WO2018035210A1

WO2018035210A1 - Methods and monitoring of treatment with an rspo antagonist

Info

Publication number: WO2018035210A1
Application number: PCT/US2017/047117
Authority: WO
Inventors: Leonardo FAORO; Rainer Brachmann; Jakob Dupont; Ann M. Kapoun; Lu Xu
Original assignee: Oncomed Pharmaceuticals, Inc.
Priority date: 2016-08-17
Filing date: 2017-08-16
Publication date: 2018-02-22

Abstract

Methods for treating diseases such as cancer or a fibrotic disease comprising administering an RSPO antagonist (e.g., anti-RSPO3 antibody), either alone or in combination with other therapeutic agents, and monitoring for skeletal-related side effects and/or toxicity.

Description

METHODS AND MONITORING OF TREATMENT WITH AN RSPO ANTAGONIST

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority benefit of U.S. Provisional Application Nos. 62/427,271, filed November 29, 2016 and 62/375,988, filed August 17, 2016, each of which is hereby incorporated by reference herein in its entirety.

FIELD OF INVENTION

[0002] The present invention relates to the field of treating diseases with an RSPO antagonist (e.g., anti-RSP03 antibody). More particularly, the invention provides methods for treating cancer or a fibrotic disease comprising administering an RSPO antagonist (e.g., anti-RSP03 antibody), either alone or in combination with other therapeuitc agents, and monitoring for side effects and/or toxicity.

BACKGROUND OF THE INVENTION

[0003] The R-Spondin (RSPO) family of proteins is conserved among vertebrates and comprises four members, RSPO l, RSP02, RSP03, and RSP04. These proteins have been referred to by a variety of names, including roof plate-specific spondins, hPWTSR (hRSP03), THS2D (RSP03), Cristin 1-4, and Futrin 1-4. The RSPOs are small secreted proteins that overall share approximately 40-60% sequence homology and domain organization. All RSPO proteins contain two furin-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).

[0004] Studies have shown that RSPO proteins have a role during vertebrate development (Kamata et al., 2004, Biochim. Biophys Acta, 1676:51-62) and in Xenopus myogenesis (Kazanskaya et al., 2004, Dev. Cell, 7:525-534). RSPO l has also been shown to function as a potent mitogen for gastrointestinal epithelial cells (Kim et al, 2005, Science, 309: 1256-1259). It has been reported that RSP03 is prominently expressed in or close to endothelial cells and their cellular precursors in Xenopus and mouse. Furthermore, it has been suggested that RSP03 can act as an angiogenic factor in embryogenesis (Kazanskaya et al., 2008, Development, 135:3655-3664).

[0005] Wnt ligands and R-spondin (RSPO) proteins have been shown to synergize to activate the canonical Wnt pathway. RSPO proteins are known to activate β-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 β-catenin signaling (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 LGR5 (U.S. Patent Publication Nos. 2009/0074782 and 2009/0191205), and these data present an alternative pathway for the activation of β-catenin signaling.

[0006] RSPO and LGR antagonists (e.g., anti-RSP03 antibodies) that disrupt β-catenin signaling are a promising source of new therapeutic agents for the treatment of cancer, as well as other β-catenin- associated diseases. See, e.g., U.S. 8, 158,757, U.S. 8,540,989, U.S. 8,802,097, and U.S 20140017253.

[0007] Wnt pathway activation is associated with colorectal cancer. Approximately 5-10% of all colorectal cancers are hereditary with one of the main forms being familial 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 identified in other Wnt pathway components including Axin and β-catenin. Individual adenomas are clonal outgrowths of epithelial cells containing a second inactivated allele, and the large number of FAP adenomas inevitably results in the development of adenocarcinomas through additional mutations in oncogenes and/or tumor suppressor genes. Furthermore, activation of the Wnt signaling pathway, including loss- of-function mutations in APC and stabilizing mutations in β-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).

[0008] Cancer therapeutic agents, including biologic agents are known to cause, or are likely to cause, side effects and/or toxicity in subjects who take them. Thus, there is a need to identify potential side effects associated with the therapeutic administration of an anti-RSP03 antibody, to monitor those side effects, and/or to mitigate those side effects so that effective cancer therapy can continue.

BRIEF SUMMARY OF THE INVENTION

[0009] In one aspect, described herein is a method of selecting a subject for treatment with an RSPO antagonist, comprising: obtaining a biological sample from the subject; determining the level of a bone formation biomarker and/or bone resorption biomarker in the sample; and selecting the subject for treatment with the RSPO antagonist if the level of the bone formation biomarker is above a predetermined level; and/or the level of bone resorption biomarker is below a predetermined level.

[0010] In another aspect, described herein is a method of identifying a subject as eligible for treatment with a RSPO antagonist, comprising: obtaining a biological sample from the subject; determining the level of a bone formation biomarker and/or bone resorption biomarker in the sample; and identifying the subject as eligible for treatment with the RSPO antagonist if the level of the bone formation biomarker is above a predetermined level; and/or the level of bone resorption biomarker is below a predetermined level.

[0011] In another aspect, described herein is a method of selecting a subject for treatment with a RSPO antagonist, comprising: determining the level of a bone formation biomarker and/or bone resorption biomarker in a sample from the subject; and selecting the subject for treatment with the RSPO antagonist if the level of the bone formation biomarker is above a predetermined level; and/or the level of bone resorption biomarker is below a predetermined level.

[0012] In another aspect, described herein is a method of identifying a subject as eligible for treatment with a RSPO antagonist, comprising: determining the level of a bone formation biomarker and/or bone resorption biomarker in a sample from the subject; and dentifying the subject as eligible for treatment with the RSPO antagonist if the level of the bone formation biomarker is above a predetermined level; and/or the level of bone resorption biomarker is below a predetermined level.

[0013] In another aspect, described herein is a method of screening for a subject at risk of a skeletal- related side effect and/or toxicity from treatment with a RSPO antagonist, comprising: obtaining a biological sample from the subject prior to treatment with the RSPO antagonist; determining the level of a bone formation biomarker and/or bone resorption biomarker in the sample; and comparing the level of the bone formation biomarker and/or bone resorption biomarker in the sample to a predetermined level of the bone formation biomarker and/or bone resorption biomarker, wherein the subject is at risk for a skeletal -related side effect and/or toxicity if the level of the bone formation biomarker in the sample is lower than the predetermined level of the bone formation biomarker; and/or the level of the bone resorption biomarker in the sample is higher than the predetermined level of the bone resorption biomarker. In another aspect, described herein is a method of screening for a subject at risk of a skeletal-related side effect and/or toxicity from treatment with a RSPO antagonist, comprising: determining the level of a bone formation biomarker and/or bone resorption biomarker in a sample from the subject; and comparing the level of the bone formation biomarker and/or bone resorption biomarker in the sample to a predetermined level of the bone formation biomarker and/or bone resorption biomarker; wherein the subject is at risk for a skeletal -related side effect and/or toxicity if the level of the bone formation biomarker in the sample is lower than the predetermined level of the bone formation biomarker; and/or the level of the bone resorption biomarker in the sample is higher than the predetermined level of the bone resorption biomarker. In one embodiment, a subject at risk for a skeletal-related side effect and/or toxicity is administered a therapeutically effective amount of an anti-resorptive medication prior to treatment with the RSPO antagonist.

[0014] In one embodiment, the biological sample is blood, serum, or plasma. In one embodiment, the bone formation biomarker is serum procollagen type 1 amino-terminal propeptide (P1NP). In one embodiment, the predetermined level of P1NP in blood, serum, or plasma is a level of P1NP determined at an earlier timepoint or at an initial screening. In one embodiment, the predetermined level of P1NP in blood, serum, or plasma is about 16 microg/ml or more, about 19 microg/ml or more, about 22 microg/ml or more, about 25 microg/ml or more, or about 30 microg/ml or more. In one embodiment, a method described herein further comprises administering the RSPO antagonist to the subject if the level of the bone formation biomarker is above the predetermined level. In one embodiment, if the P1NP level is 70% or less, 60% or less, 50% or less, 40% or less, or 30% or less than the predetermined level, then the subject is administered a therapeutically effective amount of an anti-resorptive medication prior to administering the RSPO antagonist to the subject.

[0015] In one embodiment, the bone resorption biomarker is collagen type 1 cross-linked beta C- telopeptide (β-CTX). In one embodiment, the predetermined level of β-CTX in blood, serum, or plasma is a level of β-CTX determined at an earlier timepoint or at an initial screening. In one embodiment, the predetermined level of β-CTX in blood, serum, or plasma is about 1500 pg/ml or less, about 1200 pg/ml or less, about 1000 pg/ml or less, about 800 pg/ml or less, about 600 pg/ml or less, or about 400 pg/ml or less. In one embodiment, a method described herein further comprises administering the RSPO antagonist to the subject if the level of the bone resorption biomarker is below the predetermined level. In one embodiment, if the β-CTX level is at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, or at least about 3-fold greater than the predetermined level, then the subject is administered a therapeutically effective amount of an anti-re sorptive medication prior to administering the RSPO antagonist.

[0016] In another aspect, described herein is a method of selecting a subject for treatment with a RSPO antagonist, comprising: determining bone density in the subject; and selecting the subject for treatment with the RSPO antagonist if the bone density is at or above a predetermined level.

[0017] In another aspect, described herein is a method of identifying a subject as eligible for treatment with a RSPO antagonist, comprising: determining bone density in the subject; and selecting the subject for treatment with the RSPO antagonist if the bone density is at or above a predetermined level.

[0018] In another aspect, described herein is a method of screening a subject for the risk of a skeletal- related side effect and/or toxicity from treatment with a RSPO antagonist, comprising: determining bone density in the subject; and comparing the bone density of the subject to a predetermined level of bone density; wherein the subject is at risk for a skeletal-related side effect and/or toxicity if the bone density in the subject is lower than the predetermined level of bone density. In one embodiment, a subject at risk for a skeletal-related side effect and/or toxicity is administered a therapeutically effective amount of an anti-resorptive medication prior to treatment with the RSPO antagonist.

[0019] In one embodiment, bone density is determined by dual-energy x-ray absorptiometry (DEXA). In one embodiment, bone density is expressed as a T-score. In one embodiment, the predetermined level of bone density T-score is -1, -1.5, -2, -2.5, or -3.

[0020] In another aspect, described herein is a method of monitoring a subject receiving treatment with a RSPO antagonist for the development of a skeletal-related side effect and/or toxicity, comprising: obtaining a biological sample from the subject receiving treatment; determining the level of a bone formation biomarker and/or bone resorption biomarker in the sample; and comparing the level of the bone formation biomarker and/or bone resorption biomarker in the sample to a predetermined level of the bone formation biomarker and/or bone resorption biomarker; wherein a decrease in the level of the bone formation biomarker and/or an increase in the level of the bone resorption biomarker indicates development of a skeletal-related side effect and/or toxicity.

[0021] In another aspect, described herein is a method of detecting the development of a skeletal- related side effect and/or toxicity in a subject receiving treatment with a RSPO antagonist, comprising: obtaining a biological sample from the subject receiving treatment; determining the level of a bone formation biomarker and/or bone resorption biomarker in the sample; and comparing the level of the bone formation biomarker and/or bone resorption biomarker in the sample to a predetermined level of the bone formation biomarker and/or bone resorption biomarker; wherein a decrease in the level of the bone formation biomarker and/or an increase in the level of the bone resorption biomarker indicates development of a skeletal-related side effect and/or toxicity.

[0022] In another aspect, described herein is a method for identifying a skeletal-related side effect and/or toxicity in a subject receiving treatment with a RSPO antagonist, comprising: obtaining a biological sample from the subject receiving treatment; determining the level of a bone formation biomarker and/or bone resorption biomarker in the sample; and comparing the level of the bone formation biomarker and/or bone resorption biomarker in the sample to a predetermined level of the bone formation biomarker and/or bone resorption biomarker; wherein a skeletal-related side effect and/or toxicity is indicated when the level of the bone formation biomarker is below a predetermined level; and/or the level of bone resorption biomarker is above a predetermined level.

[0023] In another aspect, described herein is a method for monitoring a skeletal-related side effect and/or toxicity in a subject receiving treatment with a RSPO antagonist, comprising: obtaining a biological sample from the subject receiving treatment; determining the level of a bone formation biomarker and/or bone resorption biomarker in the sample; and comparing the level of the bone formation biomarker and/or bone resorption biomarker in the sample to a predetermined level of the bone formation biomarker and/or bone resorption biomarker; wherein a skeletal-related side effect and/or toxicity is indicated when the level of the bone formation biomarker is below a predetermined level; and/or the level of bone resorption biomarker is above a predetermined level.

[0024] In another aspect, described herein is a method of monitoring a subject receiving treatment with a RSPO antagonist for the development of a skeletal-related side effect and/or toxicity, comprising: determining the level of a bone formation biomarker and/or bone resorption biomarker in a sample from the subject; and comparing the level of the bone formation biomarker and/or bone resorption biomarker in the sample to a predetermined level of the bone formation biomarker and/or bone resorption biomarker; wherein a decrease in the level of the bone formation biomarker and/or an increase in the level of the bone resorption biomarker indicates development of a skeletal-related side effect and/or toxicity.

