotherapy Source £00k, 4th Edition, 2008, M. C. Perry, Editor, Lippincott, Williams & s, Philadelphia, PA. herapeutic agents useful in the instant invention e, but are not limited to, ting agents such as thiotepa and cyclosphosphamide (CYTOXATN); alkyl sulfonates such as busulfan, ulfan and piposulfan; aziridines such as benzodopa, -100— carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, tricthylenethiophosphaoramide and trimethylolomelamime; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, lan, novembichin, phenesterine, prednimustine, famide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, stine; otics such as aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, omycin, calicheamicin, carabicin, caminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6—diazoox0-L—norleucine, doxorubicin, epirubicin, esorubicin; idarubicin, marcellomycin, cins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, cin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as erin, methotrexate, terin, trimetrexate; purine analogs such as fludarabine, 6- mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofilr, ne arabinoside, dideoxyuridine, ridine, enocitabine, floxuridine, S-FU; androgens such as erone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti—adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenishers such as folinic acid; aceglatone; aldophosphamide ide; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; cid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; rine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK; razoxane; sizofuran; spirogermanium; tenuazonic acid; tréaziquone; 2,2',2"-trichlorotriethylamine; urethan; Vindesine; dacarbazine; mannomustine; mitobronitol; ctol; pipobroman; gacytosine; arabinoside (Ara-C); taxoids, e.g. paclitaxel (TAXOL) and xel (TAXOTERE); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; platinum analogs such as cisplatin and carboplatin; Vinblastine; platinum; etoposide (VF-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; Vinorelbine; ine; novantrone; teniposide; daunomycin; aminopterin; ibandronate; CPTll; topoisomerase inhibitor RPS 2000; difluoromethylomithine (DMFO); retinoic acid; esperamicins; capecitabine (XELODA); and pharmaceutically acceptable salts, acids or derivatives of any of the above.

Chemotherapeutic agents also include anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens including for example tamoxifen, raloxifene, aromatase ting 4(5)—imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, stone, and toremifene (FARESTON); and ndrogens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above. In certain embodiments, the additional eutic agent is cisplatin. In certain embodiments, the additional therapeutic agent is carboplatin. In certain embodiments, the additional therapeutic agent is paclitaxel (taxol). In some embodiments, a method comprises administering anti-RSPOI antibody 89M5 or h89M5-H2L2 in combination with cisplatin.

In some embodiments, a method comprises administering anti-RSP02 antibody 130M23, hI30M23—HIL2, or h130M23-H1L6 in combination with cisplatin.

In certain embodiments, the chemotherapeutic agent is a topoisomerase inhibitor. omerase inhibitors are chemotherapy agents that ere with the action of a topoisomerase enzyme (e.g., topoisomerase I or II). Topoisomerase inhibitors include, but are not limited to, doxorubicin HCl, daunorubicin citrate, mitoxantrone HCl, actinomycin D, etoposide, topotecan HCl, teniposide (VM-26), and irinotecan, as well as ceutically acceptable salts, acids, or derivatives of any of these. In some embodiments, the additional eutic agent is irinotecan. Thus, in some embodiments, a method comprises stering a RSPOl-binding agent in combination with a topoisomerase tor. In some embodiments, a method comprises administering anti- RSPOl antibody 89M5 or h89M5-H2L2 in combination with irinotecan. In some embodiments, a method comprises administering a binding agent in combination with a topoisomerase inhibitor. In some embodiments, a method comprises administering anti-RSPO2 antibody 130M23 or h130M23-H1L2 in combination with irinotecan.

In certain ments, the chemotherapeutic agent is an anti-metabolite. An anti-metabolite is a chemical with a structure that is similar to a metabolite required for normal biochemical reactions, yet different enough to interfere with one or more normal functions of cells, such as cell on. Anti-metabolites include, but are not limited to, gemcitabine, fluorouracil, capecitabine, rexate sodium, ralitrexed, pemetrexed, tegafur, cytosine arabinoside, thioguanine, S-azacytidine, 6-mercaptopurine, azathioprine, guanine, pentostatin, fludarabine phosphate, and cladribine, as well as ceutically acceptable salts, acids, or derivatives of any of these. In certain ments, the additional therapeutic agent is gemcitabine. Thus, in some embodiments, a method comprises administering a RS‘POl-binding agent in combination with an anti-metabolite. In some embodiments, a method comprises administering anti- RSPOl antibody 89M5 or h89M5—H2L2 in combination with gemcitabine. In some ments, a method comprises administering a RSPOZ-binding agent in combination with an etabolite. In some embodiments, a method comprises administering anti- RSP02 antibody 130M23, h130M23-H1L2, or 3-H1L6 in combination with gemcitabine.

In n embodiments, the chemotherapeutic agent is an antimitotic agent, including, but not limited to, agents that bind tubulin. In some embodiments, the agent is a taxane. In certain embodiments, the agent is paclitaxel or xel, 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 ANE), DHA—paclitaxel, or PG-paclitaxel. In certain alternative embodiments, the antimitotic agent comprises a vinca alkaloid, such as vincristine, binblastine, vinorelbine, or vindesine, or pharmaceutically acceptable salts, acids, or derivatives thereof. In some embodiments, the antimitotic agent is an inhibitor of kinesin Eg5 or an inhibitor of a mitotic kinase such as Aurora A or Plkl. In certain embodiments, where the chemotherapeutic agent administered in combination with a RSPO-binding agent is an anti—mitotic agent, the cancer or tumor being treated is breast cancer or a breast tumor.

In some embodiments, an additional therapeutic agent ses an agent such as a small molecule. For example, treatment can involve the combined administration of a RSPO-binding agent (e.g. an antibody) of the present ion with a small le that acts as an inhibitor against additional tumor-associated antigens ing, but not limited to, EGFR, ErbB2, HER2, and/or VEGF. In certain embodiments, the additional therapeutic agent is a small molecule that inhibits a cancer stem cell pathway. In some embodiments, the additional therapeutic agent is an inhibitor of the Notch pathway. In some embodiments, the additional therapeutic agent is an inhibitor of the Wnt pathway. —103- In some embodiments, the additional therapeutic agent is an inhibitor of the BMP pathway. In some embodiments, the additional therapeutic agent is a molecule that inhibits B-catenin signaling.

In some embodiments, an additional therapeutic agent comprises a biological molecule, such as an antibody. For example, treatment can involve the combined administration of a RSPO-binding agent (e.g. an antibody) of the present invention with other antibodies t additional tumor-associated antigens including, but not limited to, antibodies that bind EGFR, ErbBZ, HERZ, and/or VEGF. In some embodiments, the additional therapeutic agent is a second anti-RSPO antibody. In some embodiments, the additional eutic agent is an anti-RSP02 antibody, an anti-RSPO3 antibody, and/or an anti-RSPO4 dy used in combination with an SPOI antibody. In some embodiments, the additional eutic agent is an anti-RSPOI dy, an anti-RSPO3 antibody, and/or an anti-RSPO4 antibody used in combination with an SP02 antibody. In some embodiments, an anti-RSPOI antibody is used in combination with an SP02 antibody. In certain embodiments, the additional therapeutic agent is an dy specific for an anti-cancer stem cell marker. In some embodiments, the additional eutic agent is an antibody that binds a component of the Notch pathway.

In some embodiments, the additional therapeutic agent is an dy that binds a ent of the Wnt pathway. In certain embodiments, the onal therapeutic agent is an antibody that inhibits a cancer stem cell pathway. In some embodiments, the additional therapeutic agent is an inhibitor of the Notch pathway. In some embodiments, the additional therapeutic agent is an inhibitor of the Wnt pathway. In some embodiments, the additional therapeutic agent is an inhibitor of the BMP pathway. In some embodiments, the additional therapeutic agent is an antibody that inhibits B-catenin signaling. In certain embodiments, the additional therapeutic agent is an antibody that is an angiogenesis inhibitor (e.g., an anti-VEGF or VEGF receptor antibody). In n embodiments, the additional therapeutic agent is bevacizumab (AVASTIN), trastuzumab (HERCEPTIN), panitumumab BIX), or cetuximab (ERBITUX).

In some embodiments, the methods described herein comprise administering a therapeutically effective amount of a RSPO-binding agent in combination with Wnt pathway inhibitors. In some embodiments, the Wnt pathway inhibitors are frizzled (FZD) n binding agents, “FZD-binding agents”. Non-limiting examples of FZD-binding -104— agents can be found in US. Patent No. 7,982,013, which is incorporated by reference herein in its entirety. FZD-binding agents may include, but are not limited to, anti-FZD antibodies. In some embodiments, a method comprises administering a RSPO-binding agent in combination with an anti-FZD antibody. In some ments, a method comprises administering a RSPO-binding agent in combination with the anti-FZD antibody 18R5. In some embodiments, the Wnt pathway inhibitors are Wnt protein binding , “Wm-binding agents”. Nonlimiting examples of nding agents can be found in US. Patent Nos. 7,723,477 and 7,947,277; and International Publications WO 2011/088127 and , which are incorporated by reference herein in their entirety. Wnt—binding agents may include, but are not limited to, anti-Wnt antibodies and FZD-Fc soluble receptors. In some embodiments, a method comprises administering a RSPO-binding agent in combination with a FZD-Fc soluble receptor. In some embodiments, a method comprises administering a RSPO-binding agent in combination with a FZD8-Fc e receptor. In some embodiments, a method ses administering a RSPOl-binding agent in combination with an anti-FZD antibody. In some embodiments, a method comprises administering anti-RSPOl antibody 89M5 or h89M5-H2L2 in combination with an anti-FZD antibody. In some embodiments, a method comprises administering SPOI dy 89M5 or h89M5H2L2 in combination with ZD antibody 18R5. In some embodiments, a method comprises administering anti-RSPOI antibody 89M5 or h89M5-H2L2 in ation with a FZD- Fc soluble receptor. In some embodiments, a method comprises administering anti- RSPOl antibody 89M5 or h89M5-H2L2 in combination with a FZD8-Fc soluble receptor. In some embodiments, a method comprises administering a RSPO2-binding agent in ation with an anti-FZD dy. In some embodiments, a method comprises administering anti-RSPOZ dy 130M23 or h130M23-H1L2 in combination with an anti-FZD antibody. In some embodiments, a method comprises administering anti-RSP02 antibody 130M23, h130M23-H1L2, or h130M23-H1L6 in combination with anti-FZD antibody 18R5. In some embodiments, a method comprises stering anti—RSPOZ antibody 130M23, 3-H1L2, or h130M23-H1L6 in combination with, a FZD-Fc soluble receptor. In some embodiments, a method ses administering anti-RSP02 antibody 130M23, h130M23-H1L2, or h130M23-H1L6 in combination with a FZDS-Fc soluble receptor. —105- In some embodiments, the methods described herein comprise stering a therapeutically ive amount of a RSPO-binding agent in combination with more than one additional therapeutic agent. Thus, in some embodiments, a method comprises administering a RSPO-binding agent in combination with a chemotherapeutic agent and a Wnt pathway inhibitor. In some embodiments, a method comprises administering a binding agent in combination with a chemotherapeutic agent and a Wnt pathway inhibitor. In some embodiments, a method comprises administering a binding agent in combination with a chemotherapeutic agent and anti-FZD antibody 18R5. In some embodiments, a method comprises administering a RSPOZ-binding agent in combination with a chemotherapeutic agent and a FZD8-Fc soluble receptor. In some embodiments, a method comprises administering a RSPOZ-binding agent in combination with gemcitabine and a Wnt pathway inhibitor. In some embodiments, a method comprises administering anti-RSP02 dy 130M23, hl30M23-H1L2, or h130M23- H1L6 in combination with gemcitabine and anti—FZD antibody 18K5. In some embodiments, a method comprises administering anti-RSP02 antibody 130M23, hl3OM23-H1L2, or h130M23-H1L6 in combination with gemcitabine and FZD8-Fc soluble receptor. [0272} Furthermore, treatment with a RSPO-binding agent described herein can include combination treatment With other biologic molecules, such as one or more cytokines (e.g., okines, interleukins, tumor necrosis factors, and/or growth factors) or can be accompanied by al removal of tumors, cancer cells or any other therapy deemed necessary by a treating physician. [0273} In certain embodiments, the treatment involves the administration of a RSPO- binding agent (e.g. an antibody) of the present invention in combination with radiation therapy. ent with a RSPO-binding agent can occur prior to, concurrently with, or subsequent to stration of radiation therapy. Dosing schedules for such radiation y can be determined by the skilled medical practitioner.

Combined administration can e co-administration, either in a single ceutical formulation or using separate formulations, or consecutive administration in either order but generally within a time period such that all active agents can exert their biological activities simultaneously.

It will be appreciated that the combination of a inding agent and at least one additional therapeutic agent may be administered in any order or concurrently. In some embodiments, the RSPO—binding agent will be administered to patients that have previously undergone treatment with a second therapeutic agent. In certain other embodiments, the RSPO-binding agent and a second therapeutic agent will be administered substantially simultaneously or concurrently. For example, a subject may be given a inding agent (e.g., an dy) while undergoing a course of treatment with a second therapeutic agent (e.g., chemotherapy). In certain embodiments, a RSPO-binding agent will be administered within 1 year of the treatment with a second therapeutic agent. In certain alternative embodiments, a RSPO-binding agent will be admireistered within 10, 8, 6, 4, or 2 months of any treatment with a second eutic agent. In certain other embodiments, a RSPO—binding agent will be administered within 4, 3, 2, or 1 weeks of any treatment with a second therapeutic agent. In some ments, a RSPO-binding agent will be administered within 5, 4, 3, 2, or 1 days of any treatment with a second eutic agent. It will further be appreciated that the two (or more) agents or ents may be administered to the subject within a matter of hours or minutes (i.e., substantially simultaneously).

For the treatment of a disease, the appropriate dosage of an RSPO-binding agent (e. g., an antibody) of the present invention depends on the type of disease to be treated, the severity and course of the disease, the responsiveness of the disease, whether the RSPO-binding agent or antibody is administered for therapeutic or preventative purposes, previous therapy, the patient’s clinical history, and so on, all at the discretion of the treating ian. The RSPO-binding agent or dy can be administered one time or over a series of treatments lasting from several days to l months, or until a cure is effected or a diminution of the disease state is achieved (e.g., reduction in tumor size).

Optimal dosing schedules can be calculated from measurements of drug lation in the body of the patient and will vary depending on the relative potency of an individual antibody or agent. The administering physician can easily determine optimum dosages, dosing methodologies, and tion rates. In certain ments, dosage is from 0.01ug to lOOmg/kg of body weight, from 0.1 ug to lOOmg/kg of body weight, from 1 ug to lOOmg/kg of body weight, from 1mg to 100mg/kg of body weight, 1mg to 80mg/kg of body weight from 10mg to lOOmg/kg of body weight, from 10mg to 75mg/kg of body weight, or from 10mg to g of body weight. In certain embodiments, the dosage of the antibody or other inding agent is from about 0.1mg to about 20mg/kg of body . In certain ments, dosage can be given once or more daily, weekly, monthly, or yearly. In certain embodiments, the antibody or other RSPO-binding agent is given once every week, once every two weeks or once every three weeks.

In some embodiments, a RSPO-binding agent (e.g., an antibody) may be administered at an initial higher “loading” dose, followed by one or more lower doses. In some embodiments, the frequency of administration may also . In some embodiments, a dosing regimen may comprise administering an initial dose, followed by additional doses (or “maintenance” doses) once a week, once every two weeks, once every three weeks, or once every month. For example, a dosing regimen may comprise stering an initial loading dose, followed by a weekly maintenance dose of, for e, one-half of the initial dose. Or a dosing regimen may comprise administering an initial loading dose, followed by maintenance doses of, for example one-half of the initial dose every other week. Or a closing regimen may comprise administering three initial doses for 3 weeks, followed by maintenance doses of, for example, the same amount every other week.

As is known to those of skill in the art, administration of any therapeutic agent may lead to side effects and/or toxicities. In some cases, the side effects and/or toxicities are so severe as to preclude stration of the ular agent at a eutically ive dose. In some cases, drag therapy must be discontinued, and other agents may be tried. However, many agents in the same therapeutic class often display similar side effects and/or toxicities, meaning that the patient either has to stop therapy, or if possible, suffer from the unpleasant side s associated with the therapeutic agent.

Thus, the present invention provides methods of treating cancer in a subject comprising using an intermittent dosing strategy for administering one or more agents, which may reduce side effects and/or toxicities associated with, administration of a RSPO- binding agent, chemotherapeutic agent, etc. In some embodiments, a method for treating cancer in a human t comprises administering to the subject a therapeutically effective dose of a RSPO-binding agent in combination with a therapeutically effective dose of a chemotherapeutic agent, wherein one or both of the agents are administered according to an intermittent dosing strategy. In some embodiments, the intermittent —108— dosing strategy comprises administering an initial dose of a RSPO-binding agent to the subject, and administering subsequent doses of the RSPO-binding agent about once every 2 weeks. In some embodiments, the intermittent dosing strategy comprises administering an initial dose of a RSPO-binding agent to the subject, and stering subsequent doses of the RSPO-binding agent about once every 3 weeks. In some embodiments, the intermittent dosing strategy comprises stering an initial dose of a RSPO-binding agent to the subject, and administering subsequent doses of the RSPO-binding agent about once every 4 weeks. In some embodiments, the RSPO-binding agent is administered using an intermittent dosing strategy and the chemotherapeutic agent is administered weekly.

V. Kits comprising RSPO—binding agents {928%} The present invention provides kits that comprise the RSPO-binding agents (e.g., antibodies) described herein and that can be used to perform the methods described herein. In certain embodiments, a kit comprises at least one d antibody against at least one human RSPO protein in one or more containers. In some embodiments, the kits contain all of the components necessary and/or sufficient to perform a detection assay, including all controls, directions for performing , and any ary software for analysis and presentation of results. One d in the art will readily recognize that the disclosed RSPO—binding agents of the present invention can be readily incorporated into one of the established kit formats which are well known in the art.

Further ed are kits comprising a RSPO-binding agent (e.g., an anti—RSPO antibody), as well as at least one additional therapeutic agent. In n embodiments, the second (or more) therapeutic agent is a chemotherapeutic agent. In n ments, the second (or more) therapeutic agent is a Wnt pathway inhibitor. In certain embodiments, the second (or more) therapeutic agent is an enesis inhibitor.

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

EXAMPLES Example 1 iiélxpressiou of RSR) and LGR inhuman tumors mRNA from normal , benign tumor and malignant tumor samples of a large number of human patients was analyzed by microarray analysis (Genelogic BioExpress Datasuite). This data revealed elevated expression levels of RSPOI in malignant tissue ve to normal tissue in several tumor types including kidney, endometrial, and n. RSPOl was noted to be frequently over-expressed in ovarian cancer (Fig. 1A).

In addition, this data suggested elevated expression levels of RSPO3 in malignant tissue relative to normal tissue in several tumor types including ovarian, pancreas, and lung (Fig. 1C). In addition, it was found that LGRS and LGR6 were over—expressed in malignant breast , colon tumors, lung tumors, and ovarian tumors relative to normal , while LGR4 was over—expressed in lung tumors. LGRS and LGR6 over- expression appeared to be restricted to triple-negative (ERnegPRnegHERZHCg) breast tumors relative to other breast tumor subtypes.

RNA was isolated from a series of human tumors grown in murine afts.

The RNA samples were prepared and processed using established Affymetrix protocols for the generation of labeled cRNA. The processed RNA was hybridized to Affymetrix HG—Ul33 plus 2.0 microarrays (Affymetrix, Santa Clara, CA) as outlined in the manufacturer’s technical manuals. After hybridization, the microarrays were washed, scanned, and analyzed. Scanned array background adjustment and signal intensity normalization were performed using the GCRMA algorithm (Bioconductor, www.bioconductor.org).

Particular human RSPOS and human LGRs were evaluated — RSPOl 0_at), RSP02 (1554012_at), RSPO3 (228186_s_at), RSPO4 (237423_at), LGR4 6_s_at), LGRS (210393_at) and LGR6 (227819_at). Microarray is showed that, while LGR4 and LGR6 were broadly expressed in almost all , many tumors were found to greatly over-express only particular RSPO family members and LGRS (Table 2), although these expression levels were not compared to expression levels in normal tissue.

Generally there is only a single RSPO family member that is highly expressed in a given tumor, suggesting that there may be functional ancy within the RSPO family.

Table 2 555555555555555555555555555555555555555555555555555555 W55 555555555555555555555555555555555555555555555555555555555555555555555555555555 555555555555555555555555555555555 Tumor {1181301444445 RSP02 ERSP03 {Esp04 :LGR4 {WLGRS ‘LGR6 555555555555555555555555555 55555555555555555555555555555554555555555555555555555555555555555L55555555555555555..555555.55555555555555_...._...._..._55555:5555555555555555555555555H 555555555.555555555.55555555555555555555555555555 {B34 ““““““““““‘““““““‘““‘““‘“““‘“‘“““‘“‘““‘““‘““‘““‘““‘““‘“"f‘““‘““‘““‘““‘“““‘“‘“‘““‘““‘““‘““‘““"3““‘“"‘“““““““““‘”““‘“““““““‘55555555555{4.79 493 30331 w{441 5 58888 {3260 440 .89 {6479 692.34 {.441 2678.95 E428 {5.088 -. 55.5.55 ...............................{6467.15 38525526555. 11.31 .......................................................................................... :.282246 ‘ .6234 43.94 5555555555555555555555555555455555555555555555555.5555555555 {C18 4.63 :4.95 1383 4.42 245415 :4.39 733.15 i g980.49 {4.75 g 4.40 508384 2082 3' 55.5.5.55.55.55.5555555555555 5.5.55555555555555.5.55555555555555555555555555.5555..5555555.55.555555..5..5.....555.55..... 5.55555555555555_55555555.55555_555555555555____5_55555 .55....5555555.555.555.55.555 ...5555_555555555555555555555 L""""""""""""""t""""""""""""""""1"""""""‘““““““““‘”““““““““““““““" 5555555555555555555555555555555555.55.5.555555555555555555555 55 4.43 WWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWW:15190401:4 13.95 : 14.58 f492 ’453 4.41 9995555514665755 1208.92 {.429 41089 55555555555555555555555555555{555555555555555555555555555555855555555555555555555555555555f:x l. 1 1 3-29.62 ‘4.30 ‘1.2096 :555““.m5m5555.m55555:555555555555555555““5555..55555-555555.555555555555555555555555555555555555555555555555555555555555555555555555555555555.5555“5555555555555555555555555555555555555555555555555555555555555555555555555555555555555554 l55555555555555555555555.5.55.55.55555555555555.5555555555555555555555..5.5.5.555555555555555555......555555555555.555.55.55.55555.55555555555555555.555555555..555..5..5.5..5.5.5.....55...555555.555555555555555555555555555555555555555555555555555 69.77 {209049467572...................430278 132’55.... i 37.43 3743.91 :482.33 3812.05 2578{492 = ‘ i ‘ .555.555.5555 ...............___..........................__.1‘.....__.....................:....._5.....................5..............................: 3439.88 {16.35 :.152812 :424 {19.49 ".Pancreatic tumors {*“““""“‘*‘*‘*‘*‘*“‘““‘“"‘““““‘*““*““*““‘r““"'“““‘“"‘~~‘“~~~“~~‘r""““““~~“‘~“~~~~~~~~~ 55555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555 2PN07 458 :68952 :451 {440 6:77741 :428 :746.38 ...............55..........5........5555....5........5.5.5.5.555.5..5.5..5.5.5.5....55....55...555555555..............5.......5.....................5.......4....555555555...55...55555555555555555555555555555555555555555555555555555555555555555555555 EPN18 :4.72 7 4.65 3 4.50 :.675073 E51.15 4 55555555555.5555555.555555555555555555555 555555555 Example 2 Binding ofRSPO proteins to LGRS A cell e LGRS protein was generated by ligating amino acids 22-564 of human LGRS to an N-terminal FLAG tag and to the transmembrane domain of CD4 and a C-terminal GFP protein tag using standard recombinant DNA techniques (FLAG- LGRS-CD4TM-GFP). c constructs were generated using standard recombinant DNA techniques. Specifically, full-length human RSPOI, RSPOZ, RSPO3 and RSPO4 were d in—frame to a human Fc region and the recombinant RSPO-Fc proteins were expressed in insect cells using baculovirus. The fusion proteins were purified from the insect medium using protein A tography.

HEK—293 cells were transiently transfected with the FLAG-LGRS-CD4TM-GFP construct. After 48 hours, ected cells were suspended in ice cold PBS containing 2% FBS and heparin and incubated on ice in the presence of lOug/ml RSPOl-Fc, RSPO2-Fc, RSPO3-Fc, RSPO4-Fc, or FZD8-Fc fusion ns for 15 minutes. A second tion with lOOul PE-conjugated anti-human Fc secondary antibody was performed to detect cells bound by the Fc fusion proteins. Cells were incubated with an anti-FLAG antibody (Sigma-Aldrich, St. Louis, MO) as a positive control and with an anti-PE antibody as a negative control. The cells were analyzed on a FACSCalibur instrument (BD Biosciences, San Jose, CA) and the data was processed using FlowJo re.

As shown in Figure 2, RSPOl, RSPO2, RSPO3 and RSPO4 all bound to LGRS sed on the surface of the 3 cells, while FZD8, the negative control, did not bind LGRS.

Binding affinities between RSPO proteins and LGRS were analyzed by surface plasmon resonance. A soluble LGRS-Fc construct was generated using standard recombinant DNA techniques. Specifically, amino acids 1-564 of human LGRS were ligated in frame to human Fc and the recombinant LGRS-Fc fusion protein was expressed in insect cells using baculovirus. The LGRS-Fc fusion protein was purified from the insect medium using protein A chromatography. Cleavage of the LGRS signal sequence results in a mature LGRS-Fc fusion protein containing amino acids 22-564 of LGRS.

Recombinant RSPOl-Fc, Fc, RSPO3-Fc and RSPO4—F‘c fusion proteins were immobilized on CM5 chips using standard amine-based chemistry (NHS/EDC). Two- fold dilutions of soluble LGRS-Fc were injected over the chip surface (lOOnM to 0.78nM). Kinetic data were collected over time using a Biacore 2000 system from Biacore Life Sciences (GE Healthcare) and the data were fit using the simultaneous global fit equation to yield affinity constants (KD values) for each RSPO protein (Table Table 3 ““““““““““ E........................... .-.‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘ LGRS (nM) ......................................................................................................................................

Human RSPOl, RSPO2, RSPO3 and RSPO4 all bound to LGRS, demonstrating that RSPO proteins may be ligands for LGR proteins.

Example 3 In vitro testing for inhibition of B—catenin ing To e cell suspensions, fresh human lung adenocarcinoma xenograft tumors (lung tumor #1 in Table 2) propagated in NOD/SCID mice were minced and digested in medium 199 (Invitrogen, Carlsbad, CA) containing 300U/ml collagenase type 3 (Worthington, Lakewood, NJ) and 200U/m1 DNase I (Worthington, Lakewood, NJ) for l to 2 hours at 37°C. The lung tumor cells were filtered through a 40um nylon strainer (BD Falcon, Franklin Lakes, NJ), and spun down at 82 x g for 5 minutes. Red blood cells were lysed in ACK buffer (0.8% ammonium chloride, 0.1mM EDTA, 10mM sodium bicarbonate, 0.1N HCl), washed, and centrifuged at 150 xg for 5 minutes in medium consisting of HBSS (Mediatech, as, VA), 25mM HEPES buffer (Mediatech, Manassas, VA) and 2% nactivated fetal bovine serum (HI-FBS; ogen, Carlsbad, CA). Dead cells and debris were removed by fugation on a cushion of HI—FBS at 82 x g for 8 minutes. Mouse stroma cells were depleted using SOul MagnaBind streptavidin beads (Thermo ific, Waltham, MA) per 106 cells/ml after —113— staining with Sag/mi —conjugated -i-ZKd and 2.5ug/ml anti—nieuse CD45 moneeional antibodies (BioLegend, San Diege, CA) in SM. {925123 To e ecnditioned medium, the lung tumor celis were cultured in, DMEMElQ (3:1) medium tiinvitrogen, ad, CA) supplemented with .827 suppiement (invitregen, Carisbad, CA), insulinutransferrinselenium (liivitregen, Carlsbad, CA), periieiiiimstteptcmyein (invitmgen, Carisbad, CA), 0.5ug/mi hydrcccrtiscne (Stemeeli 'iE‘echneiogies, Vancouver, ), Mag/ml EGF (MEL intematienai, Webum, MA), ZGng/mi basic FGF (MEL international, Wobum, MA) and SU/ml heparin {Sigma-Aldrich, St. iamis, M0). After 24 hours the conditioned medium. was harvested (referred t0 herein as ‘1?) {@293} STF«293 cells are stabiy ected with a 6x’l‘CF«iiicii‘erase reporter vectcr. One volume of lung tumor cell—conditioned medium (LT) or control medium was added to STF-293 cells in the presence of purified soluble LGRS-Fc, FZD8-Fc, Jag-Fe fusion proteins (lOug/ml), an anti-FZD monoclonal antibody (40ug/ml), or antibody LZl (40ug/ml). In addition, Wnt3a L-cell-conditioned medium was used as a positive control and was tested in combination with the lung tumor cell-conditioned medium (LT) at a final dilution of 1:4. The cells were incubated for 16 hours and luciferase activity was measured using -Glo® Luciferase Assay System according to the manufacturer’s instructions (Promega, Madison, WI).