[0025] In another aspect, described herein is a method of detecting the development of a skeletal- related side effect and/or toxicity in a subject receiving treatment with a RSPO antagonist, comprising: determining the level of a bone formation biomarker and/or bone resorption biomarker in a sample from the subject; and comparing the level of the bone formation biomarker and/or bone resorption biomarker in the sample to a predetermined level of the bone formation biomarker and/or bone resorption biomarker; wherein a skeletal-related side effect and/or toxicity is indicated when the level of the bone formation biomarker is below a predetermined level; and/or the level of bone resorption biomarker is above a predetermined level.

[0026] In another aspect, described herein is a method for identifying a skeletal-related side effect and/or toxicity in a subject receiving treatment with a RSPO antagonist, comprising: determining the level of a bone formation biomarker and/or bone resorption biomarker in a sample from the subject; and comparing the level of the bone formation biomarker and/or bone resorption biomarker in the sample to a predetermined level of the bone formation biomarker and/or bone resorption biomarker; wherein a skeletal-related side effect and/or toxicity is indicated when the level of the bone formation biomarker is below a predetermined level; and/or the level of bone resorption biomarker is above a predetermined level.

[0027] In another aspect, described herein is a method for monitoring a skeletal-related side effect and/or toxicity in a subject receiving treatment with a RSPO antagonist, comprising: determining the level of a bone formation biomarker and/or bone resorption biomarker in a sample from the subject; and comparing the level of the bone formation biomarker and/or bone resorption biomarker in the sample to a predetermined level of the bone formation biomarker and/or bone resorption biomarker; wherein a skeletal-related side effect and/or toxicity is indicated when the level of the bone formation biomarker is below a predetermined level; and/or the level of bone resorption biomarker is above a predetermined level.

[0028] In another aspect, described herein is a method for reducing a skeletal-related side effect and/or toxicity in a subject receiving treatment with a RSPO antagonist, comprising: obtaining a biological sample from the subject receiving treatment; determining the level of a bone formation biomarker and/or bone resorption biomarker in the sample; comparing the level of the bone formation biomarker and/or bone resorption biomarker in the sample to a predetermined level of the bone formation biomarker and/or bone resorption biomarker; and administering to the subject a therapeutically effective amount of an anti-resorptive medication if the level of the bone formation biomarker in the sample is lower than the predetermined level of the bone formation biomarker; and/or the level of the bone resorption biomarker in the sample is higher than the predetermined level of the bone resorption biomarker.

[0029] In another aspect, described herein is a method of preventing or attenuating the development of a skeletal -related side effect and/or toxicity in a subject receiving treatment with a RSPO antagonist, comprising: obtaining a biological sample from the subject prior to treatment with the RSPO antagonist; determining the level of a bone formation biomarker and/or bone resorption biomarker in the sample; comparing the level of the bone formation biomarker and/or bone resorption biomarker in the sample to a predetermined level of the bone formation biomarker and/or bone resorption biomarker; administering to the subject a therapeutically effective amount of an anti- resorptive medication; and administering to the subject the RSPO antagonist.

[0030] In another aspect, described herein is a method for reducing a skeletal-related side effect and/or toxicity in a subject receiving treatment with a RSPO antagonist, comprising: determining the level of a bone formation biomarker and/or bone resorption biomarker in a sample from the subject; comparing the level of the bone formation biomarker and/or bone resorption biomarker in the sample to a predetermined level of the bone formation biomarker and/or bone resorption biomarker; and administering to the subject a therapeutically effective amount of an anti-re sorptive medication if the level of the bone formation biomarker in the sample is lower than the predetermined level of the bone formation biomarker; and/or the level of the bone resorption biomarker in the sample is higher than the predetermined level of the bone resorption biomarker.

[0031] In another aspect, described herein is a method of preventing or attenuating the development of a skeletal-related side effect and/or toxicity in a subject receiving treatment with a RSPO antagonist, comprising: determining the level of a bone formation biomarker and/or bone resorption biomarker in a sample from the subject prior to treatment with the RSPO antagonist; comparing the level of the bone formation biomarker and/or bone resorption biomarker in the sample to a predetermined level of the bone formation biomarker and/or bone resorption biomarker; administering to the subject a therapeutically effective amount of an anti-resorptive medication; and administering to the subject the RSPO antagonist.

[0032] In another aspect, described herein is a method of treating cancer or a fibrotic disease in a subject in need thereof, comprising: administering to the subject a therapeutically effective amount of a RSPO antagonist; and determining the level of a bone formation biomarker and/or bone resorption biomarker in a sample from the subject.

[0033] In one embodiment, a method described herein further comprises: comparing the level of the bone formation biomarker and/or bone resorption biomarker in the sample to a predetermined level of the bone formation biomarker and/or bone resorption biomarker; wherein the subject is at risk for a skeletal-related side effect and/or toxicity if the level of the bone formation biomarker in the sample is lower than the predetermined level of the bone formation biomarker; and/or the level of the bone resorption biomarker in the sample is higher than the predetermined level of the bone resorption biomarker. In one embodiment, a method described herein further comprises: comparing the level of the bone formation biomarker and/or bone resorption biomarker in the sample to a predetermined level of the bone formation biomarker and/or bone resorption biomarker; wherein the subject is administered a therapeutically effective amount of an anti-re sorptive medication if the level of the bone formation biomarker in the sample is lower than the predetermined level of the bone formation biomarker; and/or the level of the bone resorption biomarker in the sample is higher than the predetermined level of the bone resorption biomarker.

[0034] In one embodiment, the sample is blood, serum, or plasma. [0035] In one embodiment, the predetermined level of the bone formation biomarker and/or bone resorption biomarker is the amount of bone formation biomarker and/or bone resorption biomarker in a sample obtained from the subject at an earlier date. In one embodiment, the predetermined level of the bone formation biomarker and/or bone resorption biomarker is the amount of bone formation biomarker and/or bone resorption biomarker in a sample obtained from the subject prior to treatment. In one embodiment, the predetermined level of the bone formation biomarker and/or bone resorption biomarker is a baseline level.

[0036] In one embodiment, the subject is administered a therapeutically effective amount of an anti- resorptive medication if the bone formation biomarker level is below a predetermined level for any one sample; and/or the bone resorption biomarker level is above a predetermined level for any one sample. In one embodiment, the subject is administered a therapeutically effective amount of an anti- resorptive medication if the bone formation biomarker level is 70% or less, 60% or less, 50% or less, 40% or less, or 30% or less than a predetermined level; and/or the bone resorption biomarker level is at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, or at least about 3-fold greater than a predetermined level.

[0037] In one embodiment, the bone formation biomarker is serum procollagen type 1 amino- terminal propeptide (P1NP). In one embodiment, the bone resorption biomarker is collagen type 1 cross-linked beta C-telopeptide (β-CTX). In one embodiment, the bone formation biomarker is serum procollagen type 1 amino-terminal propeptide (P1NP), and the predetermined level is about 16 microg/ml, about 19 microg/ml, about 22 microg/ml, about 25 microg/ml, or about 30 microg/ml. In one embodiment, the bone resorption biomarker is collagen type 1 cross-linked beta C-telopeptide (β- CTX), and the predetermined level is about 1500 pg/ml, about 1200 pg/ml, about 1000 pg/ml, about 800 pg/ml, about 600 pg/ml, or about 400 pg/ml.

[0038] In another aspect, described herein is a method of monitoring a subject receiving treatment with a RSPO antagonist for the development of a skeletal-related side effect and/or toxicity, comprising: determining bone density in the subject; and comparing the bone density in the subject to a predetermined level of bone density; wherein a decrease in bone density in the subject indicates development of a skeletal-related side effect and/or toxicity.

[0039] In another aspect, described herein is a method of detecting the development of a skeletal- related side effect and/or toxicity in a subject receiving treatment with a RSPO antagonist, comprising: determining bone density in the subject; and comparing the bone density in the subject to a predetermined level of bone density; wherein a skeletal-related side effect and/or toxicity is indicated when bone density in the subject is below the predetermined level.

[0040] In another aspect, described herein is a method for identifying a skeletal-related side effect and/or toxicity in a subject receiving treatment with a RSPO antagonist, comprising: determining bone density in the subject; and comparing the bone density in the subject to a predetermined level of bone density; wherein a skeletal-related side effect and/or toxicity is indicated when bone density in the subject is below the predetermined level.

[0041] In another aspect, described herein is a method for monitoring a skeletal-related side effect and/or toxicity in a subject receiving treatment with a RSPO antagonist, comprising: determining bone density in the subject; and comparing the bone density in the subject to a predetermined level of bone density; wherein a skeletal-related side effect and/or toxicity is indicated when bone density in the subject is below the predetermined level.

[0042] In another aspect, described herein is a method for reducing a skeletal-related side effect and/or toxicity in a subject receiving treatment with a RSPO antagonist, comprising: determining bone density in the subject; comparing the bone density in the subject to a predetermined level of bone density; and administering to the subject a therapeutically effective amount of an anti-re sorptive medication if bone density in the subject is below the predetermined level.

[0043] In another aspect, described herein is a method of preventing or attenuating the development of a skeletal -related side effect and/or toxicity in a subject receiving treatment with a RSPO antagonist, comprising: determining bone density in the subject; comparing the bone density in the subject to a predetermined level of bone density; administering to the subject a therapeutically effective amount of an anti-resorptive medication; and administering to the subject the RSPO antagonist.

[0044] In another aspect, described herein is a method of treating cancer or a fibrotic disease in a subject in need thereof, comprising: administering to the subject a therapeutically effective amount of a RSPO antagonist; and determining bone density in the subject.

[0045] In one embodiment, a method described herein further comprises: comparing the bone density in the subject to a predetermined level of bone density; wherein the subject is at risk for a skeletal- related side effect and/or toxicity if bone density in the subject is below the predetermined level. In one embodiment, a method described herein further comprises: comparing the bone density in the subject to a predetermined level of bone density; wherein the subject is administered a therapeutically effective amount of an anti-resorptive medication if bone density in the subject is below the predetermined level. In one embodiment, the predetermined level of bone density is the bone density in the subject at an earlier date. In one embodiment, the predetermined level of bone density is the bone density in the subject prior to treatment. In one embodiment, the predetermined level of bone density is a baseline level. In one embodiment, the subject is administered a therapeutically effective amount of an anti-resorptive medication if bone density in the subject is below the predetermined level. In one embodiment, bone density is determined by dual-energy x-ray absorptiometry (DEXA). In one embodiment, bone density is expressed as a T-score. In one embodiment, the predetermined level of bone density T-score is -1, -1.5, -2, -2.5, or -3.

[0046] In one embodiment, the biological sample is obtained and the bone formation biomarker and/or bone resorption biomarker is determined, the bone formation biomarker and/or bone resorption biomarker is determined, or the bone density is determined once, two or more times, three or more time, four or more times, five or more times, six or more times, seven or more times, or ten or more times. In one embodiment, the biological sample is obtained and the bone formation biomarker and/or bone resorption biomarker is determined, the bone formation biomarker and/or bone resorption biomarker is determined, or the bone density is determined no more than once, two or more times, three or more time, four or more times, five or more times, six or more times, seven or more times, or ten or more times. In one embodiment, the biological sample is obtained and the bone formation biomarker and/or bone resorption biomarker is determined, the bone formation biomarker and/or bone resorption biomarker is determined, or the bone density is determined between 1 and 5 times, between 1 and 10 times, or between 1 and 20 times. In one embodiment, the biological sample is obtained and the bone formation biomarker and/or bone resorption biomarker is determined, the bone formation biomarker and/or bone resorption biomarker is determined, or the bone density is determined once, twice or more, three times or more, or four times or more every week, every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, or every 6 weeks for a total of 2 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, 12 weeks, 4 months, 6 months or 1 year. In one embodiment, the biological sample is obtained and the bone formation biomarker and/or bone resorption biomarker is determined, the bone formation biomarker and/or bone resorption biomarker is determined, or the bone density is determined once, twice or more, three times or more, or four times or more every week, every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, or every 6 weeks for at least 2 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, 12 weeks, 4 months, 6 months or 1 year. In one embodiment, the biological sample is obtained and the bone formation biomarker and/or bone resorption biomarker is determined, the bone formation biomarker and/or bone resorption biomarker is determined, or the bone density is determined once, twice or more, three times or more, or four times or more every week, every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, or every 6 weeks for no more than 2 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, 12 weeks, 4 months, 6 months or 1 year. In one embodiment, the biological sample is obtained and the bone formation biomarker and/or bone resorption biomarker is determined, the bone formation biomarker and/or bone resorption biomarker is determined, or the bone density is determined once, twice or more, three times or more, or four times or more every week, every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, or every 6 weeks for the period of time during which the subject receives treatment with the RSPO antagonist.

[0047] In another aspect, described herein is a method for reducing a skeletal-related side effect and/or toxicity in a subject receiving treatment with a RSPO antagonist, comprising administering to the subject a therapeutically effective amount of an anti-resorptive medication.

[0048] In another aspect, described herein is a method of preventing or attenuating the development of a skeletal -related side effect and/or toxicity in a subject receiving treatment with a RSPO antagonist, comprising administering to the subject a therapeutically effective amount of an anti- resorptive medication. [0049] In one embodiment, the skeletal -related side effect and/or toxicity is related to treatment with the RSPO antagonist.