The effect of purified soluble c and FZD8-Fc fusion proteins was ed to the control c protein, and. the effect of the anti-FZD monoclonal antibody was compared to the control anti-bacterial lysozyme dy LZl. As shown in Figure 3 (left side), the lung tumor cell-conditioned medium (LT) contains an activity that potentiated the Wnt3a-induced B-catenin activity. The protein potentiating the B- catenin activity in the LT medium was inhibited by soluble LGRS-Fc which binds to RSPO proteins. This activity was also inhibited by FZD8-Fc and the anti-FZD antibody, agents that block Wnt signaling. Soluble Jag-Fe and LZldid not inhibit the activity.

Even in the absence of Wnt3a (Fig. 3, right side), the LT medium induced B-catenin signaling. Soluble Jag-Fc and LZldid not t this activity. In contrast, soluble LGRS-Fc inhibited the LT -induced B—catenin signaling, reducing the response to almost control levels, This data suggested that the lung tumor cells ed a protein (or —114— proteins) with RSPO-like activity, this activity was inhibited by LGRS, and this activity was separate from Wnt3a activity.

Similar experiments were undertaken using co-culture assays using lung tumor cell-conditioned medium and ovarian tumor cell-conditioned medium. As described above, freshly sed tumor cells depleted of stroma cells were cultured overnight.

Culture medium and cells were transferred to STF—293 cells with or without Wnt3a L- cell-conditioned medium. c fusion protein, a FZD8-Fc fusion protein, or a control Fc fusion protein was added (IOug/ml). The cells were incubated for 20 hours and luciferase activity was measured as bed above.

As shown in Figure 13, B-catenin signaling activity was induced by the tumor cells and supematants and further enhanced in combination with Wnt3a L-cell- ioned medium (Fig. 13A, lung tumor LU2; Fig. 13B, lung tumor LU25; Fig. 13C, ovarian tumor 0V3 8). FZD8-Fc, a Wnt y inhibitor, d the Wnt3a-induced B- catenin activity almost to background levels, while LGRS—Fc strongly reduced the tumor- d B-catenin activity. As above, this data suggested that the lung and ovarian tumor cells produced a protein (or proteins) with RSPO-like activity, that this activity was inhiEited by LGRS, and that this activity was te from Wnt3a activity.

Example 4 In vitro testing for inhibition ofRSPO ty by soluble LGRS Conditioned medium from human lung tumor #1 cells was prepared as described in e 3 and soluble LGRS-Fc and RSPOZ—Fc were ed as bed in Example 2.

HEK-293 cells were transfected with a 6xTCF-luciferase reporter vector (TOPflash, Millipore, Billerica, MA). After 24-48 hrs, the transfected cells were incubated with medium containing 25% lung tumor cell-conditioned medium plus 25% Wnt3a—L ceil«conditioned medium or medium containing RSPUZ (Mug/ml) pius 259i) Wnt3a-L cell-conditioned medium. Soluble LGRS was added to the cells in 4-fold serially dilutions at 20ug/ml to 0.02ug/ml. Soluble Jag-Fc protein was used as a negative control at 20ug/ml and FZD8—Fc protein was used as a positive control at 20ug/ml. The cells were incubated for 16 hours and rase activity was measured using Steady- Glo® Luciferase Assay System according to the manufacturer’s instructions ga, n, WI).

As shown in Figure 4, increasing concentrations of soluble LGRS-Fc reduced the ion of luciferase activity by the ation of RSPOZ-Fc plus Wnt3a-conditioned medium (-E|-) as well as the ion of luciferase activity by the ation of lung tumor cell-conditioned medium and Wnt3a—conditioned medium (-:-). Negative control Jag-Fc protein did not block the luciferase activity, while c, which blocks Wnt3a, blocked the luciferase activity. Importantly, LGRS displayed the same EC50 for inhibition with both the RSPOZ protein and the lung tumor cell-conditioned .

This data demonstrated that the protein(s) with RSPO-like activity produced by the lung tumor cells was inhibited by LGRS, behaved very similarly to a purified RSPO protein, and suggested that the activity in the lung tumor cell-conditioned media was due to a RSPO protein.

Example 5 Generation of anti—RSPOI monoclonal antibodies Antibodies were generated against recombinant human RSPOl protein amino acids 31-263 (R&D Systems, Minneapolis, MN). Mice (n=3) were immunized with RSPOl protein using standard techniques. Sera from individual mice were screened against RSPOl approximately 70 days after initial zation using FACS analysis.

The animal wth the highest antibody titer was selected for final antigen boost after which spleen cells were isolated for hybridoma tion. SPZ/O cells were used as fusion partners for the mouse spleen cells. Hybrédoma cells were plated at 1 cell per well in 96 well plates, and the supernatants were screened against human RSPOl by FACS analysis.

For FACS screening of anti-RSPOI antibodies a chimeric fusion protein enabling cell surface expression of the N-terminal furin-like domains of human RSPOI was constructed. As shown in Figure 5A, the fusion protein contains a N-terminal FLAG tag, followed by the two furin-like domains of RSPOI (aa 34-135) and fused to the transmembrane and intracellular domain of human CD4 and a C-terminal green fluorescent protein tag (FLAG—RSPOIfurin-CD4TM-GFP). -ll6- HEK-293 cells were transfected with FLAG—RSPOlfurin—CD4TM-GFP. After 48 hours, transfected cells were suspended in ice cold PBS containing 2% FBS and heparin and incubated on ice in the presence of SOul of hybridoma supernatants for 30 minutes.

A second tion with 100p] PIE-conjugated anti-human Fc secondary antibody was med to detect cells bound by antibody. Cells were incubated with an anti-FLAG antibody (Sigma-Aldrich, St. Louis, MO) as a positive control and an anti-PE dy as a negative control. The cells were analyzed on a FACSCalibur instrument (BD ences, San Jose, CA) and the data was processed using FlowJ0 software.

Several hybridomas were identified that bound RSPOl, including 89M2, 89M4, 89M5, 89M7, 89M19 and 89M25 (Fig. 5B). The heavy chain and light chain variable regions were sequenced from several of these antibodies. After analysis, it was found that dies 89M2, 89M4, 89M5, and 89M25 comprised the same heavy and light chain le regions. The hybridoma cell line expressing antibody 89M5 was deposited with the ATCC, 10801 University Boulevard, Manassas, VA, USA, under the conditions of the Budapest Treaty on June 30, 2011 and assigned ATCC deposit designation number PTA- 11970. The amino acid sequences of the heavy chain and light chain variable regions of 89M5 are SEQ ID NO:1O and SEQ ID NO:11. The nucleotide sequences of the heavy chain and light chain variable regions of 89M5 are SEQ ID NO:19 and SEQ ID N0220.

The heavy and light chain CDRs of 89M5 are listed in Table 1 herein. The amino acid sequences of the heavy chain and light chain of 89M5 are SEQ ID N021 and SEQ ID NO:22; the nucleotide ces of the heavy chain and light chain of 89M5 are SEQ ID N023 and SEQ ID NO:24.

Example 6 Identification of anti-RSPOI monoclonal antibodies that inhibit induction of [3- n signaling by RSPOl HER-293 cells were transfected with a 6xTCF-luciferase reporter vector (TOPflash, ore, Billerica, MA). After 24-48 hrs, the transfected HEK-293 cells were incubated with a combination of Wnt3a (Sng/ml) and human RSPOl (lOng/ml, R&D BioSystems) in the presence of anti-RSPOl dies 89M2, 89M4, 89M5, 89M7, 89M19, and 89M25, or 2 irrelevant control antibodies 254M14 and 254M26 (2-fold dilutions at 10ug/ml to 0.625ug/ml). The cells were incubated for 16 hours and luciferase —117- activity was measured using Steady-Glo® Luciferase Assay System according to the manufacturer’s instructions (Promega, n, WI). {0305] As shown in Figure 6, anti-RSPOI antibodies 89M2, 89M4, 89M5 and 89M25 each blocked signaling, s anti—RSPOI antibodies 89M7 and 89Ml9 did not block signaling. As determined by sequencing of the heavy chain and light chain varéable regions, dies 89M2, 89M4, 89M5 and 89M25 all comprise the same heavy chain and light chain variable regions and therefore, ably, the same antigen binding site.

These results trated that an anti-RSPOI antibody was able to block RSPOl- induced B-catenin signaling.

Example 7 Anti-RSFOI antibodies block g of soluble RSPOl to LGRS HEK-293 cells were ently transfected with the FLAG-LGRS-CD4TM-GFP construct (previously described in Example 2). After 48 hours, transfected cells were suspended in ice cold PBS containing 2% FBS and heparin and incubated on ice in the presence of RSPOl-Fc protein (lOug/ml) and antibodies 89M2, 89M4, 89M5, 89M7, 89Ml9 or 89M25 (lOug/ml). A second incubation with 100141 jugated anti-human Fc secondary antibody was performed to detect cells bound by the RSPOl-Fc fission protein. The cells were analyzed on a FACSCalibur instrument (BD ences, San Jose, CA) and the data was processed using FlowJo software.

As shown in Figure 7, anti—RSPOI antibodies 89M2, 89M4, 89M5 and 89M25 each blocked binding of RSPOl to LGRS, whereas anti-RSPOl antibodies 89M7 and 89Ml9 did not block binding of RSPOl to LGRS. These results correlate with the results shown in Example 6 which demonstrated the ability of antibodies 89M2, 89M4, 89M5 and 89M25 to block RSPOl signaling in an assay measuring induction of B-catenin activity in a 6xTCF luciferase reporter assay, Whereas antibodies 89M7 and 89M19 were not able to block RSPOl signaling. As discussed above, antibodies 89M2, 89M4, 89M5 and 89M25 all se the same heavy chain and light chain variable regions and presumably the same antigen binding site, therefore it would be expected that these antibodies all on in a similar, if not identical, manner.

Example 8 g affinities of anti-RSPOI antibodies The KDs of antibodies 89M4, 89M5, 89M7 and 89M25 were ined using a e 2000 system from Biacore LifeSciences (GE Healthcare). Recombinant human RSPOl-Fc or mouse RSPOl-Fc proteins were immobilized on CM5 chips using standard amine-based chemistry (NHS/EDC). The antibodies were serially diluted 2-fold from 100nM to 0.78nM in HBS-P (0.01M HEPES pH7.4, 0.15M NaCl, 0.005% v/v Surfactant P20) and were injected over the chip surface. Kinetic data were collected over time and were fit using the simultaneous global fit equation to yield affinity nts (KD values) for each antibody.

As shown in Table 4, antibodies 89M4, 89M5, 89M7 and 89M25 all had an affinity constants (KD) for human RSPOI of less than 0.1nM. These antibodies also had KD of less than 0.1nM for mouse RSPOl.

Example 9 Inhibition of ovarian tumor growth in vivo by SPOI antibodies Dissociated OV19 ovarian tumor cells (1 x 105cells) were injected in the mammary fat pads of 6-8 week old NOD/SCID mice. Tumors were allowed to grow for 45 days until they reached an average volume of 134mm3. The mice were randomized (n = 10 per group) and treated with anti-RSPOI antibody 89M5, 89M25, taxol, a combination of 89M5 and taxol, a combination of 89M25 and taxol, or control antibody 1B7.11. Antibodies were dosed at 15mg/kg once a week, and taxol was dosed at 7.5mg/ml once a week. Administration of the antibodies and taxol was performed via -ll9- injection into the intraperitoneal cavity. Tumor growth was red and tumor ‘Volumes were measured with electronic calipers at the indicated time points. Data are expressed as mean i S.E.M.

At day 35, treatment with antibody 89M5 resulted in a 40% reduction in tumor growth and 89M25 resulted in a 25% reduction in tumor growth as ed to treatment with the control antibody (Fig. 8, p = 0.37 and p = 0.19, respectively). Treatment with 89M5 or 89M25 in combination with taxol resulted in a reduction of tumor growth greater than treatment with either agent alone. Treatment with 89M5 and taxol ed in a 48% reduction in growth (p = 0.12 vs. the control group), and treatment with 89M25 and taxol ed in a 43% reduction in growth (p = 0.16 vs. the control group). Thus, dies 89M5 and 89M25 demonstrated anti-tumor growth activity in the OVl9 ovarian tumor model as a single agent, and also displayed anti—tumor growth activity in combination with taxol.

Subsequent analysis of the tumors from the mice used in this experiment (both control and treated mice) revealed that the tumors were a mixture of human ovarian tumor cells (OV19) and murine T-cell lymphoma cells.

Example 10 e mapping of anti—RSPOI monoclonal dy 89M5 To further characterize the specific region(s) of RSPOl that antibody 89M5 binds, an epitope mapping experiment was performed. A series of constructs comprising different s of human RSPOl were generated using standard recombinant DNA technology (see Fig. 9A). The constructs were fusion proteins each containing a N- terminal FLAG tag, followed by a n of RSPOl protein and fused to the transmembrane and intracellular domain of human CD4. In some versions the fusion proteins also comprise a C-terminal green cent protein tag.

HEK-293 cells were transfected with the individual constructs. After 48 hours, transfected cells were suspended in ice cold PBS containing 2% FBS and heparin and incubated on ice in the presence of anti—RSPOI antibody 89M5 for 30 minutes. A second incubation with 100111 PE—conjugated anti-human Fc secondary antibody was med to detect cells bound by antibody. Cells were incubated with an anti-FLAG antibody (Sigma-Aldrich, St. Louis, MO) as a positive l and an anti-PE antibody as a negative control. The cells were analyzed on a FACSCalibur instrument (BD Biosciences, San Jose, CA) and the data was processed using FlowJo software.