[0050] In one embodiment, the skeletal-related side effect and/or toxicity is an increased risk of bone fracture, osteopenia, or osteoporosis.

[0051] In one embodiment, the RSPO antagonist is an antibody that specifically binds at least one RSPO protein or portion thereof. In one embodiment, the antibody specifically binds at least one RSPO protein selected from the group consisting of human RSPOl, human RSP02, human RSP03, or human RSP04. In one embodiment, the antibody specifically binds RSP03.

[0052] In one embodiment, the antibody specifically binds RSP03 and comprises: a heavy chain CDR1 comprising DYSIH (SEQ ID NO:5), a heavy chain CDR2 comprising YIYPSNGDSGYNQKFK (SEQ ID NO: 6), and a heavy chain CDR3 comprising TYFANNFD (SEQ ID NO:7); and a light chain CDR1 comprising KASQSVDYDGDSYMN (SEQ ID NO:9), a light chain CDR2 comprising AASNLES (SEQ ID NO: 10), and a light chain CDR3 comprising QQSNEDPLT (SEQ ID NO: 12). In one embodiment, the antibody comprises a heavy chain variable region having at least 90% sequence identity to SEQ ID NO: 14 and a light chain variable region having at least 90% sequence identity to SEQ ID NO: 15. In one embodiment, the antibody comprises the heavy chain variable region of SEQ ID NO: 14 and the light chain variable region of SEQ ID NO: 15.

[0053] In one embodiment, the antibody is a recombinant antibody, a monoclonal antibody, a chimeric antibody, a monospecific antibody, or a bispecific antibody. In one embodiment, the antibody is a humanized antibody. In one embodiment, the antibody is a human antibody. In one embodiment, the antibody is an IgGl antibody. In one embodiment, the antibody is an IgG2 antibody. In one embodiment, the antibody is an antibody fragment comprising an antigen binding site.

[0054] In one embodiment, the antibody is OMP-131R10. In one embodiment, OMP-131R10 is administered intravenously to the subject in need thereof at a dosage of 2.5 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, or 20 mg/kg.

[0055] In one embodiment, the anti-resorptive medication is a bisphosphonate or denosumab. In one embodiment, the bisphosphonate is selected from the group consisting of: etidronate, clodronate, tiludronate, pamidronate, neridronate, olpadronate, alendronate, ibandronate, risedronate, and zoledronic acid. In one embodiment, the bisphosphonate is zoledronic acid.

[0056] In one embodiment, the subject has cancer. In one embodiment, the cancer is selected from the group consisting of: lung cancer, breast cancer, colon cancer, colorectal cancer, melanoma, pancreatic cancer, gastrointestinal cancer, renal cancer, ovarian cancer, neuroendocrine cancer, liver cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, neuroblastoma, glioma, glioblastoma multiforme, cervical cancer, stomach cancer, bladder cancer, hepatoma, and head and neck cancer. In one embodiment, the cancer is colorectal cancer, breast cancer, ovarian cancer, lung cancer, or pancreatic cancer. [0057] In one embodiment, the cancer is colorectal cancer. In one embodiment, the colorectal cancer comprises an inactivating mutation in the adenomatous polyposis coli (APC) gene. In one embodiment, the colorectal cancer does not comprise an inactivating mutation in the APC gene. In one embodiment, the colorectal cancer comprises a wild-type APC gene. In one embodiment, the colorectal cancer does not comprise an activating mutation in the β-catenin gene.

[0058] In one embodiment, the cancer has elevated expression of one or more of RSPOl, RSP02,

RSP03, and RSP04. In one embodiment, the cancer has elevated expression of RSP03.

[0059] In one embodiment, the cancer comprises a RSPO gene fusion. In one embodiment, the cancer comprises an RSP03 gene fusion. In one embodiment, the cancer comprises a fusion between

PTPRK and RSP03.

[0060] In one embodiment, the subject is treated with the RSPO antagonist in combination with one or more additional anti -cancer agents.

[0061] In one embodiment, the subject has a fibrotic disease. In one embodiment, the fibrotic disease is selected from the group consisting of pulmonary fibrosis, renal fibrosis, liver fibrosis, dermal fibrosis, cardiac fibrosis, and adhesion formation. In one embodiment, the dermal fibrosis is selected from the group consisting of scleroderma, systemic sclerosis, scleroderma-like disease, sine scleroderma, keloid formation, and hypertrophic scarring. In one embodiment, the renal fibrosis is chronic kidney disease. In one embodiment, the pulmonary fibrosis is selected from the group consisting of idiopathic pulmonary fibrosis, interstitial pulmonary fibrosis, lung fibrosis, mediastinal fibrosis, secondary pulmonary fibrosis, and pleural fibrosis. In one embodiment, the liver fibrosis is cirrhosis of the liver. In one embodiment, the cardiac fibrosis is selected from the group consisting of myocardial fibrosis, cardiac valve fibrosis, endomyocardial fibrosis, and atherosclerosis.

[0062] In some embodiments of the methods, the fibrotic disease is selected from the group consisting of: pulmonary fibrosis, renal fibrosis, liver fibrosis, dermal fibrosis, cardiac fibrosis, and adhesion formation. In some embodiments, the fibrotic disease is dermal fibrosis. In some embodiments, the dermal fibrosis includes, but is not limited to, scleroderma, systemic sclerosis, scleroderma-like disease, sine scleroderma, keloid formation, and hypertrophic scarring. In some embodiments, the fibrotic disease is renal fibrosis. In some embodiments, the renal fibrosis includes, but is not limited to, chronic kidney disease. In some embodiments, the fibrotic disease is pulmonary fibrosis. In some embodiments, the pulmonary fibrosis includes, but is not limited to, idiopathic pulmonary fibrosis, interstitial pulmonary fibrosis, lung fibrosis, mediastinal fibrosis, and pleural fibrosis. In some embodiments, the pulmonary fibrosis is primary pulmonary fibrosis. In some embodiments, the pulmonary fibrosis is secondary pulmonary fibrosis. In some embodiments, the fibrotic disease is liver fibrosis. In some embodiments, the liver fibrosis includes, but is not limited to, cirrhosis of the liver. In some embodiments, the fibrotic disease is cardiac fibrosis. In some embodiments, the cardiac fibrosis includes, but is not limited to, myocardial fibrosis, cardiac valve fibrosis, endomyocardial fibrosis, and atherosclerosis. In some embodiments, the fibrotic disease is not pulmonary fibrosis. In some embodiments, the fibrotic disease is not liver fibrosis. In some embodiments, the cardiac fibrosis is not atherosclerosis.

[0063] Where aspects or embodiments of the invention are described in terms of a Markush group or other grouping of alternatives, the present 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 explicit exclusion of one or more of any of the group members in the claimed invention.

BRIEF DESCRIPTION OF THE FIGURES

[0064] Figure 1. OMP-131R10 anti-RSP03 antibody Phase 1 clinical study outline.

[0065] Figure 2. 19 subjects have been enrolled in OMP-131R10 anti-RSP03 antibody Phase 1 clinical study, of which 18 has been treated with the drug candidate.

[0066] Figures 3A and 3B. P1NP and β-CTX markers of bone metabolism were monitored throughout the study and revealed dose-dependent changes. Figure 3A. P1NP and β-CTX bone marker expression data detected in subjects treated with OMP-131R10 anti-RSP03 antibody over the treatment period. Figure 3B. Changes in P1NP and β-CTX bone marker expression relative to base line during the treatment period. Orange bars represent a two-fold or more increase (i.e., 100% or more increase) for B-CTX, and two-fold or more decrease (i.e., 50% or more decrease) for P1NP.

[0067] Figure 4. Summary of bone changes in subjects treated with OMP-131R10 anti-RSP03 antibody shown in Figure 3A.

[0068] Figure 5. No substantial drop in DEXA values was observed in subjects treated with OMP- 131R10 anti-RSP03 antibody.

DETAILED DESCRIPTION OF THE INVENTION

[0069] The present invention relates to treating diseases with an RSPO inhibitor (e.g., an anti-RSP03 antibody). More particularly, the invention provides methods for treating cancer comprising administering an RSPO inhibitor (e.g., an anti-RSP03 antibody), either alone or in combination with other anti-cancer agents, and monitoring for skeletal-related side effects and/or toxicity, including those related to the RSPO inhibitor (e.g., an anti-RSP03 antibody). The invention also provides methods for treating fibrotic diseases and for reducing scarring resulting from wound healing comprising administering an RSPO inhibitor (e.g., an anti-RSP03 antibody) and monitoring for skeletal-related side effects and/or toxicity, including those related to the RSPO inhibitor (e.g., an anti-RSP03 antibody).

[0070] The anti-RSP03 antibody OMP-131R10 was administered to subjects in a Phase 1 single agent dose escalation trial. The data from this early trial suggests that administration of an RSPO inhibitor such as an anti-RSP03 antibody may result in skeletal-related side effects and/or toxicity in certain patients. Furthermore, the study shows that increased β-CTX levels or decreased P1NP levels may be an early indicator that a patient being treated with an RSPO inhibitor (e.g., anti-RSP03 antibody) is at risk of developing skeletal-related side effects and/or toxicities, allowing for intervention with appropriate medications.

[0071] These results make it desirable to develop risk mitigation and monitoring strategies for skeletal -related side effects and/or toxicities as described herein for subjects receiving treatment with an RSPO inhibitor (e.g., anti-RSP03 antibody) as a single agent or in combination with additional therapeutic agents, e.g., anti-cancer agents.

I. Definitions

[0072] To facilitate an understanding of the present invention, a number of terms and phrases are defined below.

[0073] The terms "antagonist" and "antagonistic" as used herein refer to any molecule that partially or fully blocks, inhibits, reduces, or neutralizes a biological activity of a target and/or signaling pathway (e.g., the RSPO-LGR pathway). The term "antagonist" is used herein to include any molecule that partially or fully blocks, inhibits, reduces, or neutralizes the activity of a protein (e.g., an RSPO protein or an LGR protein). Suitable antagonist molecules specifically include, but are not limited to, antagonist antibodies, antibody fragments, soluble receptors, or small molecules.

[0074] 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 antigen-binding site within the variable region of the immunoglobulin molecule. As used herein, the term encompasses intact polyclonal antibodies, intact monoclonal antibodies, antibody fragments comprising an antigen-binding site (such as Fab, Fab', F(ab')2, and Fv fragments), single chain Fv (scFv) antibodies, multispecific antibodies such as bispecific antibodies, monospecific antibodies, monovalent antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising an antigen-binding site of an antibody, and any other modified immunoglobulin molecule comprising an antigen-binding site as long as the antibodies exhibit the desired biological activity. An antibody can be any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (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.

[0075] The term "antibody fragment" as used herein refers to a portion of an intact antibody and generally includes the antigenic determining variable region or antigen-binding site of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, single chain antibodies, and multispecific antibodies formed from antibody fragments. "Antibody fragment" as used herein comprises at least one antigen-binding site or epitope -binding site.

[0076] The term "variable region" of an antibody as used herein 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 region of the heavy or light chain generally consists of four framework regions 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, contribute to the formation of the antigen-binding site of the antibody. There are at least two techniques for determining CDRs: ( 1) an approach based on cross-species sequence variability (i.e., Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Edition, National Institutes of Health, Bethesda MD), and (2) an approach based on crystallographic studies of antigen-antibody complexes (Al-Lazikani et al., 1997, J. Mol. Biol., 273 :927-948). In addition, combinations of these two approaches are sometimes used in the art to determine CDRs.

[0077] The term "monoclonal antibody" as used herein refers to a homogenous antibody 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 against different antigenic determinants. The term "monoclonal antibody" encompasses both intact and full-length 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 at least one antigen-binding site. Furthermore, "monoclonal antibody" refers to such antibodies made by any number of techniques, including but not limited to, hybridoma production, phage selection, recombinant expression, and transgenic animals.

[0078] The term "humanized antibody" as used herein refers to antibodies that are specific immunoglobulin chains, chimeric immunoglobulins, or fragments thereof that contain minimal non- human sequences. Typically, humanized antibodies are human immunoglobulins in which amino acid residues of the CDRs are replaced by amino acid residues from the CDRs of a non-human species (e.g., mouse, rat, rabbit, or hamster) that have the desired specificity, affinity, and/or binding capability.

[0079] The term "human antibody" as used herein refers to an antibody 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.

[0080] The term "chimeric antibody" as used herein refers to an antibody wherein the amino acid sequence of the immunoglobulin 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 derived from one species of mammals (e.g., mouse, rat, rabbit, etc.) with the desired specificity, affinity, and/or binding capability, while the constant regions are homologous to the sequences in antibodies derived from another species (usually human).

[0081] The term "affinity-matured antibody" as used herein refers to an antibody with one or more alterations in one or more CDRs that result in an improvement in the affinity of the antibody for antigen, compared to a parent antibody that does not possess those alterations(s). Preferred affinity- matured antibodies will have nanomolar or even picomolar affinities for the target antigen. Affinity- matured antibodies are produced by procedures known in the art including heavy chain and light chain variable region shuffling, random mutagenesis of CDR and/or framework residues, or site-directed mutagenesis of CDR and/or framework residues.