As shown in Figure 9B, the FACS analysis suggests that amino acids within the furin2 domain of RSPOI are involved in the binding site for anti-RSPOI antibody 89M5 (Example 10, Fig. 9). These preliminary results do not preclude that fact that amino acids in other RSPOI domains may be involved in the binding site.

Example 1 1 tion of anti—RSP02 monoclonal antibodies Antibodies were generated t recombinant human RSP02 protein amino acids 22-205 (R&D Systems, Minneapolis, MN)“ Mice (n=3) were immunized with RSP02 protein using standard techniques. Sera from individual mice were screened t RSP02 imately 70 days after initial zation using FACS analysis.

The animal with the t antibody titer was selected for final antigen boost after which spleen cells were isolated for hybridoma production. SP2/0 cells were used as fusion partners for the mouse spleen cells. Hybridoma cells were plated at 1 cell per well in 96 well plates, and the supematants were screened against human RSP02 by FACS analysis.

As described in Example 5, for FACS screening of anti-RSP02 antibodies a chimeric fusion protein enabling cell surface expression of the N-terminal furin—like domains of human RSP02 was constructed. Similar to what is depicted in Figure 5A for RSPOI, the RSP02 fusion protein contains a N-terminal. FLAG tag, ed by the furin-like s of RSP02 (aa 31-146) and fused to the transmembrane and intracellular domain of human CD4 and a inal green fluorescent n tag (FLAG-RSP02furin-CD4TM-GFP).

HEK-293 cells were transfected with FLAG-RSPOqurin-CD4TM-GFP. After 48 hours, transfected cells were suspended in ice cold PBS containing 2% FBS and heparin and incubated on ice in the presence of 50p] of hybridoma supematants for 30 minutes.

A second incubation with 100ul PE—conjugated anti-human Fc secondary antibody was performed to detect cells bound by antibody. Cells were incubated with an anti-FLAG antibody (Sigma—Aldrich, St. Louis, MO) as a positive control and an anti-PE antibody as -121— a negative control. The cells were analyzed on a FACSCalibur instrument (BD Biosciences, San Jose, CA) and the data was processed using FlowJ0 re.

Several omas were identified that bound RSPOZ, including , 130M24, 130M25, 130M26, 130M27, and 130M28 (Fig. 10). The heavy chain and light chain variable regions were sequenced from several of these antibodies. The hybridoma cell line expressing antibody 130M23 was deposited with the ATCC, 10801 University Boulevard, Manassas, VA, USA, under the ions of the Budapest Treaty on August , 2011 and assigned ATCC t designation number PTA-12021. The amino acid sequences of the heavy chain variable region and light chain variable region of 130M23 are SEQ ID N027 and SEQ ID NOz28. The nucleotide sequences of the heavy chain and light chain variable regions of 130M23 are SEQ ID NO:35 and SEQ ID NO:36. The heavy chain and light chain CDRS of 130M23 are listed in Table 1 herein. The amino acid sequences of the heavy chain and light chain of 130M23 are SEQ ID N037 and SEQ ID NO:38; the nucleotide sequences of the heavy chain and light chain of 130M23 are SEQ ID NO:39 and SEQ ID N0240.

Example 12 Identification of anti-RSP02 monoclonal antibodies that inhibit induction of S— catenin signaling by RSP02 HEK-293 cells were transfected with a 6xTCF-luciferase reporter vector (TOPflash, Millipore, Billerica, MA). After 24-48 hrs, the ected HEK-293 cells were incubated with a combination of Wnt3a (Sng/ml) and human RSP02 (lOng/ml, R&D BioSystems) or human RSPO3 (10ng/ml, R&D BioSystems) in the presence of anti-RSP02 antibodies 130M23, 130M24, , 130M26, 130M27, and 130M28.

Cells were incubated with a combination of Wnt3a and RSPO, Wnt3a only or with no addition as ls. The cells were incubated for 16 hours and Iuciferase activity was measured using Steady-Glo® Luciferase Assay System ing to the manufacturer’s instructions (Promega, Madison, WI).

As shown in Figure 11, anti-RSP02 antibodies 130M23, 130M24, 130M25, 130M26, 130M27, and 130M28 each reduced RSPOZ-induced B-catenin signaling, and anti-RSP02 antibodies 130M23, 130M24 completely blocked RSPOZ-induced B-catenin signaling. In contrast these dies did not block B-catenin ing induced by —122- RSPO3. These results demonstrated that antibodies 130M23, 130M24, 130M25, 130M26, 130M27, and 130M28 are specific inhibitors of RSP02 and are capable of reducing and/or completely blocking RSP02—induced B-catenin signaling.

Example 13 SP02 antibodies block binding of soluble RSP02 to LGRS 3 cells were transiently transfected with the FLAG-LGRS-CD4TM-GFP construct (previously described in Example 2). After 48 hours, transfected cells were suspended in ice cold PBS containing 2% FBS and heparin and ted on ice in the presence of RSP02-Fe protein (IOug/ml) and antibodies 130M23, 130M24, 130M25, 130M26, 130M27, and 130M28. A second incubation with lOOul PE—conjugated anti- human Fc secondary antibody was med to detect cells bound by the RSP02-Fe fusion protein. The cells were analyzed on a FACSCalibur instrument (BD Biosciences, San Jose, CA) and the data was processed using FlowJo software.

As shown in Figure 12, anti-RSP02 antibodies 130M23 and 130M24 each blocked binding of RSP02 to LGRS, whereas SP02 antibodies 130M25, 130M26, 130M27, and 130M28 only weakly blocked or did not block binding of RSP02 to LGRS.

These results coréfelate with the s shown in Example 11 which demonstrated the y of antibodies 130M23 and 130M24 to completely block RSP02-induced nin ing, whereas antibodies 130M25, 130M26, 130M27, and 130M28 were less potent inhibitors of RSP02-induced B-catenin signaling.

Example 14 Generation of anti-RSPO3 monoclonal dies Antibodies are generated against recombinant human RSPO3 protein amino acids 22-272 (R&D Systems, Minneapolis, MN). Mice (11:3) are immunized with RSPO3 n using standard techniques. Sera fzom individual mice are screened against RSPO3 approximately 70 days after initial immunization using FACS analysis. The animal with the highest antibody titer is ed for final antigen boost after which spleen cells are isolated for hybridoma production, SP2/0 cells are used as fusion partners for the mouse spleen cells. Hybridoma cells are plated at 1 cell per well in 96 well plates, and the supematants are screened against human RSPO3 by FACS analysis.

As described in Example 5, for FACS screening of anti-RSPO3 antibodies a chimeric fusion n enabling cell surface expression of the N-terminal like domains of human RSPO was constructed. Similar to what is depicted in Figure 5A for RSPOl, the RSPO3 fusion protein contains a N—terminal FLAG tag, followed by the furin-like domains of RSPO3 (aa 32-141) and fused to the transmembrane and intracellular domain of human CD4 and a C-terminal green fluorescent protein tag (FLAG-RSPO3furin-CD4TM-GFP).

HEK-293 cells are transfected with FLAG-RSPO3furin-CD4TM—GFP. After 48 hours, transfected cells are suspended in ice cold PBS containing 2% FBS and heparin and incubated on ice in the presence of 50p] of hybridoma supernatants for 30 minutes.

A second incubation with 100ul PE—conjugated anti—human Fc secondary antibody is performed to detect cells bound by antibody. Cells are incubated with an anti-FLAG antibody (Sigma-Aldrich, St. Louis, MO) as a positive control and an anti-PE antibody as a negative control. The cells are analyzed on a libur ment (BD Biosciences, San Jose, CA) and the data is processed using Flow]0 software.

Example 15 Binding affinity of anti-RSP02 antibody 130M23 The KB of 130M23 was determined using a e 2000 system from Biacore LifeSciences (GE Healthcare). inant human RSP02-Fe or mouse RSP02—Fe proteins were immobilized on, CMS chips using standard based chemistry (NHS/EDC). The antibodies were serially diluted 2-fold from 100nM to 0.78nM in HBS- P (0.01M HEPES pH7.4, 0.15M NaCl, 0.005% V/v tant P20) and were injected over the chip surface. Kinetic data were collected over time and were fit using the simultaneous global fit equation to yield affinity nts (KD ) for each antibody.

Antibody 130M23 had an affinity constant (KD) for human RSP02 of 0. l4nM and a KD of 0.35nM for mouse RSP02. — 124 — Example 16 In vitro testing for inhibition of RSPO ty by anti—RSP02 antibody Conditioned medium from human lung tumor LU2 cells was prepared as described in e 3 and soluble LGRS—Fc was ed as described in Example 2.

STF-293 cells were incubated with LU2 cells plus 25% lung tumor cell- conditioned medium plus 25% Wnt3a-L onditioned medium. dy 130M23 and soluble LGRS-Fc were added to the cells in 5-fold serially dilutions from SOug/ml to 0.0006ug/ml. An irrelevant monoclonal antibody, similarly diluted, and a control Fc fusion protein (50ug/ml) were used as negative controls. The cells were incubated for 20 hours and luciferase activity was measured using Steady-Glo® Luciferase Assay System according to the manufacturer’s instructions (Promega, Madison, WI).

As shown in Figure 14, sing trations of soluble c () reduced the induction of luciferase activity by the combination of lung tumor cell- conditioned medium and Wnt3a-conditioned medium. Increasing concentrations of anti- RSPO2 dy 130M23 (-I—) also reduced the induction of luciferase activity by the combination of lung tumor cell-conditioned medium and Wnt3a-conditioned medium. 130M23 blocked conditioned medium induced activity with an IC50 of 129nM and was greater than 100—fold more potent than LGRS-Fc. A control Fc fusion protein (-A-), as well as an irrelevant antibody () did not block the luciferase activity.

Example 17 Inhibition of pancreatic tumor growth in vivo by anti-RSPO antibodies Dissociated PN31 atic tumor cells (1 x 1050ells) were injected subcutaneously into the flanks of 6-8 week old NOD/SCID mice. Tumors were allowed to grow for 61 days until they reached an average volume of 120mm3. The mice were ized (n = 10 per group) and treated with anti-RSPOI antibody 89M5, anti-RSPO2 antibody 130M23, gemcitabine, a combination of 89M5 and gemcitabine, a combination of 130M23 and gemcitabine, or control antibody . Antibodies were dosed at 15mg/kg once a week, and gemcitabine was dosed at 30mg/ml once a week.

Administration of the antibodies and gemcitabine was performed via injection into the — 1'25 — eritoneal cavity. Tumor growth was monitored and tumor volumes were measured with electronic calipers at c time points. Data are sed as mean i S.E.M.

As shown in Figure 15, treatment with anti-RSPOI dy 89M5 or anti-RSP02 antibody 130M23 as single agents had only a minimal effect on tumor growth. Treatment with gemcitabine alone d tumor growth by 49% as ed to the controls (p = 0.09). However, treatment with 89M5 or 130M23 in combination with gemcitabine resulted in a reduction of tumor growth greater than treatment with either agent alone.

Treatment with 89M5 and gemcitabine resulted in a 59% reduction in growth (p = 0.015 vs. the control group), and treatment with 130M23 and gemcitabine ed in a 58% reduction in growth (p = 0.016 vs. the control group). Thus, anti-RSPOI antibody 89M5 and anti-RSP02 antibody 130M23 demonstrated strong anti—tumor growth activity in combination with gemcitabine in a pancreatic xenograft model.

Example 18 Inhibition of pancreatic tumor grth in vivo by anti-RSPO antibodies in combination with Wnt pathway inhibitors Dissociated PN7 pancreatic tumor cells (1 x 105cells) were injected subcutaneously into the flanks of 6-8 week old NOD/SCID mice. Tumors were d to grow for 25 days until they reached an average volume of 130mm3. The mice were randomized (11 _ 10 per group) and treated with anti-RSPO2 antibody 130M23, ZD antibody 18R5, gemcitabine, a combination of 130M23 and gemcitabine, a combination of 18R5 and abine, a combination of 130M23 and 18R5, a combination of 130M23, 18R5 and gemcitabine, or control antibody 1B7.11. SPO2 antibody 130M23 was dosed at g once a week, anti—FZD antibody 18R5 was dosed at 20mg/kg once a week, and gemcitabine was dosed at 30mg/ml once a week.

Administration of the antibodies and gemcitabine was performed via injection into the intraperitoneal cavity. Tumor growth was monitored and tumor volumes were measured with electronic calipers at specific time points. Data are expressed as mean i S.E.M. A parallel set of experiments included mice treated with a FZDS-Fc soluble receptor (10mg/kg) in combination with gemcitabine and FZD8-Fc in combination with 130M23 and gemcitabine. —126- Treatment with antibody 130M23 or antibody 18R5 as a single agent resulted in approximately 55% reduction in tumor growth as compared to treatment with the control antibody (Fig. 16A, p < 0.001). Treatment with 130M23 or 18R5 in combination with gemcitabine resulted in a reduction of tumor growth r than treatment with either agent alone. Treatment with 130M23 and gemcitabine resulted in a 68% reduction in growth (p < 0.001 vs. the l group), and treatment with 18R5 and gemcitabine resulted in a 75% reduction in growth (p < 0.001 vs. the control group). Furthermore, a combination of 130M23, gemcitabine and 18R5 resulted in almost complete inhibition of growth of the PN7 tumors (Fig. 16A). Similar results were seen with a combination of 130M23, gemcitabine and a FZD8-Fc soluble receptor (Fig. 16B). Thus, an anti—RSPOZ antibody such as 130M23 has single agent ty in inhibiting pancreatic tumor growth.

Furthermore, combination of an anti—RSP02 antibody with gemcitabine, or a ation of an anti—RSPO2 dy wéth gemcitabine and a Wnt y inhibitor such as anti- FZD dy 18R5 or a FZDS-Fc e receptor, was shown to be a very effective therapy for inhibiting tumor growth in a pancreatic tumor model.

IHC studies showed that the anti-RSP02 antibody 130M23 d morphological changes in the PN7 tumors of treated mice as ed to untreated mice.

These cells also displayed a significant decrease in proliferation using an anti-Ki67 antibody. These results possibly reflect a loss in tumor cells and an increase in stroma. {0337] The PN7 tumors» described above were processed to yield single cell suspensions.

Mouse cells were depleted from the cell mixtures using biotinylated anti-H2Kd and anti- CD45 antibodies and streptavidin-conjugated magnetic beads. The remaining human tumor cells were serially transplanted into a new cohort of mice. 90 tumor cells from each treatment group were injected into the flanks of NOD—SCID mice (n = 10 mice per group). Tumors were allowed to grow for 40 days with no treatment and tumor volumes were measured with electronic calipers.

Figure 16C shows the tumor volume from individual mice in each group. Cells isolated from mice treated with anti-RSE~‘02 dy 130M23 or anti-FZD antibody 18R5 as single agents or in a combination had greatly decreased tumorigenicity as compared to cells isolated from mice treated with control antibody. This d tumorigenicity was much greater than the decrease in tumorégenicity observed with gemcitabine alone. Cells from mice treated with combinations of gemcitaEine and —127- 130M23, gemcitabine and 18R5, or gemcitabine and FZD8—Fc showed tumorigenicity that was only slightly reduced as compared to cell isolated from mice treated “éth control antibody. Interestingly, cells isolated from mice treated with a combination of , 18R5 and abine or 130M23, FZD8-Fc and gemcitabine demonstrated a significant and striking lack of tumor growth, greater than any of the agents alone or in two agent combinations. These results showed that inhibiting multiple pathways in addition to standard chemotherapy has an additive, and possibly a synergistic effect in reducing tumorigenicity and cancer stem cells.

Example 19 Humanization ofRSPO antibodies Humanized antibodies against human RSPOI and RSP02 were generated. The heavy chain variable region and the light chain variable region of the murine onal antibodies 89M5 and 130M23 were isolated and sequenced from the hybrédoma line using degenerate PCR essentially as described in Larrick, J.M., et al., 1989, Biochem.

Biophys. Res. Comm. 160: 1250 and Jones, S.T. & Bendig, M.M., 1991, Bio/Technology 9: 88. Human heavy and light chain variable framework regions likely to be structurally similar to the parental 89M5 or 130M23 antibody amino acid sequences were then considered as reference human framework s to help guide the design of novel synthetic frameworks. To identify the human framework regions bearing similarity to muréne orks, the predicted protein sequences encoded by the murine heavy chain and light chain variable domains of 89M5 and 130M23 were compared with human antibody sequences encoded by expressed human cDNA using BLAST es for human ce deposited in Genbank. The amino acid ences between candidate humanized ork heavy chains and the parent murine monoclonal antibody heavy chain le regions and light chain variable regions were evaluated for likely importance, and a judgment made as to whether each difference in position contributes to proper folding and function of the variable . This analysis was guided by examination of solved crystal structures of other antibody fragments (e.g., the ure of Fab 2E8 as described in Trakhanov et a1, Acta Crystallogr D Biol llogr, 1999, 55:122-28, as well as other protein crystal structures (e.g., protein data bank structures lADQ and lGIG)). Structures were modeled using computer software including Jmol, quick PDB, and Pymol. Consideration was given to the potential impact of an amino acid at a given position on the packing of the t framework, the interaction between the heavy and light chain variable regions, the degree of solvent exposure of the amino acid side chain, and the likelihood that an amino acid would impact the positioning of the CDR loops. From this analysis, candidate heavy chain variable regions fused me to the human IgG2 constant region and candidate light chain variable s fused in frame with the human IgKCl constant region were conceived and chemically synthesized. The candidate heavy chains and light chains se: i) a synthetic framework designed to resemble natural human frameworks and ii) the parental 89M5 or 130M23 murine antibody CDRs.

The functionality of each candidate variant humanized heavy chain and light chain was tested by cotransfection into mammalian cells. Each candidate humanized heavy chain described above was cotransfected into HEK-293 cells with the murine light chain cDNA, and conditioned media was d by FACS for RSPO g activity.

Humanized 89M5 heavy chain variant “89M5-H2” (SEQ ID NO:68) ted the most robust binding and was selected. The 89M5-H2 humanized heavy chain was cotransfected into 3 cells with each of the candidate humanized light chains, and conditioned media was again assayed for antigen binding by FACS. Light chain variant “89M5-L2” (SEQ ID N0269) exhibited the most robust g and was selected.

Similarly the humanized 130M23 heavy chain variant “130M23—H1” (SEQ ID NO:70) exhibited the most robust binding and was ed. The 130M23-H1 humanized heavy chain was cotransfected into HEK-293 cells with each of the ate humanized light chains, and conditioned media was again assayed for antigen binding by FACS. Light chain variant “130M23-L2” (SEQ ID NO:71) exhibited the most robust binding and was selected.

To increase antibody production, a variant of 130M23-H1L2 was generated. The variant comprises the same heavy chain as 130M23-H1 L2, but has a modified light chain and is referred to as hl30M23-H1L6.

Example 20 Binding affinity of humanized 89M5 and humanized 130M23 The KB of h89M5-H2L2 was determined using a e 2000 system from Biacore LifeSciences (GE Healthcare). Recombinant human RSPOl-Fc or mouse RSPOl—Fc ns were immobilized on CMS chips using standard amine-based chemistry (NHS/EDC). The antibodies were serially diluted 2-fold from 100nM to 0.78nM in HBS-P (0.01M HEPES pH7.4, 0.15M NaCl, 0.005% v/v Surfactant P20) and were injected over the chip surface. Kinetic data were collected over time and were fit using the simultaneous global fit equation to yield affinity constants (KD values) for each antibody.

H2L2 had an affinity constant (KD) for human RSPOl and mouse RSPOl ofless than 0.1nM.

The KD of h130M23—H1L2 and hl30M23-H1L6 were determined using a e 2000 system from Biacore LifeSciences (GE Healthcare). Recombinant human RSP02- Fc protein was immobilized on CMS chips using standard based try (NHS/EDC). The antibodies were serially diluted 2-fold from 100nM to 0.78nM in HBS- P (0.01M HEPES pH7.4, 0.15M NaCl, 0.005% V/v Surfactant P20) and were injected over the chip surface. Kinetic data were collected over time and were fit using the simultaneous global fit equation to yield affinity constants (KD values) for each antibody. h130M23-H1L2 had an affinity constant (KD) for human RSP02 of 0.13nM and h130M23-H1L6 had an affinity constant (KD) for human RSP02 of 0.15nM.

Example 21 FACS binding ofanti-RSPO dies HEK293 cells were transiently transfected with an expression vector encoding FLAG-RSPOlfurin-CD4TM-GFP. As described in Example 5, FLAG-RSPOlfurin- CD4TM-GFES3 is a chimeric fusion protein ng cell surface expression of the N- terminal furin-like domains of human RSPOl. FLAG-RSPOIfarin-CD4TM-GFP transfected cells were ted in the presence of anti-RSPOI antibody 89M5 or humanized anti-RSPOI antibody H2L2. Five-fold serial dilutions of each -130— antibody were ed for their ability to bind to the RSPOl expressing cells. The cells were d with rythrin, conjugated anti-IgG to reveal bound antibody. The cells were analyzed on a FACSCalibur ment (BD Biosciences, San Jose, CA) and the data was processed using FlowJo software.

As shown in Figure 17A, these studies indicate that both anti-RSPOI antibody 89M5 and humanized anti-RSPOl antibody h89M5-H2L2 bind to human ESPOI.

HEK293 cells were transiently transfected with an expression vector ng FLAG-RSP02furin-CD4TM-GFP. As described in Example 11, FLAG-RSPO2furin- GFP is a chimeric fusion n enabling cell e expression of the N- terrninal furin—like domains of human RSP02. FLAG-RSP02furin-CD4TM-GFP transfected cells were incubated in the presence of anti-RSP02 antibody 130M23 or humanized anti-RSP02 antibody h130M5-H1L2. Five-fold serial dilutions of each antibody were examined for their ability to bind to the RSP02 expressing cells. The cells were stained with Phycoerythrin ated anti-IgG to reveal bound antibody. The cells were analyzed on a libur instrument (BD Biosciences, San Jose, CA) and the data was sed using FlowJo software.

As shown in Figure 17B, these studies indicate that both anti-RSP02 antibody 130M23 and humanized anti-RSP02 antibody hl 30M23-H1L2 bind to human RSP02.

Example 22 Inhibition of breast tumor growth in vivo by anti—RSPO antibodies in combination with a chemotherapeutic agent Dissociated OMP-B39 breast tumor cells (4 x ls) were injected subcutaneously into the flanks of 6—8 week old NOD/SCID mice. OMP—O39 is a triple negative breast cancer tumor with a high level of RSP02 expression. In addition, the level of RSPOl is higher than other breast tumors characterized in Example 1 (see Table 2). Tumors were allowed to grow for 39 days until they reached an average volume of 120mm3. The mice were randomized (n = 10 per group) and treated with a combination of anti-RSP01 antibody 89M5 and anti—RSP02 antibody 130M23, cisplatin, a combination of anti-RSPOI and RSP02 antibodies and cisplatin, or a control antibody.

Antibodies were dosed at lSmg/kg once a week and cisplatin was dosed at 1.5mg/kg twice a week. Administration of the antibodies and cisplatin was performed via injection into the intraperitoneal cavity. Tumor growth was monitored and tumor volumes were measured with electronic rs on the indicated days. Data are expressed as mean i S.E.M.

As shown in Figure 18, a combination of anti-RSPOlantibody 89M5 and anti- RSP02 antibody 130M23 with cisplatin inhibited tumor growth better than cisplatin alone (p = 0.04, combination group vs tin alone). The triple combination had a significant , despite the fact that the combination of antibodies 89M5 and 130M25 Without tin had only a l effect on this tumor.