[0082] 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, epitopes can be formed both from contiguous amino acids and non-contiguous amino acids juxtaposed 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 typically includes at least 3, and more usually, at least 5, or 8-10 amino acids in a unique spatial conformation.

[0083] The terms "selectively binds" or "specifically binds" as used herein mean that a binding agent or an antibody reacts or associates more frequently, more rapidly, with greater duration, with greater affinity, or with some combination of the above to the epitope, protein, or target molecule than with alternative substances, including unrelated or related proteins. In certain embodiments "specifically binds" means, for instance, that an antibody binds a target with a K_D of about 0. ImM or less, but more usually less than about ΙμΜ. In certain embodiments, "specifically binds" means that an antibody binds a target with a K_D of at least about 0. ΙμΜ or less, at least about 0.0 ΙμΜ or less, or at least about InM 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 RSPO protein and mouse RSPO protein). 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 RSPO l and human RSP03). It is understood that, in certain embodiments, an antibody or binding agent that specifically binds a first target can or cannot 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 can, in certain embodiments, specifically bind more than one target. In certain embodiments, multiple targets can be bound by the same antigen-binding site on the antibody. For example, an antibody can, 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 RSP03). In certain alternative embodiments, an antibody can be bispecific and comprise at least two antigen-binding sites with differing specificities. By way of non-limiting example, a bispecific antibody can comprise one antigen-binding site that recognizes an epitope on one protein (e.g., a human RSPO protein) and further comprise a second, different antigen-binding site that recognizes a different epitope on a second protein. Generally, but not necessarily, reference to binding means specific binding.

[0084] The term "soluble receptor" as used herein refers to an extracellular fragment (or a portion thereof) of a receptor protein preceding the first transmembrane domain of the receptor that can be secreted from a cell in soluble form.

[0085] The term "LGR soluble receptor" as used herein refers to an extracellular fragment of an LGR receptor protein (e.g., LGR5) preceding the first transmembrane domain of the receptor that can be secreted from a cell in soluble form. LGR soluble receptors comprising the entire extracellular domain (ECD) as well as smaller fragments of the ECD are encompassed by the term. In certain embodiments, the extracellular fragment is capable of binding at least one human RSPO protein.

[0086] The terms "polypeptide" and "peptide" and "protein" are used interchangeably herein and refer to polymers of amino acids of any length. The polymer can be linear or branched, it can comprise modified amino acids, and it can 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 formation, 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 containing one or more analogs of an amino acid (including, for example, unnatural amino acids), as well as other modifications known in the art. It is understood that, because the polypeptides used in the methods described herein can be based upon antibodies, in certain embodiments, the polypeptides can occur as single chains or associated chains.

[0087] The term "amino acid" as used herein refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine. The phrase "amino acid analog" refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, e.g., an alpha carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs can have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. The phrase "amino acid mimetic" refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that function similarly to a naturally occurring amino acid.

[0088] The terms "polynucleotide" and "nucleic acid" are used interchangeably herein and refer to polymers of nucleotides 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.

[0089] The terms "identical" or percent "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 nucleotides or amino acid residues that are the same, when compared and aligned (introducing gaps, if necessary) for maximum correspondence, not considering any conservative amino acid substitutions as part of the sequence identity. The percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that can be used to obtain alignments of amino acid or nucleotide sequences are well-known in the art. These include, but are not limited to, BLAST and BLAST variations, ALIGN and ALIGN variations, Megalign, BestFit, GCG Wisconsin Package, etc. In some embodiments, two nucleic acids or polypeptides are substantially identical, meaning 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 sequence comparison algorithm 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 nucleotides or residues, at least about 60-80 nucleotides or residues in length or any integral value therebetween. In some embodiments, identity exists over a longer region than 60-80 nucleotides or residues, such as at least about 80-100 nucleotides or residues, and in some embodiments the sequences are substantially identical over the full length of the sequences being compared, such as the coding region of a nucleotide sequence.

[0090] The term "conservative amino acid substitution" as used herein refers to a substitution in which one amino acid residue is replaced with another amino acid residue having a similar 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, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For example, substitution of a phenylalanine for a tyrosine is a conservative substitution. Preferably, conservative substitutions in the sequences of the polypeptides and antibodies do not abrogate the binding of the polypeptide or antibody containing the amino acid sequence to the antigen(s). Methods of identifying amino acid conservative substitutions which do not eliminate antigen binding are well-known in the art.

[0091] 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 include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid, or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, and DNA or RNA expression vectors encapsulated in liposomes.

[0092] As used herein, 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.

[0093] 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.

[0094] The terms "cancer" and "cancerous" as used herein refer to or describe the physiological condition in mammals in which a population of cells is characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, blastema, sarcoma, and hematologic cancers such as lymphoma and leukemia.

[0095] The terms "proliferative disorder" and "proliferative disease" as used herein refer to disorders associated with abnormal cell proliferation such as cancer.

[0096] The terms "tumor" and "neoplasm" as used herein refer to any mass of tissue that results from excessive cell growth or proliferation, either benign (non-cancerous) or malignant (cancerous), including pre-cancerous lesions.

[0097] The term "metastasis" 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 "metastatic" or "metastasizing" cell is generally one that loses adhesive contacts with neighboring cells and migrates from the primary site of disease to invade neighboring body structures.

[0098] 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 proliferative 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 transplantation 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.

[0099] The terms "cancer cell" and "tumor cell" as used herein refer to the total population of cells derived from a cancer or tumor or pre-cancerous lesion, including both non-tumorigenic cells, which comprise the bulk of the cancer cell population, and tumorigenic cells (cancer 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.

[00100] The term "tumorigenic" as used herein refers to the functional features of a cancer stem cell including the properties of self-renewal (giving 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).

[00101] The term "tumorigenicity" as used herein refers to the ability of a sample of cells from a tumor to form palpable tumors upon serial transplantation into immunocompromised hosts (e.g., mice).

[00102] The term "fibrotic diseases" as used herein includes but is not limited to connective tissue diseases. Those of skill in the art generally believe fibrosis to be the formation or development of excess fibrous connective tissue in an organ or tissue. In some embodiments, fibrosis occurs as a reparative or reactive process. In some embodiments, fibrosis occurs in response to damage or injury. The term "fibrosis" is to be understood as the formation or development of excess fibrous connective tissue in an organ or tissue as a reparative or reactive process, as opposed to a formation of fibrous tissue as a normal constituent of an organ or tissue.

[00103] The term "subject" as used herein 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.

[00104] The term "pharmaceutically acceptable" refers to an agent, compound, molecule, etc. approved or approvable by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, including humans.

[00105] The phrases "pharmaceutically acceptable excipient, carrier or adjuvant" and "acceptable pharmaceutical carrier" refer to an excipient, carrier, or adjuvant that can be administered to a subject, together with a therapeutic agent, and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic effect. In general, those of skill in the art and the FDA consider a pharmaceutically acceptable excipient, carrier, or adjuvant to be an inactive ingredient of any formulation or pharmaceutical composition.

[00106] The terms "effective amount" and "therapeutically effective amount" and "therapeutic effect" as used herein refer to an amount of a binding agent, an antibody, a polypeptide, a polynucleotide, a small molecule, or other therapeutic agent effective to "treat" a disease or disorder in a subject or mammal. In the case of cancer, the therapeutically effective amount of an agent (e.g., an antibody) has a therapeutic effect and as such can reduce the number of cancer cells; decrease tumorigenicity, tumorigenic frequency, or tumorigenic capacity; reduce the number or frequency of cancer stem cells; reduce tumor size; reduce the cancer cell population; inhibit and/or stop cancer cell infiltration 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/or stop tumor or cancer cell growth; relieve to some extent one or more of the symptoms associated with the cancer; reduce morbidity and mortality; improve quality of life; or a combination of such effects. To the extent the agent prevents growth and/or kills existing cancer cells, it can be referred to as cytostatic and/or cytotoxic. In the case of fibrotic disease, the therapeutically effective amount of an agent (e.g., an antibody) has a therapeutic effect and as such can prevent the development of a fibrotic disease; slow down the development of a fibrotic disease; slow down the progression of a fibrotic disease; reduce the amount of fibrosis in a disease; reduce pathological deposits of fibrotic material in an organ; reduce pathological deposits of connective tissue or extracellular matrix in an organ; relieve to some extent one or more of the symptoms associated with a fibrotic disease; reduce morbidity and mortality; improve quality of life; or a combination of such effects.

[00107] The terms "treating" and "treatment" and "to treat" and "alleviating" and "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 measures that prevent or slow the development of a targeted pathologic condition or disorder. Thus, those in need of treatment include those who already have a disorder; those prone to have a disorder; and those in whom a disorder is to be prevented. In some embodiments, a subject is successfully "treated" according to the methods described herein if the patient shows one or more of the following: a reduction in the number of or complete absence of cancer cells; a reduction in tumor size; inhibition of or an absence of cancer cell infiltration into peripheral organs including the spread of cancer cells into soft tissue and bone; inhibition 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. In the case of fibrotic disease, in some embodiments, a subject is successfully "treated" according to the methods of the present invention if the patient shows one or more of the following: prevention of the development of a fibrotic disease; the slowing of development of a fibrotic disease; the slowing of progression of a fibrotic disease; reduction in the amount of fibrosis in a disease; reduction of pathological deposits of fibrotic material in an organ; reduction of pathological deposits of connective tissue or extracellular matrix in an organ; the relief to some extent of one or more symptoms associated with a fibrotic disease; reduction of morbidity and mortality; improvement of quality of life; or some combination of such effects.

[00108] As used in the present disclosure and claims, the singular forms "a", "an", and "the" include plural forms unless the context clearly dictates otherwise.

[00109] It is understood that wherever embodiments are described herein with the language "comprising" otherwise analogous embodiments described in terms of "consisting of and/or "consisting essentially of are also provided. It is also understood that wherever embodiments are described herein with the language "consisting essentially of otherwise analogous embodiments described in terms of "consisting of are also provided.

[00110] As used herein, reference to "about" or "approximately" a value or parameter includes (and describes) embodiments that are directed to that value or parameter. For example, description referring to "about X" includes description of "X".

[00111] The term "and/or" as used in a phrase such as "A and/or B" herein is intended 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 following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

II. RSPO antagonists

[00112] Described herein are methods comprising administering an RSPO antagonist (e.g., anti- RSP03 antibody). In certain embodiments, the RSPO antagonist is an agent that binds one or more soluble extracellular components of the RSPO-LGR pathway. In certain embodiments, the RSPO antagonist is an agent that binds one or more extracellular region(s) of an RSPO protein, e.g., RSP03. In certain embodiments, the RSPO antagonist is an agent that directly modulates the activity of one or more RSPO proteins, e.g., RSP03.

[00113] In certain embodiments, the RSPO antagonist is an agent that inhibits β-catenin signaling. In certain embodiments, the RSPO antagonist is an agent that modulates Wnt-mediated β-catenin signaling.

[00114] In certain embodiments, the RSPO antagonist is an agent that binds one or more human RSPO proteins, e.g., human RSP03. These agents are referred to herein as "RSPO-binding agents". Non-limiting examples of RSPO-binding agents can be found in U.S. Patent Nos. 8158758, 8158757, 8802097, 8088374, and U.S. Patent Publication Nos. 2014/0017253, 2014/0134703, 2013/0337533, 2014/0186917, 2012/0263730, 2012/0039912, 2009/0220495, 2012/0088727, 2014/0056894, and 20150147333, each of which is hereby incorporated by reference herein in its entirety for all purposes.