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

All publications, patents, patent applications, intemet sites, and accession numbers/database sequences including both polynucleotide and polypeptide sequences cited herein are hereby incorporated by reference herein in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, intemet site, or accession number/database sequence were specifically and individually ted to be so incorporated by reference. —132- Human RSPOl protein sequence with signal sequence (SEQ ID N021) MRLGLCVVALVLSWTHLTISSRGIKGKRQRRESAEGSQACAKGCELCSEVNGCLKCSPKL FILLERNDIRQVGVCLPSCPPGYFDARNPDMNKCIKCKIEHCEACFSHNFCTKCKEGLYL HKGRCYPACPEGSSAANGTMECSSPAQCEMSEWSPWGPCSKKQQLCGFRRGSEERTRRVL HAPVGDHAACSDTKETRRCTVRRVPCPEGQKRRKGGQGRRENANRNLARKESKEAGAGSR RRKGQQQQQQQGTVGPLTSAGPA Human RSP02 protein sequence with signal sequence (SEQ ID N0z2) MQFRLFSFALIILNCMDYSHCQGNRWRRSKRASYVSNPICKGCLSCSKDNGCSRCQQKLF FFLRREGMRQYGECLHSCPSGYYGHRAPDMNRCARCRIENCDSCFSKDFCTKCKVGFYLH RGRCFDECPDGFAPLEETMECVEGCEVGHWSEWGTCSRNNRTCGFKWGLETRTRQIVKKP VKDTIPCPTIAESRRCKMTMRHCPGGKRTPKAKEKRNKKKKRKLIERAQEQHSVFLATDR Human RSP03 protein sequence with signal sequence (SEQ ID N0:3) MHLRLISWLFIILNFMEYIGSQNASRGRRQRRMHPNVSQGCQGGCATCSDYNGCLSCKPR LFFALERIGMKQIGVCLSSCPSGYYGTRYPDINKCTKCKADCDTCFNKNFCTKCKSGFYL HLGKCLDNCPEGLEANNHTMECVSIVHCEVSEWNEWSPCTKKGKTCGFKRGTETRVREII QHPSAKGNLCPPTNETRKCTVQRKKCQKGERGKKGRERKRKKPNKGESKEAIPDSKSLES QRENKQQQKKRKVQDKQKSVSVSTVH Human RSP04 protein sequence with signal sequence (SEQ ID N0:4) MRAPLCLLLLVAHAVDMLALNRRKKQVGTGLGGNCTGCIICSEENGCSTCQQRLFLFIRR EGIRQYGKCLHDCPPGYFGIRGQEVNRCKKCGATCESCFSQDFCIRCKRQFYLYKGKCLP TCPPGTLAHQNTRECQGECELGPWGGWSPCTHNGKTCGSAWGLESRVREAGRAGHEEAAT SRKCPIQRPCPGERSPGQKKGRKDRRPRKDRKLDRRLDVRPRQPGLQP Human RSPOl protein sequence without ted signal sequence (SEQ ID N0:5) SRGIKGKRQRRISAEGSQACAKGCELCSEVNGCLKCSPKLFILLERNDIRQVGVCLPSCP PGYFDARNPDMNKCIKCKIEHCEACFSHNFCTKCKEGLYLHKGRCYPACPEGSSAANGTM ECSSPAQCEMSEWSPWGPCSKKQQLCGFRRGSEERTRRVLHAPVGDHAACSDTKETRRCT VRRVPCPEGQKRRKGGQGRRENHNRNLARKESKEHGAGSRRRKGQQQQQQQGTVGPLTSA Human RSPOl furin-like domain 1 (SEQ ID NO:6) AEGSQACAKGCELCSEVNGCLKCSPKLFILLERNDIRQVGVCLPSCPPGYFD Human RSP01 furin-like domain 2 (SEQ ID N027) MNKCIKCKIEHCEACFSHNFCTKCKEGLYLHKGRCYPACPEGSSA Human RSPOl thrombospondin domain (SEQ ID N0:8) QCEMSEWSPWGPCSKKQQLCGFRRGSEERTRRVLHAPVGDHAACSDTKETRRCTVRRVPCP Human RSPOI amino acids 31 263 (SEQ ID N019) RISAEGSQACAKGCELCSEVNGCLKCSPKLFELLERNDIRQVGVCLPSCPPGYFDARNPD MNKCIKCKIEHCEACFSHNFCTKCKEGLYLHKGRCYPACPEGSSAANGTMECSSPAQCEM SEWSPWGPCSKKQQLCGFRRGSEERTRRVLHAPVGDHAACSDTKETRRCTVRRVPCPEGQ KRRKGGQGRRENANRNLARKESKEAGAGSRRRKGQQQQQQQGTVGPLTSAGPA 89M5 Heavy chain variable region (SEQ ID NO:10) EVQLQQSGPELVKPGASVKISCKTSGYTFTGYTMHWVRQSHGKTLEWIGGINPNNGGTTY NQNFKGKATLTVEKSSTTAYLELRSLTSEDSALYYCARKEFSDGYYFFAYWGQGTLVTVSA 89M5 Light chain variable region (SEQ ID NO:11) DIVMTQSHKFMSTSVGDRVNITCKASQDVIFAVAWYQQKPGQSPKLLIYWASTRHTGVPD RFTGSVSGTDYTLTISSVQAEDLALYYCQQHYSTPWTFGGGTKLEIK 89M5 Heavy chain CDRl (SEQ ID NO: 12) TGYTMH 89M5 Heavy chain CDR2 (SEQ ID NO:13) GINPNNGGTTYNQNFKG 89M5 Heavy chain CDR3 (SEQ ID NO:14) KEFSDGYYFFAY 89M5 Light chain CDRl (SEQ ID NO:15) KASQDVIFAVA 89M5 Light chain CDR2 (SEQ ID NO:16) WASTRHT 89M5 Light chain CDR3 (SEQ ID NO:17) QQHYSTPW FLAG Tag (SEQ ID NO:18) DYKDDDDK 89M5 Heavy chain variable region nucleotide sequence (SEQ ID NO:19) GAGGTCCAGCTGCAACAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGATA TCCTGCAAGACTTCTGGATACACATTCACTGGATACACCATGCACTGGGTGAGGCAGAGC CATGGAAAGACCCTTGAGTGGATTGGAGGTATTAATCCTAACAATGGTGGTACTACTTAC AACTTCAAGGGCAAGGCCACATTGACTGTAGAGAAGTCCTCCACCACAGCCTAC TTGGAGCTCCGCAGCCTGACATCTGAGGATTCTGCACTCTATTACTGTGCAAGAAAGGAG TTCTCTGATGGTTACTACTTTTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCT 89M5 Light chain variable region nucleotide sequence (SEQ ID NO:20) GACATTGTGATGACCCAGTCTCACAAATTCATGTCCACATCAGTGGGAGACAGGGTCAAC TGCAAGGCCAGTCAGGATGTGATTTTTGCTGTAGCCTGGTATCAACAGAAACCA GGACAATCTCCTAAACTACTGATTTACTGGGCATCCACCCGGCACACTGGAGTCCCTGAT CGCTTCACAGGCAGTGTATCTGGGACAGATTATACTCTCACCATCAGCAGTGTGCAGGCT CTGGCACTTTATTACTGTCAGCAACATTATAGCACTCCGTGGACGTTCGGTGGA GGCACCAAGCTGGAAATCAAA 89M5 Heavy chain amino acid sequence with predicted signal sequence underlined (SEQ ID NO:21) —134- GKTLEWIGGINPNNGGTTYNQNFKGKATLTVEKSSTTAYLELRSL?SEDSALYYCARKEF SDGYYFFAYWGQGTLVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVT SSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPR DCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVE VHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRP KAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGS YFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK 89M5 Light chain amino acid sequence with predicted signal sequence underlined (SEQ ID NO:22) MGFKMESQIQQEYEXEQWgSGVDGDIVMTQSHKFMSTSVGDRVNITCKASQDVI FAVAWY SPKLLIYWASTRHTGVPDRFTGSVSGTDYTLTISSVQAEDLALYYCQQHYSTPW TFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQ NGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC 89M5 Heavy chain nucleotide sequence (SEQ ID NO:23) TGGAGCTGGATCTTTCTCTTTCTCCTGTCAGGAACTGCAGGTGTCCTCTCTGAG GTCCAGCTGCAACAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGATATCC ACTTCTGGATACACATTCACTGGATACACCATGCACTGGGTGAGGCAGAGCCAT GGAAAGACCCTTGAGTGGATTGGAGGTATTAATCCTAACAATGGTGGTACTACTTACAAC CAGAACTTCAAGGGCAAGGCCACATTGACTGTAGAGAAGTCCTCCACCACAGCCTACTTG GAGCTCCGCAGCCTGACATCTGAGGATTCTGCACTCTATTACTGTGCAAGAAAGGAGTTC TCTGATGGTTACTACTTTTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTTCA GCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAAC TCCETGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACC TGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGAC CTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTC ACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGG GATTGTGGTTGTAAGCCTTGCATATGTACAGTCCCAGAAGTATCATCTGTCTTCATCTTC CCCCCAAAGCCCAAGGATGTGCTCACCATTACTCTGACTCCTAAGGTCACGTGTGTTGTG GTAGACATCAGCAAGGATGATCCCGAGGTCCAGTTCAGCTGGTTTGTAGATGATGTGGAG GTGCACACAGCTCAGACGCAACCCCGGGAGGAGCAGTTCAACAGCACTTTCCGCTCAGTC AGTGAACTTCCCfiTCATGCACCAGGACTGGCTCAATGGCAAGGfiGTTCAAATGCAGGGTC AACAGTGCAGCTTTCCCTGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGCAGACCG AAGGCTCCACAGGTGTACACCATTCCACCTCCCAAGGAGCAGATGGCCAAGGATAAAGTC AGTCTGACCTGCATGATAACAGACTTCTTCCCTGAAGACATTACTGTGGAGTGGCAGTGG AATGGGCAGCCAGCGGAGAACTACAAGAACACTCAGCCCATCATGGACACAGATGGCTCT GTCTACAGCAAGCTCAATGTGCAGAAGAGCAACTGGGAGGCAGGAAATACTTTC ACCTGCTCTGTGTTACATGAGGGCCTGCACAACCACCATACTGAGAAGAGCCTCTCCCAC TCTCCTGGTAAATGATAA 89M5 Light chain nucleotide sequence (SEQ ID NO:24) ATGGGCTTCAAGATGGAGTCACAGATTCAGGCATTTGTATTCGTGTTTCTCTGGTTGTCT GGTGTTGACGGAGACATTGTGATGACCCAGTCTCACAAATTCATGTCCACATCAGTGGGA GACAGGGTCAACATCACCTGCAAGGCCAGTCAGGATGTGATTTTTGCTGTAGCCTGGTAT CAACAGAAACCAGGACAATCTCCTAAACTACTGATTTACTGGGCATCCACCCGGCACACT GGAGTCCCTGATCGCTTCACAGGCAGTGTATCTGGGACAGATTATACTCTCACCATCAGC AGTGTGCAGGCTGAAGACCTGGCACTTTATTACTGTCAGCAACATTATAGCACTCCGTGG ACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGGGCTGATGCTGCACCAACTGTATCC ATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTG AACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAA AATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGC AGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCC ACTCACAAGACATCAACTTCACCCRTTGTCAAGAGCTTCAACAGGAATGAGTGTTAGTGA 89M5 Heavy chain amino acid sequence without predicted signal sequence (SEQ ID NO:25) EVQLQQSGPELVKPGASVKISCKTSGYTFTGYTMHWVRQSHGKTLEWIGGINPNNGGTTY NQNFKGKATLTVEKSSTTAYLELRSLTSEDSALYYCARKEFSDGYYFFAYWGQGTLVTVS SAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQS DLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFI FPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRS VSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDK VSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNT HEGLHNHHTEKSLSHSPGK 89M5 Light chain amino acid sequence without predicted signal sequence (SEQ ID NO:26) DIVMTQSHKFMSTSVGDRVNITCKASQDVIFAVAWYQQKPGQSPKLLIYWASTRHTGVPD RFTGSVSGTDYTLTISSVQAEDLALYYCQQHYSTPWTFGGGTKLEEKRADAAPTVSIFPP SSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLT LTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC 130M23 Heavy chain variable region (SEQ ID NO:27) EVKLVESGGGLVKPGGSLKFSCAASGFSFSSYAMSWVRQTPEKRLEWVASISSGGSTYYP DSVKGRFTISRDNVRNILYLQMSSLRSEDTAMYFCARGGDPGVYNGDYEDAMDYWGQGTS VTVSS 130M23 Light chain varéable region (SEQ ID NO:28) DIVMTQSHKFMSTSVGDRVSITCKASQDVSSAVAWYQQKPGQSPKLLIYWASTRHTGVPD RETNSGSGTDYTLTISSVQAEDLALYYCQQHYSTPWTFGGGTKLEIK 130M23 Heavy chain CDRl (SEQ ID NO:29) SSYAMS 130M23 Heavy chain CDRZ (SEQ ID NO:30) SISSGGSTYYPDSVKG 130M23 Heavy chair; CDR3 (SEQ ID N03 1) RGGDPGVYNGDYEDAMDY 130M23 Light chain CDRl (SEQ ID NO:32) SSAVA 130M23 Light chain CDRZ (SEQ ID NO:33) WASTRHT 130M23 Light chain CDR3 (SEQ ID NO:34) QQHYSTP 130M23 Heavy chain variable region nucleotide sequence (SEQ ID NO:35) GAAGTGAAGCTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAATTT GCAGCCTCTGGATTCAGTTTCAGTAGTTATGCCATGTCTTGGGTTCGCCAGACT CCAGAGAAGAGGCTGGAGTGGGTCGCATCCATTAGTAGTGGTGGTAGTACCTACTATCCA GACAGTGTGAAGGGCCGATTCACCATCTCCAGAGATAATGTCAGGAACATCCTGTACCTG CAAATGAGCAGTCTGAGGTCTGAGGACACGGCCATGTATTTCTGTGCACGAGGCGGGGAT CCGGGGGTCTACAATGGTGACTACGAAGATGCTATGGACTACTGGGGTCAAGGAACCTCA GTCACCGTCTCCTCA 130M23 Light chain variable region nucleotide sequence (SEQ ID NO:36) —l36— GACATTGTGATGACCCAGTCTCACAAATTCATGTCCACATCAGTCGGAGACAGGGTCAGC ATCACCTGCAAGGCCAGTCAGGATGTGAGTTCTGCTGTAGCCTGGTRTCAACAAAAACCA GGGCAATCTCCTAAACTACTGATTTACTGGGCATCCACCCGGCACACTGGAGTCCCTGAT CGCTTCACAAACAGTGGATCTGGGACAGATTATACTCTCACCATCAGTAGTGTGCAGGCT GAAGACCTGGCACTTTATTACTGTCAGCAACATTATAGCACTCCGTGGACGTTCGGTGGA GGCACCAAGCTGGAAATCAAA 130M23 Heavy chain amino acid sequence with predicted signal sequence underlined (SEQ ID N03 7) MNFGLRLVFLVLVLKGVQCEVKLVESGGGLVKPGGSLKFSCAASGFSFSSYAMSWVRQTP EKRLEWVASISSGGSTYYPDSVKGRFTISRDNVRNILYLQMSSLRSEDTAMYFCARGGDP GVYNGDYEDAMDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEP VTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKK IVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFV DDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKT PQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMD VYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK 130M23 Light chain amino acid sequence with predicted signal sequence underlined (SEQ ID NO:3 8) MGIKMESQIQAFVFVFLWLSGVDGDIVMTQSHKFMSTSVGDRVSlTCKASQDVSSAVAWY QQKPGQSPKLLIYWASTRHTGVPDRFTNSGSGTDYTLTISSVQAEDLALYYCQQHYSTPW TFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQ NGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC l30M23 Heavy chain nucleotide sequence (SEQ ID NO:39) ATGAACTTCGGGCTGAGATTGGTTTTCCTTGTCCTTGTTTTAAAAGGTGTCCAGTGTGAA GTGAAGCTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAATTTTCC TGTGCAGCCTCTGGATTCAGTTTCAGTAGTTATGCCATGTCTTGGGTTCGCCAGACTCCA GAGAAGAGGCTGGAGTGGGTCGCATCCATTAGTAGTGGTGGTAGTACCTACTATCCAGAC AAGGGCCGATTCACCATCTCCAGAGATAATGTCAGGAACATCCTGTACCTGCAA ATGAGCAGTCTGAGGTCTGAGGACACGGCCATGTATTTCTGTGCACGAGGCGGGGATCCG GGGGTCTACAATGGTGACTACGAAGATGCTATGGACTACTGGGGTCAAGGAACCTCAGTC ACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCT GCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCA GTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTC CTGCAGTCTGACCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCC ACCGTCACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAA CCCAGGGATTGTGGTTGTAAGCCTTGCATATGTACAGTCCCAGAAGTATCATCT GTCTTCATCTTCCCCCCAAAGCCCAAGGATGTGCTCACCATTACTCTGACTCCTAAGGTC ACGTGTGTTGTGGTAGACATCAGCAAGGATGATCCCGAGGTCCAGTTCAGCTGGTTTGTA GATGATGTGGAGGTGCACACAGCTCAGACGCAACCCCGGGAGGAGCAGTTCAACAGCACT TTCCGCTCAGTCAGTGAACTTCCCATCATGCACCAGGACTGGCTCAATGGCAAGGAGTTC AAATGCAGGGTCAACAGTGCAGCTTTCCCTGCCCCCATCGAGAAAACCATCTCCAAAACC AAAGGCAGACCGAAGGCTCCACAGGTGTACACCATTCCACCTCCCAAGGAGCAGATGGCC AAGGATAAAGTCAGTCTGACCTGCATGATAACAGACTTCTTCCCTGAAGACATTACTGTG GAGTGGCAGTGGAATGGGCAGCCAGCGGAGAACTACAAGAACACTCAGCCCATCATGGAC ACAGATGGCTCTTACTTCGTCTACAGCAAGCTCAATGTGCAGAAGAGCAACTGGGAGGCA GGAAATACTTTCACCTGCTCTGTGTTACATGAGGGCCTGCACAACCACCATACTGAGAAG AGCCTCTCCCECTCTCCTGGTAAATGA 130M23 Light chain nucleotide sequence (SEQ ID NO:40) ATGGGCATCAAGATGGAGTCACAGATTCAGGCATTTGTATTCGTGTTTCTCTGGTTGTCT GGTGTTGACGGAGACATTGTGATGACCCAGTCTCACAAATTCATGTCCACATCAGTCGGA GACAGGGTCAGCATCACCTGCAAGGCCAGTCAGGATGTGAGTTCTGCTGTAGCCTGGTAT CAACAAAAACCAGGGCAATCTCCTAAACTACTGATTTACTGGGCATCCACCCGGCACACT GGAGTCCCTGATCGCTTCACAAACAGTGGATCTGGGACAGATTATACTCTCACCATCAGT AGTGTGCAGGCTGAAGACCTGGCACTTTATTACTGTCAGCAACATTATAGCACTCCGTGG ACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGGGCTGATGCTGCACCAACTGTATCC CCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTG AACAACTTCTACCCCAAAGACATCEfiTGTCAAGTGGAAGATTGATGGCAGTGAACGACAA AATGGCGTCCTGAACAGTTGGACTGETCAGGACAGCAAAGACAGCACCTACAGCATGAGC CTCACGTTGACCAAGGACGAG?ATGAACGACATAACAGCTATACCTGTGAGGCC ACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGTTAG 130M23 Heavy chain amino acid sequence without predicted signal sequence (SEQ ID NO:41) EVKLVESGGGLVKPGGSLKFSCAASGFSFSSYAMSWVRQTPEKRLEWVASISSGGSTYYP DSVKGRFTISRDNVRNILYLQMSSLRSEDTAMYFCARGGDPGVYNGDYEDAMDYWGQGTS VTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPA VLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVS SVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNS ELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQM LTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWE AGNTFTCSVLHEGLHNHHTEKSLSHSPGK 130M23 Light chain amino acid sequence without predicted signal sequence (SEQ ID NO:42) DIVMTQSHKFMSTSVGDRVSITCKASQDVSSAVAWYQQKPGQSPKLLIYWASTRHTGVPD RFTNSGSGTDYTLTISSVQAEDLALYYCQQHYSTPWTFGGGTKLEIKRADAAPTVSIFPP SSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLT LTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC Human RSP02 protein sequence without predicted signal sequence (SEQ ID NO:43) QGNRWRRSKRASYVSNPICKGCLSCSKDNGCSRCQQKLFFFLRREGMRQYGECLHSCPSG YYGHRAPDMNRCARCRIENCDSCFSKDFCTKCKVGFYLHRGRCFDECPDGFAPLEETMEC VEGCEVGHWSEWGTCSRNNRTCGFKWGLETRTRQIVKKPVKDTIPCPTIAESRRCKMTMR HCPGGKRTPKAKEKRNKKKKRKLIERAQEQHSVFLATDRANQ Human RSP02 amino acids 22—205 (SEQ ID NO:44) QGNRWRRSKRASYVSNPICKGCLSCSKDNGCSRCQQKLFFFLRREGMRQYGECLHSCPSG YYGHRAPDMNRCARCRIENCDSCFSKDFCTKCKVGFYLHRGRCFDECPDGFAPLEETMEC VEGCEVGHWSEWGTCSRNNRTCGFKWGLETRTRQIVKKPVKDTIPCPTIAESRRCKMTMR HCPG Human RSP02 furin-like domain 1 (SEQ ID NO:45) YVSNPICKGCLSCSKDNGCSRCQQKLFFFLRREGMRQYGECLHSCPSGYYG Human RSP02 furin-like domain 2 (SEQ ID NO:46) MNRCARCRIENCDSCFSKDFCTKCKVGFYLHRGRCFDECPDGFAP Human RSP02 thrombospondin domain (SEQ ID NO:47) —138- GCEVGHWSEWGTCSRNNRTCGFKWGLETRTRQIVKKPVKDTIPCPTIAESRRCKMTMRHCP Human RSPO3 n ce without predicted signal sequence (SEQ ID NO:48) QNASRGRRQRRMHPNVSQGCQGGCATCSDYNGCLSCKPRLFFALERIGMKQIGVCLSSCP SGYYGTRYPDINKCTKCKADCDTCFNKNFCTKCKSGFYLHLGKCLDNCPEGLEANNHTME CVSIVHCEVSEWNPWSPCTKKGKTCGFKRGTETRVREIIQHPSAKGNLCPPTNETRKCTV QRKKCQKGERGKKGRERKRKKPNKGESKEAIPDSKSLESSKEIPEQRENKQQQKKRKVQD KQKSVSVSTVH Human RSPO3 furin-like domain 1 (SEQ ID NO:49) PNVSQGCQGGCATCSDYNGCLSCKPRLFFALERIGMKQIGVCLSSCPSGYYG Human RSPO3 furén-like domain 2 (SEQ ID N0250) INKCTKCKADCDTCFNKNFCTKCKSGFYLHLGKCLDNCPEGLEA Human RSPO3 thrombospondin domain (SEQ ID N0251) HCEVSEWNPWSPCTKKGKTCGFKRGTETRVREIIQHPSAKGNLCPPTNETRKCTVQRKKCQ h89M5-H2L2 Heavy chain nucleotide sequence (SEQ ID NO:52) ATGGACTGGACCTGGAGGATACTCTTTCTCGTGGCAGCAGCCACAGGAGCCCACTCCCAG GTCCAGCTCGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCTGTGAAGGTTTCC ACTTCTGGATACACCTTCACTGGATACACCATGCACTGGGTTAGACAGGCCCCC GGACAAAGGCTGGAGTGGATGGGAGGTATTAATCCTAACAATGGTGGTACTACTTACAAC CAGAACTTCAAGGGCAGAGTCACCATTACCAGGGACACATCCGCAAGCACAGCCTACATG TCCAGCCTGAGATCTGAAGACACAGCTGTGTATTACTGTGCAAGAAAGGAGTTC TCTGATGGATACTACTTTTTTGCTTACTGGGGCCAAGGGACCCTGGTCACCGTCAGCTCA GCCAGCACAAAGGGCCCTAGCGTCTTCCCTCTGGCTCCCTGCAGCAGGAGCACCAGCGAG AGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCG TGGAACTCAGGCGCTCTGACCAGCGGCGTGCACACCTTCCCAGCTGTCCTACAGTCCTCA GGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAACTTCGGCACCCAGACC TACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGACAGTTGAGCGC AAATGTTGTGTCGAGTGCCCACCGTGCCCAGCACCACCTGTGGCAGGACCGTCAGTCTTC CTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACGTGC GTGGTGGTGGACGTGAGCCACGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGC GTGGAGGTGCATAATGCCAAGACAAAGCCACGGGAGGAGCAGTTCAACAGCACGTTCCGT GTGGTCAGCGTCCTCACCGTTGTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGC AAGGTCTCCAACAAAGGCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGG CAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAAC CAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGG GAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACACCTCCCATGCTGGACTCCGAC GGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAAC GTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCRCAACCACTACACGCAGAAGAGCCTC TCCCTGTCTCCGGGTAAATGA h89M5-H2L2 Heavy chain amino acid sequence with predicted signal sequence underlined (SEQ ID NO:53) IO<WSCDOZJWTWRILFLVAAATGAHSQVQLVQSGAEVKKPGASVKVSCKTSGYTFTGYTMHWVRQAPQRLEWMGGINPNNGGTTYNQNFKGRVTlTRDTSASTAYMELSSLRSEDTAVYYCARKEFDGYYFFAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSVSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK h89M5-H2L2 Heavy chain le region nucleotide sequence (SEQ ID NO:54) CAGGTCCAGCTCGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCTGTGAAGGTT TCCTGCAAGACTTCTGGATACACCTTCACTGGATACACCATGCACTGGGTTAGACAGGCC CCCGGACAAAGGCTGGAGTGGATGGGAGGTATTAATCCTAACAATGGTGGTACTACTTAC AACCAGAACTTCAAGGGCAGAGTCACCATTACCAGGGACACATCCGCAAGCACAGCCTAC ATGGAGCTGTCCAGCCTGAGATCTGAAGACACAGCTGTGTATTACTGTGCAAGAAAGGAG TTCTCTGATGGATACTACTTTTTTGCTTACTGGGGCCAAGGGACCCTGGTCACCGTCAGC h89M5-H2L2 Heavy chain variable region amino acid sequence (SEQ ID NO:55) QVQLVQSGAEVKKPGASVKVSCKTSGYTFTGYTMHWVRQAPGQRLEWMGGINPNNGGTTY NQNFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCARKEFSDGYYFFAYWGQGTLVTVS H2L2 Light chain nucleotide sequence (SEQ ID NO:56) ATGGACATGAGGGTCCCCGCACAGCTCCTGGGGCTCCTGCTCCTCTGGCTCCGGGGTGCC GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTCGGAGACAGA GTCACCATCACTTGCAAGGCCTCCCAGGATGTGATTTTTGCTGTTGCCTGGTATCAGCAG AAACCAGGGAAAGCCCCTAAGCTCCTGATCTATTGGGCATCCACCCGGCACACTGGGGTC CCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTACACTCTCACCATCAGCAGTCTG CAACCTGAAGATTTTGCE‘“CTTACTACTGTCAGCAACATTATAGCACTCCTTGGACTTTC GGCGGAGGGACCAAGGTGGAGATCAAACGGACTGTGGCTGCACCATCTGTCTTCATCTTC CCTCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAAC TTCTATCCCAGAGAGGCCAAAGTCCAGTGGAAGGTGGATAACGCCCTCCAATCCGGTAAC TCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAACACC CTGACACTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCAT CAGGGCCTGAGCTCCCCCGTCACAAAGAGCTTCAACAGGGGfiGAGTGCTAA h89M5-H2L2 Light chain amino acid sequence with predicted signal sequence underlined (SEQ ID NO:57) MDMRVPAQLLGLLLLWLRGARCD;QMTQSPSSLSASVGDRVTITCKASQDVIFAVAWYQQ KPGKAPKLLIYWASTRHTGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQHYSTPWTF GGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN SQESVTEQDSKDSTYSLSNTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC h89M5-H2L2 Light chain variable region nucleotide sequence (SEQ ID NO:58) GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTCGGAGACAGAGTCACC ATCACTTGCAAGGCCTCCCAGGATGTGATTTTTGCTGTTGCCTGGTATCAGCAGAAACCA GGGAAAGCCCCTAAGCTCCTGATCTATTGGGCATCCACCCGGCACACTGGGGTCCCATCA AGGTTCAGTGGCAGTGGATCTGGGACAGATTACACTCTCACCATCAGCAGTCTGCAACCT GAAGATTTTGCAACTTACTACTGTCAGCAACATTATAGCACTCCTTGGACTTTCGGCGGA AAGGTGGAGATCAAA h89M5-H2L2 Light chain variable region amino acid sequence (SEQ ID NO:59) DIQMTQSPSSLSASVGDRVTITCKASQDVIFAVAWYQQKPGKAPKLLIYWASTRETGVPS —14o— RFSGSGSGTDYTLTISSLQPEDFATYYCQQHYSTPWTFGGGTKVEIK hl30M23-H1L2 Heavy chain nucleotide sequence (SEQ ID NO:60) CTGGGACTCAGATGGGTTTTCCTCGTTGCTATTCTGGAAGGAGTCCAGTGTGAG GTGCAGCTGGTGGAGTCTGGGGGAGGCCTGGTCAAGCCTGGAGGATCTCTGCGGCTCTCC TGTGCAGCCTCTGGATTCACCTTCTCCTCTTATGCCATGTCTTGGGTCCGGCAGGCTCCA GGGARGGGGCTGGAATGGGTCTCATCCATTTCTAGTGGAGGTAGCACATATTATCCTGAC AGCGTGAAGGGCCGGTTCACCATCTCCAGAGACAACGCCAAGAACAGCCTGTATCTGCAA ATGAACAGCCTGAGAGCCGAGGACACAGCTGTGTATTACTGTGCTAGAGGTGGAGATCCT GGGGTCTACAATGGAGATTACGAAGATGCTATGGACTACTGGGGGCAAGGAACAACAGTC AGCTCAGCCAGCACAAAGGGCCCTAGCGTCTTCCCTCTGGCTCCCTGCAGCAGG AGCACCAGCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCG GTGACGGTGTCGTGGAACTCAGGCGCTCTGACCAGCGGCGTGCACACCTTCCCAGCTGTC CTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAACTTC GGCACCCAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAG ACAGTTGAGCGCAAATGTTGTGTCGAGTGCCCACCGTGCCCAGCACCACCTGTGGCAGGA CCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCT GAGGTCACGTGCGTGGTGGTGGACGTGAGCCACGAAGACCCCGAGGTCCAGTTCAACTGG TACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCACGGGAGGAGCAGTTCAAC AGCACGTTCCGTGTGGTCAGCGTCCTCACCGTTGTGCACCAGGACTGGCTGAACGGCAAG AAGYGCAAGGTCTCCAACAAAGGCCTCCCAGCCCCCATCGAGAAAACCATCTCC AAAACCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAG ATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATC GCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACACCTCCCATG CTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGG CAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACG CAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA h130M23-H1L2 Heavy chain amino acid sequence with predicted signal sequence underlined (SEQ ID NO:61) MEEQERWVFLVAILEGVQCEVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMSWVRQAP GKGLEWVSSISSGGSTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGGDP YEDAMDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDK TVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNW YVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTIS KTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPM LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 3-H1L2 Heavy chain variable region nucleotide sequence (SEQ ID NO;62) GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCCTGGTCAAGCCTGGAGGATCTCTGCGGCTC TCCTGTGCAGCCTCTGGATTCACCTTCTCCTCTTATGCCATGTCTTGGGTCCGGCAGGCT CCAGGGAAGGGGCTGGAATGGGTCTCATCCATTTCTAGTGGAGGTAGCACATATTATCCT GACAGCGTGAAGGGCCGGTTCACCATCTCCAGAGACAACGCCAAGAACAGCCTGTATCTG CAAATGAACAGCCTGAGAGCCGAGGACACAGCTGTGTATTACTGTGCTAGAGGTGGAGAT CCTGGGGTCTACAATGGAGATTACGfiRGATGCTATGGACTACTGGGGGCAAGGAACAACA GTCACAGTCAGCTCA h130M23-Hl L2 Heavy chain variable region amino acid sequence (SEQ ID NO:63) EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSISSGGSTYYP FTISRDNAKNSLYLQMNSLRAEDTAVYYCARGGDPGVYNGDYEDAMDYWGQGTT VTVSS h130M23-H1L2 Light chain nucleotide sequence (SEQ ID NO:64) -141— TACCTCCTCCCTACAGCTGCCGCTGGACTCCTCCTCCTCGCTGCCCAGCCTGCC ATGGCCGACATCCAGATGACCCAGTCCCCTTCCTCCCTGTCTGCTTCCGTCGGAGACAGA GTCACCATCACTTGCAAGGCCTCCCAGGATGTGTCCTCTGCTGTCGCTTGGTATCAGCAG AAACCAGGAAAAGCTCCTAAGCTCCTGATCTATTGGGCATCCACCAGGCACACAGGAGTC CCTTCCAGGTTCTCCGGCTCTGGATCTGGGACAGATTTCACTCTCACCATCAGCTCCGTG CAAGCTGAAGATTTTGCAACTTACTACTGTCAGCAACATTATAGCACTCCTTGGACATTC GGACAAGGGACCAAGGTGGAAATCAAAAGAACTGTGGCTGCACCTTCTGTCTTCATCTTC CCTCCATCTGATGAGCAGCTCAAATCTGGAACTGCCTCCGTTGTGTGCCTGCTGAATAAC TTCTATCCTAGAGAGGCCAAAGTCCAGTGGAAGGTGGATAACGCCCTCCAATCCGGTAAC GAGTCTGTCACAGAGCAGGACTCCAAGGACAGCACCTACTCCCTCAGCAACACC CTGACACTGTCTAAAGCTGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCAT CTGAGCTCCCCCGTCACAAAATCCTTCAACAGGGGAGAGTGCTAA h130M23-H1 L2 Light chain amino acid sequence with predicted signal sequence underlined (SEQ ID N026$ MKYLLPTAAAQLLLLEIAQ‘EA‘MADIQMTQSPSSLSASVGDRVTTTCKASQDVSSAVAWYQQ KPGKAPKLLIYWASTRHTGVPSRFSGSGSGTDFTLTISSVQAEDFATYYCQQHYSTPWTF GQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN SQESVTEQDSKDSTYSLSNTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC h130M23-H1 L2 Light chain variable region nucleotide sequence (SEQ ID NO:66) GfiCATCCAGATGACCCAGTCCCCTTCCTCCCTGTCTGCTTCCGTCGGAGACAGAGTCACC ATCACTTGCAAGGCCTCCCAGGATGTGTCCTCTGCTGTCGCTTGGTATCAGCAGAAACCA GGAAAAGCTCCTAAGCTCCTGATCTATTGGGCATCCACCAGGCACACAGGAGTCCCTTCC AGGTTCTCCGGCTCTGGATCTGGGACAGATTTCACTCTCACCATCAGCTCCGTGCAAGCT GAAGATTTTGCAACTTACTACTGTCAGCAACATTATAGCACTCCTTGGACATTCGGACAA GGGACCAAGGTGGAAATCAAA h130M23-H1 L2 Light chain variable region amino acid sequence (SEQ ID NO:67) DIQMTQSPSSLSASVGDRVTITCKASQDVSSAVAWYQQKPGKAPKLLIYWASTRHTGVPS RFSGSGSGTDFTLTISSVQAEDFATYYCQQHYSTPWTFGQGTKVEIK h89M5-H2L2 Heavy chain amino acid sequence without predicted signal sequence (SEQ ID NO:68) QVQLVQSGAEVKKPGASVKVSCKTSGYTFTGYTMHWVRQAPGQRLEWMGGINPNNGGTTY NQNFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCARKEFSDGYYFFAYWGQGTLVTVS SASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSV PKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTF TVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK h89M5—H2L2 Light chain amino acid sequence without predicted signal sequence underlined (SEQ ID N0269) DIQMTQSPSSLSASVGDRVTITCKASQDVIFAVAWYQQKPGKAPKLLIYWASTRHTGVPS SGTDYTLTISSLQPEDFATYYCQQHYSTPWTFGGGTKVEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSNTLT LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC h130M23-H1L2 Heavy chain amino acid sequence without predicted signal sequence underlined (SEQ ID NO:70) EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSISSGGSTYYP DSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGGDPGVYNGDYEDAMDYWGQGTT VTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA -142— VLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVA GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQF NSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSRE VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSR FSCSVMHEALHNHYTQKSLSLSPGK h130M23-H1L2 Light chain amino acid sequence without predicted signal sequence underlined (SEQ ID NO:7l) DIQMTQSPSSLSASVGDRVTITCKASQDVSSAVAWYQQKPGKAPKLLIYWASTRHTGVPS RFSGSGSGTEFTLTISSVQAEDFATYYCQQHYSTPWTFGQGTKVEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSNTLT LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC h130M23-H1L6 Light chain nucleotide sequence (SEQ ID NO:72) ATGGGCATCAAGATGGgGiCELC'thATICAGGCATTTGTATTCGTGTTTCTCTGGTTGTCT GGTGTTGACGGAGACATCCAGATGACCCAGTCCCCTTCCTCCCTGTCTGCTTCCGTCGGA GACAGAGTCACCATCACTTGCAAGGCCTCCCAGGATGTGTCCTCTGCTGTCGCTTGGTAT CAGCAGAAACCAGGAAAAGCTCCTAAGCTCCTGATCTATTGGGCATCCACCAGGCACACA GGAGTCCCTTCCAGGTTCTCCGGCTCTGGATCTGGGACAGATTTCACTCTCACCATCAGC TCCCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAGCAACATTATAGCACTCCTTGG ACATTCGGACAAGGGACCAAGGTGGAAATCAAAAGAACTGTGGCTGCACCTTCTGTCTTC ATCTTCCCTCCATCTGATGAGCAGCTCAAATCTGGAACTGCCTCCGTTGTGTGCCTGCTG AATAACTTCTATCCTAGAGAGGCCAAAGTCCAGTGGAAGGTGGATAACGCCCTCCAATCC GGTAACTCCCAGGAGTCTGTCACAGAGCAGGACTCCAAGGACAGCACCTACTCCCTCAGC CTGACACTGTCTAAAGCTGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTC ACCCATCAGGGACTGAGCTCCCCCGTCACAAAATCCTTCAACAGGGGAGAGTGCTAA hl30M23-H1L6 Light chain amino acid sequence with predicted signal sequence underlined (SEQ ID NO:73) MGIKMESQIQAFVFVFLWLSGVDGDIQMTQSPSSLSASVGDRVTITCKASQDVSSAVAWY QQKPGKAPKLLIYWASTRHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHYSTPW TFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSNTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC hl30M23-H1L6 Light chain amino acid ce without predicted signal sequence underlined (SEQ ID NO:74) DIQMTQSPSSLSASVGDRVTITCKASQDVSSAVAWYQQKPGKAPKLLIYWASTRHTGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQHYSTPWTFGQGTKVEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSNTLT EKHKVYACEVTHQGLSSPVTKSFNRGEC hl30M23-H1L6 Light chain variable region nucleotide sequence (SEQ ID NO:75) GACATCCAGATGACCCAGTCCCCTTCCTCCCTGTCTGCTTCCGTCGGRGACAGAGTCACC ATCACTTGCAAGGCCTCCCAGGATGTGTCCTCTGCTGTCGCTTGGTATCAGCAGAAACCA GGAAAAGCTCCTAAGCTCCTGATCTATTGGGCATCCACCAGGCACACAGGAGTCCCTTCC AGGTTCTCCGGCTCTGGATCTGGGACAGATTTCACTCTCACCATCAGCTCCCTGCAACCT GAAGATTTTGCAACTTACTACTGTCfiGCAACATTATAGCACTCCTTGGACATTCGGACAA GGGACCAAGGTGGAAATCAAA h130M23-H1L6 Light chain variable region amino acid sequence (SEQ ID NO:76) SPSSLSASVGDRVTITCKASQDVSSAVAWYQQKPGKAPKLLIYWASTRHTGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQHYSTPWTFGQGTKVEIK I/WE