[00115] In some embodiments, the RSP03-binding agent is an antibody disclosed in U.S. Patent Publication No. 2015/0147333. For example, the anti-RSP03 -binding agent can comprise the 4H1, 4D4, 5C2, 5D6, 5E11, 6E9, 21C2, or 26E11 antibody. In some embodiments, the anti- RSP03- binding agent is a chimeric antibody comprising the VH or VL domain of the 4H1, 4D4, 5C2, 5D6, 5E11, 6E9, 21C2, or 26E11 antibody. In some embodiments, the anti-RSP03 -binding agent is a chimeric antibody comprising the VH domain of the 4H1, 4D4, 5C2, 5D6, 5E11, 6E9, 21C2, or 26E11 antibody, and the VL domain of the 4H1, 4D4, 5C2, 5D6, 5E11, 6E9, 21C2, or 26E11 antibody. In some embodiments, the anti- RSP03-binding agent is a chimeric antibody comprising the VH and VL domains of the 4H1, 4D4, 5C2, 5D6, 5E11, 6E9, 21C2, or 26E11 antibody. In some embodiments, the anti-RSP03-binding agent is a humanized antibody comprising the 3 VH CDRs or the 3 VL CDRs of the 4H1, 4D4, 5C2, 5D6, 5E11, 6E9, 21C2, or 26E11 antibody. In some embodiments, the anti- RSP03-binding agent is a chimeric antibody comprising the VH and VL domains of the 5D6 antibody. In some embodiments, the anti -RSP03 -binding agent is a humanized antibody comprising the 3 VH CDRs of the 4H1, 4D4, 5C2, 5D6, 5E11, 6E9, 21C2, or 26E11 antibody and the 3 VL CDRs of the 4H1, 4D4, 5C2, 5D6, 5E11, 6E9, 21C2, or 26E11 antibody. In some embodiments, the anti -RSP03 -binding agent is a humanized antibody comprising the 3 VH CDRs and the 3 VL CDRs of the 4H1, 4D4, 5C2, 5D6, 5E11, 6E9, 21C2, or 26E11 antibody. In some embodiments, the anti -RSP03 -binding agent is a humanized antibody comprising the 3 VH CDRs and the 3 VL CDRs of the 4H1, 4D4, 5C2, 5D6, 5E11, 6E9, 21C2, or 26E11 antibody or a variant of the 3 VH CDRs and/or the 3 VL CDRs of the 4H1, 4D4, 5C2, 5D6, 5E11, 6E9, 21C2, or 26E11 antibody. In some embodiments, the anti -RSP03 -binding agent is a humanized antibody comprising the 3 VH CDRs and the 3 VL CDRs of the 5D6 antibody. In some embodiments, the anti- RSP03-binding agent is an antibody comprising the 3 VH CDRs or the 3 VL CDRs of the 5D6, 5D6vl, 5D6v2.1, 5D6v2.2, 5D6v2.3, 5D6v2.4, 5D6v2.8, 5D6v2.10, 5D6v3.2, 5D6v3.3, 5D6v4.1, 5D6v4.3, 5D6v5.1, or 5D6v5.2 antibody. In some embodiments, the anti-RSP03-binding agent is an antibody comprising the 3 VH CDRs of the 5D6, 5D6vl, 5D6v2.1, 5D6v2.2, 5D6v2.3, 5D6v2.4, 5D6v2.8, 5D6v2.10, 5D6v3.2, 5D6v3.3, 5D6v4.1, 5D6v4.3, 5D6v5.1, or 5D6v5.2 antibody and the 3 VL CDRs of the 5D6, 5D6vl, 5D6v2.1, 5D6v2.2, 5D6v2.3, 5D6v2.4, 5D6v2.8, 5D6v2.10, 5D6v3.2, 5D6v3.3, 5D6v4.1, 5D6v4.3, 5D6v5.1, or 5D6v5.2 antibody. In some embodiments, the anti-RSP03- binding agent is an antibody comprising the 3 VH CDRs and the 3 VL CDRs of the 5D6, 5D6vl, 5D6v2.1, 5D6v2.2, 5D6v2.3, 5D6v2.4, 5D6v2.8, 5D6v2.10, 5D6v3.2, 5D6v3.3, 5D6v4.1, 5D6v4.3, 5D6v5.1, or 5D6v5.2 antibody. In some embodiments, the anti -RSP03 -binding agent is an antibody comprising the VH or VL domain of the 5D6, 5D6vl, 5D6v2.1, 5D6v2.2, 5D6v2.3, 5D6v2.4, 5D6v2.8, 5D6v2.10, 5D6v3.2, 5D6v3.3, 5D6v4.1, 5D6v4.3, 5D6v5.1, or 5D6v5.2 antibody. In some embodiments, the anti-RSP03-binding agent is an antibody comprising the VH domain of the 5D6, 5D6vl, 5D6v2.1, 5D6v2.2, 5D6v2.3, 5D6v2.4, 5D6v2.8, 5D6v2.10, 5D6v3.2, 5D6v3.3, 5D6v4.1, 5D6v4.3, 5D6v5.1, or 5D6v5.2 antibody and the VL domain of the 5D6, 5D6vl, 5D6v2.1, 5D6v2.2, 5D6v2.3, 5D6v2.4, 5D6v2.8, 5D6v2.10, 5D6v3.2, 5D6v3.3, 5D6v4.1, 5D6v4.3, 5D6v5.1, or 5D6v5.2 antibody. In some embodiments, the anti-RSP03-binding agent is an antibody comprising the VH and VL domains of the 5D6, 5D6vl, 5D6v2.1, 5D6v2.2, 5D6v2.3, 5D6v2.4, 5D6v2.8, 5D6v2.10, 5D6v3.2, 5D6v3.3, 5D6v4.1, 5D6v4.3, 5D6v5.1, or 5D6v5.2 antibody. In some embodiments, the anti -RSP03 -binding agent comprises an antibody selected from the group consisting of 5D6vl, 5D6v2.1, 5D6v2.2, 5D6v2.3, 5D6v2.4, 5D6v2.8, 5D6v2.10, 5D6v3.2, 5D6v3.3, 5D6v4.1, 5D6v4.3, 5D6v5.1, and 5D6v5.2. In some embodiments, the anti-RSP03 -binding agent comprises the VH and/or VL domains of the 5D6v4.1 antibody. In some embodiments, the anti-RSP03 -binding agent comprises the he VH and/or VL domains of the 5D6v4.3 antibody. In some embodiments, the anti- RSP03-binding agent comprises the he VH and/or VL domains of the 5D6v5.1 antibody. In some embodiments, the anti-RSP03 -binding agent comprises the he VH and/or VL domains of the 5D6v5.2 antibody. In some embodiments, the anti-RSP03 -binding agent comprises the 5D6v4.1 antibody. In some embodiments, the anti-RSP03 -binding agent comprises the 5D6v4.3 antibody. In some embodiments, the anti-RSP03-binding agent comprises the 5D6v5.1 antibody. In some embodiments, the anti-RSP03-binding agent comprises the 5D6v5.2 antibody.

[00116] In some embodiments, the RSPO-binding agent is an antibody. In some embodiments, the RSPO-binding agent is a polypeptide. In certain embodiments, the RSPO-binding agent binds RSP03 ("RSP03-binding agents"). In certain embodiments, the RSPO-binding agent specifically binds one or more human RSPO proteins. The full-length amino acid (aa) sequences for human RSPO l, RSP02, RSP03, and RSP04 are known in the art and are provided herein as SEQ ID NO: 1 (RSPOl), SEQ ID NO:2 (RSP02), SEQ ID NO:3 (RSP03), and SEQ ID NO:4 (RSP04).

[00117] In certain embodiments, the antigen-binding site of an RSPO-binding agent (e.g., an antibody or a bispecific antibody) is capable of binding (or binds) one, two, three, or four RSPOs. In certain embodiments, the antigen-binding site of an RSPO-binding agent (e.g., an antibody or a bispecific antibody) is capable of binding (or binds) a first RSPO protein (e.g., RSP03) as well as one, two, or three other RSPOs (e.g., RSPOl, RSP02, and/or RSP04). In some embodiments, the RSPO-binding agent (e.g., antibody) specifically binds both human RSPO and mouse RSPO.

[00118] In certain embodiments, the RSPO-binding agent is an antibody that specifically binds within amino acids 22-272 of human RSP03 (SEQ ID NO:3). In certain embodiments, the RSPO-binding agent is an antibody that specifically binds within amino acids 22-207 of human RSP03 (SEQ ID NO: 3). In certain embodiments, the RSPO-binding agent is an antibody that specifically binds within amino acids 35-135 of human RSP03 (SEQ ID NO:3). In certain embodiments, the RSPO-binding agent is an antibody that specifically binds within amino acids 35-86 of human RSP03 (SEQ ID NO: 3). In certain embodiments, the RSPO-binding agent is an antibody that specifically binds within amino acids 92-135 of human RSP03 (SEQ ID NO:3). In certain embodiments, the RSPO-binding agent binds a furin-like cysteine-rich domain of RSP03. In some embodiments, the RSPO-binding agent binds at least one amino acid within a furin-like cysteine-rich domain of RSP03. In some embodiments, the RSPO-binding agent binds the thrombospondin domain of RSP03. In some embodiments, the RSPO-binding agent binds at least one amino acid within the thrombospondin domain of RSP03.

[00119] In certain embodiments, the RSPO-binding agent or antibody binds at least one RSPO protein (e.g., RSP03) with a dissociation constant (K_D) of about luM or less, about lOOnM or less, about 40nM or less, about 20nM or less, about ΙΟηΜ or less, about InM or less, or about O. lnM or less. In certain embodiments, an RSPO-binding agent or antibody binds at least one RSPO protein (e.g., RSP03) with a dissociation constant (K_D) of about luM or less, about lOOnM or less, about 40nM or less, about 20nM or less, about ΙΟηΜ or less, about InM or less, or about O. lnM or less. In some embodiments, an RSPO-binding agent or antibody binds at least one RSPO protein with a K_D of about 20nM or less. In some embodiments, an RSPO-binding agent or antibody binds at least one RSPO protein with a K_D of about lOnM or less. In some embodiments, an RSPO-binding agent or antibody binds at least one RSPO protein with a K_D of about InM or less. In some embodiments, an RSPO- binding agent or antibody binds at least one RSPO protein with a K_D of about 0.5nM or less. In some embodiments, an RSPO-binding agent or antibody binds at least one RSPO protein with a K_D of about 0. InM or less. In certain embodiments, an RSPO-binding agent or antibody described herein binds at least two RSPO proteins. In some embodiments, the RSPO-binding agent binds both human RSPO and mouse RSPO with a K_D of about lOnM or less. In some embodiments, an RSPO-binding agent binds both human RSPO and mouse RSPO with a K_D of about InM or less. In some embodiments, an RSPO-binding agent binds both human RSPO and mouse RSPO with a K_D of about 0. InM or less. In some embodiments, the dissociation constant of a binding agent (e.g., an antibody) to an RSPO protein is the dissociation constant determined using an RSPO fusion protein comprising at least a portion of the RSPO protein immobilized on a Biacore chip. In some embodiments, the dissociation constant of a binding agent (e.g., an antibody) to an RSPO protein is the dissociation constant determined using the binding agent captured by an anti-human IgG antibody on a Biacore chip and an RSPO protein.

[00120] In certain embodiments, the RSPO-binding agent (e.g., an antibody) binds to at least one human RSPO protein (e.g., RSP03) with a half maximal effective concentration (EC₅₀) of about ΙμΜ or less, about ΙΟΟηΜ or less, about 40nM or less, about 20nM or less, about lOnM or less, about InM or less, or about O. lnM or less. In certain embodiments, an RSPO-binding agent (e.g., an antibody) binds to at least one human RSPO with a half maximal effective concentration (EC₅₀) of about 1 μΜ or less, about ΙΟΟηΜ or less, about 40nM or less, about 20nM or less, about lOnM or less, about InM or less, or about O. lnM or less.

Table 1

(SEQ ID NO:9)

AASNLES

(SEQ ID NO: 10) or

CDR2

AAS

(SEQ ID NO: 11)

QQSNEDPLT

(SEQ ID NO: 12) or

CDR3

QQSNEDPLTF

(SEQ ID NO: 13)

[00121] In certain embodiments of the methods described herein, the RSPO-binding agent is an RSP03-binding agent (e.g., an antibody) that specifically binds human RSP03, wherein the RSP03- binding agent (e.g., an antibody) comprises one, two, three, four, five, and/or six of the CDRs of antibody 131R010 (see Table 1 herein).

[00122] In certain embodiments, the RSPO-binding agent is an RSP03-binding agent (e.g., an antibody) that specifically binds human RSP03, wherein the RSP03-binding agent comprises a heavy chain CDRl comprising DYSIH (SEQ ID NO:5), a heavy chain CDR2 comprising YIYPSNGDSGYNQKFK (SEQ ID NO: 6), and a heavy chain CDR3 comprising TYFANNFD (SEQ ID NO:7) or ATYFANNFDY (SEQ ID NO:8). In some embodiments, the RSP03-binding agent further comprises a light chain CDRl comprising KASQSVDYDGDSYMN (SEQ ID NO: 9), a light chain CDR2 comprising AASNLES (SEQ ID NO: 10) or AAS (SEQ ID NO: 11), and a light chain CDR3 comprising QQSNEDPLT (SEQ ID NO: 12) or QQSNEDPLTF (SEQ ID NO: 13). In some embodiments, the RSP03-binding agent comprises a light chain CDRl comprising KASQSVDYDGDSYMN (SEQ ID NO: 9), a light chain CDR2 comprising AASNLES (SEQ ID NO: 10) or AAS (SEQ ID NO: 11), and a light chain CDR3 comprising QQSNEDPLT (SEQ ID NO: 12) or QQSNEDPLTF (SEQ ID NO: 13). In certain embodiments, the RSP03-binding agent comprises: (a) a heavy chain CDRl comprising DYSIH (SEQ ID NO:5), a heavy chain CDR2 comprising YIYPSNGDSGYNQKFK (SEQ ID NO:6), and a heavy chain CDR3 comprising TYFANNFD (SEQ ID NO: 7); and (b) a light chain CDRl comprising KASQSVDYDGDSYMN (SEQ ID NO:9), a light chain CDR2 comprising AASNLES (SEQ ID NO: 10), and a light chain CDR3 comprising QQSNEDPLT (SEQ ID NO: 12). In certain embodiments, the RSP03-binding agent comprises: (a) a heavy chain CDRl comprising DYSIH (SEQ ID NO:5), a heavy chain CDR2 comprising YIYPSNGDSGYNQKFK (SEQ ID NO:6), and a heavy chain CDR3 comprising ATYFANNFDY (SEQ ID NO: 8); and (b) a light chain CDRl comprising KASQSVDYDGDSYMN (SEQ ID NO:9), a light chain CDR2 comprising AASNLES (SEQ ID NO: 10), and a light chain CDR3 comprising QQSNEDPLT (SEQ ID NO: 12).