Claims (25)

CLAIM :

1. An isolated monoclonal antibody that specifically binds human R-spondin2 (RSPO2), which comprises: (a) a heavy chain CDR1 comprising SSYAMS (SEQ ID NO:29), a heavy chain CDR2 comprising SISSGGSTYYPDSVKG (SEQ ID NO:30), and a heavy chain CDR3 comprising RGGDPGVYNGDYEDAMDY (SEQ ID NO:31); and (b) a light chain CDR1 comprising KASQDVSSAVA (SEQ ID NO:32), a light chain CDR2 sing WASTRHT (SEQ ID NO:33), and a light chain CDR3 comprising QQHYSTP (SEQ ID .

2. The antibody of claim 1, which comprises: (a) a heavy chain variable region having at least 90% sequence ty to SEQ ID NO:27; and (b) a light chain variable region having at least 90% sequence identity to SEQ ID NO:28.

3. The antibody of claim 1, which comprises: (a) a heavy chain le region having at least 95% sequence identity to SEQ ID NO:27; and (b) a light chain variable region having at least 95% sequence identity to SEQ ID NO:28.

4. The antibody of claim 1, which comprises: (a) a heavy chain variable region comprising SEQ ID NO:27; and (b) a light chain variable region comprising SEQ ID NO:28.

5. The antibody of claim 1, which comprises: (a) a heavy chain variable region having at least 90% sequence identity to SEQ ID NO:63; and (b) a light chain variable region having at least 90% sequence ty to SEQ ID NO:67 or SEQ ID NO:76.

6. The antibody of claim 1, which comprises: AH26(11355570_1):EOR (a) a heavy chain variable region having at least 95% sequence identity to SEQ ID NO:63; and (b) a light chain variable region having at least 95% sequence identity to SEQ ID NO:67 or SEQ ID NO:76.

7. The antibody of claim 1, which comprises: (a) a heavy chain variable region comprising SEQ ID NO:63; and (b) a light chain variable region comprising SEQ ID NO:67.

8. The antibody of claim 1, which comprises: (a) a heavy chain le region comprising SEQ ID NO:63; and (b) a light chain variable region comprising SEQ ID NO:76.

9. An isolated monoclonal antibody that competes with a second antibody for specific binding to RSPO2, wherein the second antibody comprises (a) a heavy chain variable region comprising SEQ ID NO:27 and a light chain variable region comprising SEQ ID NO:28; (b) a heavy chain variable region comprising SEQ ID NO:63 and a light chain variable region sing SEQ ID NO:67; or (c) a heavy chain variable region comprising SEQ ID NO:63 and a light chain le region comprising SEQ ID NO:76.

10. An ed monoclonal antibody that binds the same epitope on RSPO2 as the antibody ing to any one of claims 1-8.

11. The antibody according to any one of claims 1-10, which is a recombinant dy, a chimeric antibody, a ific antibody, a humanized antibody, an IgG1 antibody, an IgG2 antibody, or an antibody fragment comprising an antigen binding site.

12. A monoclonal antibody produced by the hybridoma cell line having ATCC deposit number PTA-12021. AH26(11355570_1):EOR

13. A monoclonal humanized antibody sing the same heavy chain CDR1, CDR2, and CDR3, and light chain CDR1, CDR2, and CDR3 as the antibody produced by the hybridoma cell line having ATCC deposit number PTA-12021.

14. The antibody according to any one of claims 1-13, which inhibits g of RSPO2 to at least one leucine-rich repeat containing G protein coupled receptor (LGR).

15. The antibody of claim 14, wherein the LGR is selected from the group consisting of LGR4, LGR5, and LGR6.

16. The antibody of claim 15, n the LGR is LGR5.

17. The antibody according to any one of claims 1-16, which (a) inhibits RSPO2 signaling; (b) inhibits activation of β-catenin; (c) inhibits nin signaling; (d) inhibits tumor growth; (e) induces expression of differentiation markers in a tumor; (f) induces cells in a tumor to differentiate; and/or (g) reduces the frequency of cancer stem cells in a tumor.

18. A cell comprising or producing the antibody according to any one of claims 1-13, wherein the cell is not within a human body.

19. A hybridoma cell line having ATCC deposit number 021.

20. An isolated polynucleotide molecule comprising a nucleotide sequence that encodes an antibody according to any one of claims 1-13.

21. A cell comprising the polynucleotide of claim 20, wherein the cell is not within a human body.

22. A pharmaceutical composition comprising the antibody according to any one of claims 1- 13 and a pharmaceutically able carrier. AH26(11355570_1):EOR

23. Use of an antibody according to any one of claims 1-13 in the preparation of a medicament for inhibiting growth of a tumor.

24. Use of an antibody ing to any one of claims 1-13 in the preparation of a ment for inhibiting nin signaling in a cell. 25. The use of claim 24, wherein the cell is a tumor cell. 26. The use according to claim 24 or claim 25, wherein the tumor is selected from the group consisting of colorectal tumor, ovarian tumor, atic tumor, lung tumor, liver tumor, breast tumor, kidney tumor, prostate tumor, gastrointestinal tumor, melanoma, cervical tumor, bladder tumor, glioblastoma, and head and neck tumor. 27. Use of an antibody according to any one of claims 1-13 in the ation of a medicament for ent of cancer. 28. The use of claim 27, wherein the cancer is selected from the group consisting of colorectal cancer, ovarian cancer, pancreatic cancer, lung cancer, liver cancer, breast cancer, kidney , prostate cancer, gastrointestinal cancer, melanoma, cervical cancer, bladder cancer, astoma, and head and neck cancer. 29. Use of the antibody according to any one of claims 1-13 in the preparation of a medicament for treatment of a disease associated with activation of β-catenin. 30. The use according to any one of claims 23-29, wherein inhibiting growth of a tumor, inhibiting β-catenin signaling, treatment of cancer, or treatment of a disease associated with activation of β-catenin comprises the use of at least one additional eutic agent. 31. The use according to any one of claims 23-29, wherein the medicament is for use with at least one additional therapeutic agent. 32. The use of claim 30 or claim 31, wherein the at least one additional therapeutic agent is a chemotherapeutic agent. AH26(11355570_1):EOR 33. The use of claim 30 or claim 31, wherein the at least one additional eutic agent is a Wnt pathway inhibitor. 34. An isolated monoclonal antibody that specifically binds human R-spondin2 (RSPO2), which comprises a heavy chain amino acid sequence of SEQ ID NO:41 and a light chain amino acid sequence of SEQ ID NO:42. 35. An isolated monoclonal antibody that specifically binds human R-spondin2 (RSPO2), which comprises a heavy chain amino acid ce of SEQ ID NO:70 and a light chain amino acid sequence of SEQ ID NO:71. 36. An isolated monoclonal antibody that specifically binds human din2 (RSPO2), which comprises a heavy chain amino acid sequence of SEQ ID NO:70 and a light chain amino acid sequence of SEQ ID NO:74. OncoMed Pharmaceuticals, Inc. By the Attorneys for the Applicant SPRUSON & FERGUSON Per: AH26(11355570_1):EOR 1 I23 EE... \— <r l\ ONQOmfi'le-C’ NNV’ C") 0 <1" LO N LO C9 N o (O \—C\l (000‘— l!) |\ (O C") N N N l\ C0 ‘— ‘— “a\—\— a. N m > <1- CON cox—coax— ‘_ F . .°Q QN‘f“. N‘fN‘f“. C) (’00 OZN gal EEI EEI Baal ififil EEEI HEEI ifigl ifigl Eifigl EEEEI iififil ifigl gal 20 oocxuoco oooo LOONOO o (0 m><o9m x: ommmommnmc» _. <r<r<r<rloco<rlo<r<r <<n 03$.» ElElElElgl 26E. a) o. .0."—

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