[00123] In certain embodiments, the RSPO-binding agent is an RSP03-binding agent (e.g., an antibody or bispecific antibody) that specifically binds human RSP03, wherein the RSP03-binding agent comprises: (a) a heavy chain CDRl comprising DYSIH (SEQ ID NO:5) or a variant thereof comprising 1, 2, 3, or 4 amino acid substitutions; (b) a heavy chain CDR2 comprising YIYPSNGDSGYNQKFK (SEQ ID NO:6) or a variant thereof comprising 1, 2, 3, or 4 amino acid substitutions; (c) a heavy chain CDR3 comprising TYFANNFD (SEQ ID NO: 7), ATYFANNFDY (SEQ ID NO:8), or a variant thereof comprising 1, 2, 3, or 4 amino acid substitutions; (d) a light chain CDRl comprising KASQSVDYDGDSYMN (SEQ ID NO:9) or a variant thereof comprising 1, 2, 3, or 4 amino acid substitutions; (e) a light chain CDR2 comprising AASNLES (SEQ ID NO: 10), AAS (SEQ ID NO: 11), or a variant thereof comprising 1, 2, 3, or 4 amino acid substitutions; and (f) a light chain CDR3 comprising QQSNEDPLT (SEQ ID NO: 12), QQSNEDPLTF (SEQ ID NO: 13), or a variant thereof comprising 1, 2, 3, or 4 amino acid substitutions. In certain embodiments, the amino acid substitutions are conservative substitutions. In some embodiments, the substitutions are made as part of a germline humanization process.

[00124] In certain embodiments, the RSPO-binding agent is an RSP03-binding agent (e.g., an antibody) that specifically binds RSP03, wherein the RSP03-binding agent comprises a heavy chain variable region having at least about 80% sequence identity to SEQ ID NO: 14 and/or a light chain variable region having at least 80% sequence identity to SEQ ID NO: 15. In certain embodiments, the RSP03-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: 14. In certain embodiments, the RSP03-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: 15. In certain embodiments, the RSP03 -binding agent comprises a heavy chain variable region having at least about 95% sequence identity to SEQ ID NO: 14 and/or a light chain variable region having at least about 95% sequence identity to SEQ ID NO: 15. In certain embodiments, the RSP03-binding agent comprises a heavy chain variable region comprising SEQ ID NO: 14 and/or a light chain variable region comprising SEQ ID NO: 15. In certain embodiments, the RSP03-binding agent comprises a heavy chain variable region comprising SEQ ID NO: 14 and a light chain variable region comprising SEQ ID NO: 15. In certain embodiments, the RSP03-binding agent comprises a heavy chain variable region consisting of SEQ ID NO: 14 and a light chain variable region consisting of SEQ ID NO: 15.

[00125] In certain embodiments, the RSPO-binding agent is an RSP03-binding agent (e.g., an antibody) that specifically binds RSP03, wherein the RSP03-binding agent comprises: (a) a heavy chain having at least 90% sequence identity to SEQ ID NO: 16 or SEQ ID NO: 17; and/or (b) a light chain having at least 90% sequence identity to SEQ ID NO: 18 or SEQ ID NO: 19. In some embodiments, the RSP03-binding agent comprises: (a) a heavy chain having at least 95% sequence identity to SEQ ID NO: 16 or SEQ ID NO: 17; and/or (b) a light chain having at least 95% sequence identity to SEQ ID NO: 18 or SEQ ID NO: 19. In some embodiments, the RSP03-binding agent comprises a heavy chain comprising SEQ ID NO: 17 and/or a light chain comprising SEQ ID NO: 19. In some embodiments, the RSP03-binding agent comprises a heavy chain comprising SEQ ID NO: 17 and a light chain comprising SEQ ID NO: 19.

[00126] In certain embodiments, an RSP03 -binding agent comprises the heavy chain variable region and light chain variable region of antibody 131R010. In certain embodiments, an RSP03 -binding agent comprises the heavy chain and light chain of antibody 131R010 (with or without the leader sequence). In certain embodiments, an RSP03-binding agent is antibody 131R010. In certain embodiments, an RSP03-binding agent comprises the heavy chain variable region and/or light chain variable region of antibody 131R010 in a chimeric form of the antibody. In certain embodiments, an RSP03-binding agent comprises the heavy chain CDRs and/or light chain CDRs of antibody 131R010. In some embodiments, the anti-RSP03 antibody is 131R010.

[00127] Plasmids encoding the heavy chain and light chain of antibody 131R010 were deposited with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, VA, USA, under the conditions of the Budapest Treaty on June 18, 2013 and assigned ATCC deposit designation number PTA-120420 and PTA-120421. In some embodiments, the RSP03-binding agent comprises a heavy chain variable region encoded by the plasmid deposited with ATCC and designated PTA- 120420. In some embodiments, the RSP03 -binding agent comprises a light chain variable region encoded by the plasmid deposited with ATCC and designated PTA-120421. In some embodiments, the RSP03-binding agent comprises a heavy chain variable region encoded by the plasmid deposited with ATCC and designated PTA-120420 and a light chain variable region encoded by the plasmid deposited with ATCC and designated PTA-120421. In some embodiments, the RSP03-binding agent comprises a heavy chain encoded by the plasmid deposited with ATCC and designated PTA-120420. In some embodiments, the RSP03-binding agent comprises a light chain encoded by the plasmid deposited with ATCC and designated PTA-120421. In some embodiments, the RSP03-binding agent comprises a heavy chain encoded by the plasmid deposited with ATCC and designated PTA-120420 and a light chain encoded by the plasmid deposited with ATCC and designated PTA-120421.

[00128] In certain embodiments, an RSP03-binding agent comprises, consists essentially of, or consists of, antibody 131R010. In certain embodiments, an RSP03-binding agent comprises, consists essentially of, or consists of, a variant of antibody 131R010.

[00129] Described herein are methods comprising polypeptides, including, but not limited to, antibodies that specifically bind at least one human RSPO protein, for example, RSP03.

[00130] In certain embodiments, the polypeptide comprises one, two, three, four, five, and/or six of the CDRs of antibody 131R010 (see Table 1 herein). In some embodiments, the polypeptide comprises CDRs with up to four (i.e., 0, 1, 2, 3, or 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 region.

[00131] In some embodiments, the RSPO-binding agent is a polypeptide that specifically binds a human RSP03, wherein the polypeptide comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO: 14 and/or SEQ ID NO: 15. In some embodiments, the polypeptide comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO: 16 and/or an amino acid sequence having at least about 80% sequence identity to SEQ ID NO: 18. In some embodiments, the polypeptide comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO: 17 and/or an amino acid sequence having at least about 80% sequence identity to SEQ ID NO: 19. 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: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO: 19. In certain embodiments, the polypeptide comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 14 and/or an amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 15. In certain embodiments, the polypeptide comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 16 and/or an amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 18. In certain embodiments, the polypeptide comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 17 and/or an amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 19. In certain embodiments, the polypeptide comprises an amino acid sequence of SEQ ID NO: 14 and/or an amino acid sequence of SEQ ID NO: 15. In certain embodiments, the polypeptide comprises an amino acid sequence of SEQ ID NO: 16 and/or an amino acid sequence of SEQ ID NO: 18. In certain embodiments, the polypeptide comprises an amino acid sequence of SEQ ID NO: 17 and/or an amino acid sequence of SEQ ID NO: 19.

[00132] In certain embodiments, the RSPO-binding agent is an RSP03-binding agent (e.g., antibody) that competes for specific binding to RSP03 with an antibody that comprises the CDRs of antibody 131R010.

[00133] In certain embodiments, the RSPO-binding agent is an RSP03-binding agent (e.g., an antibody) that binds the same epitope, or essentially the same epitope on RSP03, as an antibody that comprises the CDRs of antibody 131R010.

[00134] In certain embodiments, the RSPO-binding agent is an RSP03-binding agent (e.g., an antibody) that binds an epitope on RSP03 that overlaps with the epitope on RSP03 bound by an antibody comprising the CDRs of antibody 131R010.

[00135] In certain embodiments, the RSPO-binding agent is an RSP03-binding agent (e.g., an antibody) disclosed in U.S. Patent Publication No. 20150147333, each of which is hereby incorporated by reference herein in its entirety for all purposes. In certain embodiments, the RSPO- binding agent is anti-RSP03 antibody 4H1, 4D4, 5C2, 5D6, 5E11, 6E9, 21C2, or 26E11 disclosed in U.S. Patent Publication No. 20150147333. In certain embodiments, the RSPO-binding agent is an anti-RSP03 antibody comprising the 6 CDRs of anti-RSP03 antibody 4H1, 4D4, 5C2, 5D6, 5E11, 6E9, 21C2, or 26E11. In certain embodiments, the RSPO-binding agent is an anti-RSP03 antibody comprising the VH and/or VL region(s) of anti-RSP03 antibody 4H1, 4D4, 5C2, 5D6, 5E11, 6E9, 21C2, or 26E11. In certain embodiments, the RSPO-binding agent is an RSP03-binding agent (e.g., an antibody) that binds the same epitope, or essentially the same epitope on RSP03 as anti-RSP03 antibody 4H1, 4D4, 5C2, 5D6, 5E11, 6E9, 21C2, or 26E11.

[00136] In certain embodiments of the methods described herein, an RSPO-binding agent (e.g., an antibody) binds at least one human RSPO protein and modulates RSPO activity. In some embodiments, the RSPO-binding agent is an RSPO antagonist and decreases RSPO activity. In some embodiments, the RSPO-binding agent is an RSPO antagonist and decreases β-catenin activity.

[00137] In certain embodiments, an RSP03-binding agent (e.g., an antibody) binds human RSP03 and modulates RSP03 activity. In some embodiments, an RSP03-binding agent is an RSP03 antagonist and decreases RSP03 activity. In some embodiments, an RSP03-binding agent is an RSP03 antagonist and decreases β-catenin activity.

[00138] In certain embodiments, the RSPO-binding agent (e.g., an anti-RSP03 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 embodiments, the RSPO-binding agent inhibits activity of human RSP03.

[00139] In certain embodiments, the RSPO-binding agent (e.g., anti-RSP03 antibody) is an antagonist of at least one human RSPO protein. In certain embodiments, the RSPO-binding agent inhibits 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 signaling of human RSP03.

[00140] In certain embodiments, the RSPO-binding agent (e.g., anti-RSP03 antibody) is an antagonist of β-catenin signaling. In certain embodiments, the RSPO-binding agent inhibits β-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%.

[00141] In certain embodiments, the RSPO-binding agent (e.g., anti-RSP03 antibody) inhibits binding of at least one RSPO (e.g., RSP03) 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 an RSPO protein to at least one LGR protein. In some embodiments, the RSPO-binding agent inhibits binding of an RSPO protein to LGR4 (SEQ ID NO:20), LGR5 (SEQ ID NO:21), and/or LGR6 (SEQ ID NO:22). In certain embodiments, the inhibition of binding of an 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, an RSPO-binding agent that inhibits binding of at least one RSPO to at least one LGR protein further inhibits β-catenin signaling.

[00142] In certain embodiments, the RSPO-binding agent (e.g., anti-RSP03 antibody) blocks binding of at least one RSPO (e.g., RSP03) to a receptor. In certain embodiments, the RSPO-binding 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 an RSPO to at least one LGR protein. In some embodiments, the RSPO-binding agent blocks binding of at least one RSPO protein to LGR4 (SEQ ID NO: 20), LGR5 (SEQ ID NO:21), and/or LGR6 (SEQ ID NO:22). In certain embodiments, the blocking of binding of an 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, an RSPO-binding agent that blocks binding of at least one RSPO protein to at least one LGR protein further inhibits β-catenin signaling.

[00143] In certain embodiments, the RSPO-binding agent (e.g., an anti-RSP03 antibody) inhibits β- catenin signaling. It is understood that an RSPO-binding agent that inhibits β-catenin signaling can, in certain embodiments, inhibit signaling by one or more receptors in the β-catenin signaling pathway but not necessarily inhibit signaling by all receptors. In certain alternative embodiments, β-catenin signaling by all human receptors can be inhibited. In certain embodiments, β-catenin signaling by one or more receptors selected from the group consisting of LGR4 (SEQ ID NO:20), LGR5 (SEQ ID NO:21), and/or LGR6 (SEQ ID NO:22) is inhibited. In certain embodiments, the inhibition of β- catenin signaling by an RSPO-binding agent is a reduction in the level of β-catenin 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%.

[00144] In certain embodiments, the RSPO-binding agent (e.g., an anti-RSP03 antibody) inhibits activation of β-catenin. It is understood that an RSPO-binding agent that inhibits activation of β- catenin can, in certain embodiments, inhibit activation of β-catenin by one or more receptors, but not necessarily inhibit activation of β-catenin by all receptors. In certain alternative embodiments, activation of β-catenin by all human receptors can be inhibited. In certain embodiments, activation of β-catenin by one or more receptors selected from the group consisting of LGR4 (SEQ ID NO:20), LGR5 (SEQ ID NO:21), and LGR6 (SEQ ID NO:22) is inhibited. In certain embodiments, the inhibition of activation of β-catenin by an RSPO-binding agent is a reduction in the level of activation of β-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%.

[00145] In certain embodiments, the RSPO-binding agent (e.g., anti-RSP03 antibody) is an antibody. In some embodiments, the antibody is a recombinant antibody. In some embodiments, the antibody is a monoclonal 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 antibody. In some embodiments, the antibody is an IgA, IgD, IgE, IgG, or IgM antibody. In certain embodiments, the antibody is an IgGl antibody. In certain embodiments, the antibody is an IgG2 antibody. In some embodiments, the antibody is an IgG4 antibody. In certain embodiments, the antibody is an antibody fragment comprising an antigen-binding site. In some embodiments, the antibody is a bispecific antibody or a multispecific antibody. In some embodiments, the antibody is a monovalent antibody. In some embodiments, the antibody is a monospecific antibody. In some embodiments, the antibody is a bivalent antibody. In some embodiments, the antibody is conjugated to a cytotoxic moiety. In some embodiments, the antibody is isolated. In some embodiments, the antibody is substantially pure.

[00146] RSPO-binding agents (e.g., anti-RSP03 antibodies) can be assayed for specific binding by any method known in the art. The immunoassays which can be used include, but are not limited to, competitive and non-competitive assay systems using techniques such as Biacore analysis, FACS analysis, immunofluorescence, immunocytochemistry, Western blot analysis, radioimmunoassay, ELISA, "sandwich" immunoassay, immunoprecipitation assay, precipitation reaction, gel diffusion precipitin reaction, immunodiffusion assay, agglutination assay, complement-fixation assay, immunoradiometric assay, fluorescent immunoassay, and protein A immunoassay. 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).

[00147] For example, the specific binding of an agent (e.g., anti-RSP03 antibody) to a human RSPO protein (e.g., RSP03) can be determined using ELISA. An ELISA assay comprises preparing antigen, coating wells of a 96 well microtiter plate with antigen, adding the RSPO-binding agent conjugated to a detectable compound such as an enzymatic substrate (e.g. horseradish peroxidase or alkaline phosphatase) to the well, incubating for a period of time, and detecting the presence of the agent bound to the antigen. In some embodiments, the RSPO-binding agent is not conjugated to a detectable compound, but instead a second antibody that recognizes the RSPO-binding agent (e.g., an anti-Fc antibody) and is conjugated to a detectable compound is added to the well. In some embodiments, instead of coating the well with the antigen, the RSPO-binding agent can be coated to the well and a second antibody 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 know the parameters that can be modified to increase the signal detected as well as other variations of ELISAs known in the art.

[00148] In another example, the specific binding of an agent (e.g., anti-RSP03 antibody) to a human RSPO protein (e.g., RSP03) can be determined using FACS. A FACS screening assay can comprise generating a cDNA construct that expresses an antigen (e.g., RSP03), optionally as a fusion protein (e.g., RSP03-CD4TM), transfecting the construct into cells, expressing the antigen on the surface of the cells, mixing the RSPO-binding agent with the transfected cells, and incubating for a period of time. The cells bound by the RSPO-binding agent can be identified using a secondary antibody conjugated to a detectable compound (e.g., PE-conjugated anti-Fc antibody) and a flow cytometer. One of skill in the art would know the parameters that can be modified to optimize the signal detected as well as other variations of FACS that can enhance screening (e.g., screening for blocking antibodies).

[00149] The binding affinity of an agent (e.g., anti-RSP03 antibody) to an antigen and the off-rate of an agent-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., labeled with H or ¹²⁵I), or fragment or variant thereof, with a binding agent of interest in the presence of increasing amounts of unlabeled antigen followed by the detection of the agent bound to the labeled antigen. The affinity of the agent for the antigen and the binding off-rates can be determined from the data by Scatchard plot analysis. In some embodiments, Biacore kinetic analysis is used to determine the binding on and off rates of agents that bind an antigen. In some embodiments, Biacore kinetic analysis comprises analyzing the binding and dissociation of antibodies from chips with immobilized antigen on their surface. In some embodiments, Biacore kinetic analysis comprises analyzing the binding and dissociation of antigen from chips with immobilized binding agent on their surface.

[00150] In vivo and in vitro assays for determining whether an RSPO-binding agent (e.g., anti-RSP03 antibody) agent inhibits β-catenin signaling are known in the art. For example, cell-based, luciferase reporter assays utilizing a TCF/Luc reporter vector containing multiple copies of the TCF-binding domain upstream of a firefly luciferase reporter gene can be used to measure β-catenin signaling levels in vitro (Gazit et al., 1999, Oncogene, 18; 5959-66; TOPflash, Millipore, Billerica MA). The level of β-catenin signaling in the presence of one or more Wnts (e.g., Wnt(s) expressed by transfected cells or provided by Wnt-conditioned media) with or without an RSPO protein or RSPO- conditioned media in the presence of an 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 an RSPO-binding agent on β-catenin signaling can be measured in vitro or in vivo by measuring the effect of the agent on the level of expression of β-catenin-regulated genes, such as c-myc (He et al., 1998, Science, 281 : 1509-12), cyclin Dl (Tetsu et al, 1999, Nature, 398:422-6) and/or fibronectin (Gradl et al. 1999, Mol. Cell Biol, 19:5576-87). In certain embodiments, the effect of an RSPO- binding agent on β-catenin signaling can also be assessed by measuring the effect of the agent on the phosphorylation state of Dishevelled- 1, Dishevelled-2, Dishevelled-3, LRP5, LRP6, and/or β-catenin.

[00151] In some embodiments, the RSPO antagonists (e.g., anti-RSP03 antibodies) are polyclonal antibodies, which can be prepared by any known method.

[00152] In some embodiments, the RSPO antagonists (e.g., anti-RSP03 antibodies) are monoclonal antibodies. Monoclonal antibodies can be prepared, for example, using hybridoma methods or recombinant DNA methods that are known to one of skill in the art.

[00153] The polynucleotide(s) encoding a monoclonal antibody can be further 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 monoclonal antibody can be substituted for those regions of, for example, a human antibody to generate a chimeric antibody, or for a non-immunoglobulin polypeptide to generate a fusion antibody. In some embodiments, the constant regions are truncated or removed to generate the desired antibody fragment of a monoclonal antibody. In some embodiments, site-directed or high-density mutagenesis of the variable region can be used to optimize specificity, affinity, etc. of a monoclonal antibody.

[00154] In some embodiments, the RSPO antagonist (e.g., anti-RSP03 antibody) is a humanized antibody. Typically, humanized antibodies are human immunoglobulins in which residues from the CDRs are replaced by residues from CDRs of a non-human species (e.g., mouse, rat, rabbit, hamster, etc.) that have the desired specificity, affinity, and/or binding capability using methods known to one skilled in the art. In some embodiments, the framework region residues of a human immunoglobulin are replaced with the corresponding residues in an antibody from a non-human species. In some embodiments, the humanized antibody can be further modified by the substitution of additional residues either in the framework region and/or within the replaced non-human residues to refine and optimize antibody specificity, affinity, and/or capability. In general, the humanized antibody will comprise variable domain regions 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 sequence. In some embodiments, the humanized antibody can also comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. In certain embodiments, such humanized antibodies are used therapeutically because they can reduce antigenicity and HAMA (human anti-mouse antibody) responses when administered to a human subject.

[00155] In certain embodiments, the RSPO antagonist (e.g., anti-RSP03 antibody) is a human antibody. Human antibodies can be directly prepared using any technique known in the art, including the use of immortalized human B lymphocytes, phage libraries that express human antibodies, and transgeneic mice that contain human immunoglobulin loci. These antibodies can optionally be affinity matured using strategies including, but not limited to, chain shuffling and site-directed mutagenesis, which are known in the art and can be employed to generate high affinity human antibodies.

[00156] 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.

[00157] In certain embodiments, the RSPO antagonist (e.g., anti-RSP03 antibody) is a bispecific antibody that specifically recognizes at least one human RSPO protein (e.g., RSP03). 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 different epitopes on human RSP03) or on different molecules (e.g., one epitope on RSP03 and a different epitope on a second protein). In some embodiments, the bispecific antibodies are monoclonal human or humanized antibodies. In some embodiments, the bispecific antibodies are intact antibodies. In some embodiments, the bispecific antibodies are antibody fragments. In certain embodiments, the antibodies are multispecific. In some embodiments, the antibodies can specifically recognize and bind a first antigen target, (e.g., an RSPO or LGR protein) as well as a second antigen target, such as an effector molecule on a leukocyte (e.g., CD2, CD3, CD28, CD80, or CD86) or a Fc receptor (e.g., CD64, CD32, or CD 16) 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 possess an antigen-binding arm and an arm which binds a cytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA. Techniques for making bispecific or multispecific antibodies are known by those skilled in the art.

[00158] In certain embodiments, the RSPO antagonist (e.g., anti-RSP03 antibody) is a monospecific antibody. For example, in certain embodiments, each of the one or more antigen-binding sites that an antibody contains is capable of binding (or binds) a homologous epitope on different proteins.

[00159] In certain embodiments, the RSPO antagonist (e.g., anti-RSP03 antibody) is an antibody fragment comprising an antigen-binding site. Antibody fragments can have different functions or capabilities than intact antibodies; for example, antibody fragments can have increased 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 antibody molecule. In some embodiments, antibody fragments include a Fab fragment generated by reducing the disulfide bridges of an F(ab')2 fragment. In other embodiments, antibody fragments include 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. These fragments can be produced using any method known in the art, e.g., expressed in and secreted from E. coli or other host cells, or isolated from antibody phage libraries as discussed herein. In some embodiments, antibody fragments are linear antibody fragments. In certain embodiments, antibody fragments are monospecific or bispecific. In certain embodiments, the RSPO antagonist is a scFv.

[00160] It can further be desirable, especially in the case of antibody fragments, to modify an antibody in order to increase its serum half-life. This can be achieved, for example, by incorporation of a salvage receptor binding epitope into the antibody fragment by mutation of the appropriate region in the antibody fragment or by incorporating the epitope into a peptide tag that is then fused to the antibody fragment at either end or in the middle (e.g., by DNA or peptide synthesis). In some embodiments, an antibody is modified to decrease its serum half-life.

[00161] In certain embodiments, the RSPO antagonist (e.g., anti-RSP03 antibody) is a heteroconjugate antibody. Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune cells to unwanted cells. The heteroconjugate antibodies can be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl -4 -mercaptobutyrimidate.

[00162] Modified antibodies can comprise any type of variable region that provides for the association of the antibody with the target (i.e., a human RSP03 protein). In this regard, the variable region can comprise or be derived from any type of mammal that can be induced to mount a humoral response and generate immunoglobulins 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 origin. In some embodiments, both the variable and constant regions of the modified immunoglobulins are human. In other embodiments, the variable regions of compatible antibodies (usually derived from a non-human source) can be engineered or specifically tailored to improve the binding properties or reduce the immunogenicity of the molecule. In this respect, variable regions can be humanized or otherwise altered through the inclusion of imported amino acid sequences.

[00163] In certain embodiments, the variable domains in both the heavy and light chains are altered 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 can 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 preferably from an antibody from a different species. It may not be necessary to replace 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.

[00164] Alterations to the variable region notwithstanding, the modified antibodies can comprise antibodies (e.g., full-length antibodies or immunoreactive fragments thereof) 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 desired biochemical characteristics such as increased tumor localization and/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 modified antibodies will comprise a human constant region. Modifications to the constant region comprise additions, deletions or substitutions of one or more amino acids in one or more domains. The modified antibodies disclosed herein can comprise alterations or modifications to one or more of the three heavy chain constant domains (CHI, 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 comprise domain deleted constructs or variants wherein the entire CH2 domain has been removed (ΔΟΗ2 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 constant region.

[00165] In some embodiments, the modified antibodies are engineered to fuse the CH3 domain directly to the hinge region of the antibody. In other embodiments, a peptide spacer is inserted between the hinge region and the modified CH2 and/or CH3 domains. For example, constructs can 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 5-20 amino acid spacer. Such a spacer can be added to ensure that the regulatory elements of the constant domain remain free and accessible or that the hinge region remains flexible. However, amino acid spacers can, 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 will be relatively non-immunogenic so as to maintain the desired biological qualities of the modified antibodies.

[00166] In some embodiments, the modified antibodies can 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 can be enough to substantially reduce Fc binding and thereby increase cancer cell localization and/or tumor penetration. Similarly, one can simply delete the part of one or more constant region domains that control a specific effector function (e.g. complement Clq binding). Such partial deletions of the constant regions can improve selected characteristics of the antibody (serum half-life) while leaving other desirable functions associated with the subject constant region domain intact. Moreover, the constant regions of the disclosed antibodies can be modified through the mutation or substitution of one or more amino acids that enhances the profile of the resulting construct. In this respect it can 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 modified antibody. In certain embodiments, the modified antibodies comprise the addition of one or more amino acids to the constant region to enhance desirable characteristics such as decreasing or increasing effector function or provide for more cytotoxin or carbohydrate attachment sites.

[00167] The constant region mediates several effector functions. For example, binding of the CI component of complement to the Fc region of IgG or IgM antibodies (bound to antigen) activates the complement system. Activation of complement is important in the opsonization and lysis of cell pathogens. The activation of complement also stimulates the inflammatory response and can also be involved in autoimmune hypersensitivity. In addition, the Fc region of an antibody can bind a cell expressing an Fc receptor (FcR). There are a number of Fc receptors which are specific for different classes of antibody, including IgG (gamma receptors), IgE (epsilon receptors), IgA (alpha receptors) and IgM (mu receptors). Binding 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, release of inflammatory mediators, placental transfer, and control of immunoglobulin production.

[00168] In certain embodiments, the RSPO antagonists (e.g., anti-RSP03 antibody) are antibodies that provide for altered effector functions. These altered effector functions can affect the biological profile of the administered antibody. For example, in some embodiments, the deletion or inactivation (through point mutations or other means) of a constant region domain can reduce Fc receptor binding of the circulating modified antibody (e.g., anti-RSPO 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 can easily be made using well known biochemical or molecular engineering techniques.

[00169] In certain embodiments, an RSPO antagonist (e.g., anti-RSP03 antibody) is an antibody that does not have one or more effector functions. For instance, in some embodiments, the antibody has no ADCC activity, and/or no CDC activity. In certain embodiments, the antibody does not bind an Fc receptor, and/or complement factors. In certain embodiments, the antibody has no effector function.

[00170] Variants and equivalents which are substantially homologous to the chimeric, humanized, and human antibodies, or antibody fragments thereof, set forth herein can also be used in the methods described herein. These can contain, for example, conservative substitution mutations.

[00171] In certain embodiments, the antibodies described herein are isolated. In certain embodiments, the antibodies described herein are substantially pure.

[00172] In certain embodiments, an anti-RSP03 antibody does not have one or more effector functions normally associated with an Fc region. In some embodiments, the agent has no antibody- dependent cell-mediated cytotoxicity (ADCC) activity, and/or no complement-dependent cytotoxicity (CDC) activity. In certain embodiments, the agent does not bind to the Fc receptor and/or complement factors. In certain embodiments, the agent has no effector function.

[00173] In some embodiments, the RSPO antagonists are polypeptides. 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 (e.g., RSP03). It will be recognized in the art that some amino acid sequences can be varied without significant effect on the structure or function of the protein. Thus, the methods described herein further encompass using 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 RSPO-binding polypeptides can include deletions, insertions, inversions, repeats, and/or other types of substitutions.

[00174] 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 polypeptides and variants. An overview for chemical moieties can be found in Remington: The Science and Practice of Pharmacy, 2T^d Edition, 2012, Pharmaceutical Press, London.

[00175] 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 N-terminus of nascent polypeptides (e.g., amino acids 1-21 of human LGR5 (SEQ ID NO:21)). 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 sequences are cleaved 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 dependent upon amino acid residues within the signal sequence. Although there is usually one specific cleavage site, more than one cleavage site can be recognized and/or can be used by a signal peptidase resulting in a non-homogenous N-terminus of the polypeptide. 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 can 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 embodiments, 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 compared to a "native" or "parental" signal sequence. In some embodiments, the signal sequence of the polypeptide comprises amino acid substitutions and/or deletions that allow one cleavage site to be dominant, thereby resulting in a substantially homogeneous polypeptide with one N-terminus. In some embodiments, a signal sequence of the polypeptide affects the expression level of the polypeptide, e.g., increased expression or decreased expression.

[00176] The isolated polypeptides described herein can be produced by any suitable method known in the art. Such methods range from direct protein synthesis methods to constructing a DNA sequence encoding polypeptide sequences and expressing those sequences in a suitable host. In some embodiments, a DNA sequence is constructed using recombinant technology by isolating or synthesizing a DNA sequence encoding a wild-type protein of interest. Optionally, the sequence can be mutagenized by site-specific mutagenesis to provide functional analogs thereof. The skilled artisan can use any appropriate method to produce such polypeptides.

[00177] In certain embodiments, the polypeptide can be used in any one of a number of conjugated (i.e. an immunoconjugate or radioconjugate) or non-conjugated forms. In certain embodiments, antibodies can be used in a non-conjugated form to harness the subject's natural defense mechanisms including complement-dependent cytotoxicity and antibody dependent cellular toxicity to eliminate the malignant or cancer cells.

[00178] In some embodiments, the polypeptide is conjugated to a cytotoxic agent. In some embodiments, the cytotoxic agent is a chemotherapeutic agent including, but not limited to, methotrexate, adriamicin, doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents. 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 inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. 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, ¹²⁵I, ¹³¹I, ¹² I, ^mIn, ^{1 1}In, ¹⁰⁵Rh, ¹⁵ Sm, ⁶⁷Cu, ⁶⁷Ga, ¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re and ²¹²Bi. In some embodiments, 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 be produced. In certain embodiments, conjugates of an antibody and a cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyidithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p- diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as l,5-difluoro-2,4-dinitrobenzene).

[00179] In certain embodiments, the RSPO antagonist (e.g., anti-RSP03 antibody) is an antagonist of at least one RSPO protein (i.e., 1, 2, 3, or 4 RSPO proteins). In certain embodiments, the RSPO antagonist inhibits activity of the RSPO protein(s) to which it binds. In certain embodiments, the RSPO antagonist inhibits 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% of the activity of the human RSPO protein(s) to which it binds. In certain embodiments, the RSPO antagonist inhibits activity of RSP03.

[00180] In certain embodiments, the RSPO antagonist (e.g., anti-RSP03 antibody) inhibits binding of at least one human RSPO to an appropriate receptor. In certain embodiments, the RSPO antagonist inhibits binding of at least one human RSPO protein to one or more human LGR proteins. In some embodiments, the at least one RSPO protein is RSP03. In some embodiments, the one or more human LGR proteins are selected from the group consisting of: LGR4, LGR5, and LGR6. In certain embodiments, the RSPO antagonist inhibits binding of one or more RSPO proteins to LGR4, LGR5, and/or LGR6. In certain embodiments, the inhibition of binding of a particular RSPO to a LGR protein by an RSPO antagonist 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, an RSPO antagonist that inhibits binding of an RSPO to a LGR protein also inhibits RSPO-LGR pathway signaling. In certain embodiments, an RSPO antagonist that inhibits human RSPO pathway signaling is an antibody. In certain embodiments, an RSPO antagonist that inhibits human RSPO-LGR pathway signaling is an anti-RSPO antibody. In certain embodiments, an RSPO antagonist that inhibits human RSPO-LGR pathway signaling is an anti-RSP03 antibody. In certain embodiments, an RSPO antagonist that inhibits human RSPO-LGR pathway signaling is OMP-131R010. In certain embodiments, an RSPO antagonist that inhibits human RSPO-LGR pathway signaling is an antibody comprising the 6 CDRs of OMP-131R010.

[00181] In certain embodiments, the RSPO antagonists (e.g., an anti-RSP03 antibody) described herein are antagonists of at least one human RSPO protein and inhibit RSPO activity. In certain embodiments, the RSPO antagonist 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 antagonist inhibits activity of one, two, three, or four RSPO proteins. In some embodiments, the RSPO-binding agent binds at least RSP03. In certain embodiments, an RSPO antagonist that inhibits human RSPO activity is an antibody. In certain embodiments, an RSPO antagonist that inhibits human RSPO activity is an anti-RSPO antibody. In certain embodiments, an RSPO antagonist that inhibits human RSPO activity is an anti-RSP03 antibody. In certain embodiments, an RSPO antagonist that inhibits human RSPO activity is OMP-131R010. In certain embodiments, an RSPO antagonist that inhibits human RSPO activity is an antibody comprising the 6 CDRs of OMP-131R010.

[00182] In certain embodiments, the RSPO antagonist described herein is an antagonist of at least one human RSPO protein and inhibits RSPO signaling. In certain embodiments, the RSPO antagonist inhibits 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 antagonist inhibits signaling by one, two, three, or four RSPO proteins. In some embodiments, the RSPO antagonist inhibits signaling of at least RSP03. In certain embodiments, an RSPO antagonist that inhibits RSPO signaling is an antibody. In certain embodiments, an RSPO antagonist that inhibits RSPO signaling is an anti-RSPO antibody. In certain embodiments, an RSPO antagonist that inhibits RSPO signaling is an anti-RSP03 antibody. In certain embodiments, an RSPO antagonist that inhibits RSPO signaling is OMP-131R010. In certain embodiments, an RSPO antagonist that inhibits RSPO signaling is an antibody comprising the 6 CDRs of OMP-131R010.

[00183] In certain embodiments, an RSPO antagonist described herein is an antagonist of β-catenin signaling. In certain embodiments, the RSPO antagonist inhibits β-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, an RSPO antagonist that inhibits β-catenin signaling is an antibody. In certain embodiments, an RSPO antagonist that inhibits β-catenin signaling is an anti- RSPO antibody. In certain embodiments, an RSPO antagonist that inhibits β-catenin signaling is an anti-RSP03 antibody. In certain embodiments, an RSPO antagonist that inhibits β-catenin signaling is OMP-131R010. In certain embodiments, an RSPO antagonist that inhibits β-catenin signaling is an antibody comprising the 6 CDRs of OMP-131R010.

[00184] In certain embodiments, the RSPO antagonist described herein inhibits binding of at least one RSPO protein to a receptor. In certain embodiments, the RSPO antagonist inhibits binding of at least one human RSPO protein (e.g., RSP03) to one or more of its receptors. In some embodiments, the RSPO antagonist inhibits binding of at least one RSPO protein to at least one LGR protein. In some embodiments, the RSPO-binding agent inhibits binding of at least one RSPO protein to LGR4, LGR5, and/or LGR6. In certain embodiments, the inhibition of binding of at least one RSPO (e.g., RSP03) 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, an RSPO antagonist that inhibits binding of at least one RSPO to at least one LGR protein further inhibits RSPO-LGR pathway signaling and/or β-catenin signaling. In certain embodiments, an RSPO antagonist that inhibits binding of at least one human RSPO to at least one LGR protein is an antibody.

[00185] In certain embodiments, an RSPO antagonist (e.g., anti-RSP03 antibody) has one or more of the following effects: inhibit proliferation of tumor cells, inhibit tumor growth, reduce the frequency of cancer stem cells in a tumor, reduce the tumorigenicity of a tumor, reduce the tumorigenicity of a tumor by reducing the frequency of cancer stem cells in the tumor, 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 markers in the tumor cells, prevent metastasis of tumor cells, or decrease survival of tumor cells.

[00186] In certain embodiments, an RSPO antagonist (e.g., anti-RSP03 antibody) is capable of inhibiting tumor growth and/or reducing tumor size. In certain embodiments, an RSPO antagonist is capable of inhibiting tumor growth and/or reducing tumor size in vivo (e.g., in a xenograft mouse model and/or in a human having cancer). In some embodiments, the tumor is a tumor selected from the group consisting of colorectal tumor, colon tumor, pancreatic tumor, lung tumor, ovarian tumor, liver tumor, breast tumor, kidney tumor, prostate tumor, gastrointestinal tumor, melanoma, cervical tumor, bladder tumor, glioblastoma, and head and neck tumor. In certain embodiments, the tumor is a breast tumor. In certain embodiments, the tumor is an ovarian tumor. In certain embodiments, the tumor is a lung tumor. In certain embodiments, the tumor is a pancreatic tumor. In another embodiment, the cancer is colorectal cancer. In some embodiments, Wnt signaling is activated in the colorectal cancer (e.g., by an inactivating mutation in the APC gene or an activating mutation in the β- catenin gene). In certain embodiments, the colorectal cancer is third-line colorectal cancer. In some embodiments, the colorectal cancer is resistant to treatment with chemotherapy comprising 5- fluorouracil, irinotecan, and/or oxaliplatin. In certain embodiments, the tumor is an RSPO -dependent tumor, LGR-dependent tumor, or β-catenin-dependent tumor.

[00187] In certain embodiments, an RSPO antagonist (e.g., anti-RSP03 antibody) is capable of reducing the tumorigenicity of a tumor. In certain embodiments, an RSPO antagonist is capable of reducing the tumorigenicity of a tumor comprising 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 100-fold, or about 1000-fold. In certain embodiments, the reduction in the number or frequency of cancer stem cells is determined by limiting dilution assay using an animal model. Additional examples and guidance regarding the use of limiting dilution assays to determine a reduction in the number or frequency of cancer stem cells in a tumor can be found, e.g., in International Publication Number WO 2008/042236, and U.S. Patent Publication Nos. 2008/0064049, and 2008/0178305, each of which is hereby incorporated by reference herein in its entirety for all purposes.

[00188] In certain embodiments, an RSPO antagonist (e.g., anti-RSP03 antibody) is active in vivo for at least 1 hour, at least about 2 hours, at least about 5 hours, at least about 10 hours, at least about 24 hours, at least about 2 days, at least about 3 days, at least about 1 week, or at least about 2 weeks. In certain embodiments, the RSPO antagonist is an IgG (e.g., IgGl or IgG2) antibody that is active in vivo for at least 1 hour, at least about 2 hours, at least about 5 hours, at least about 10 hours, at least about 24 hours, at least about 2 days, at least about 3 days, at least about 1 week, or at least about 2 weeks.

[00189] In certain embodiments, an RSPO antagonist (e.g., anti-RSP03 antibody) 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 2 days, at least about 3 days, at least about 1 week, or at least about 2 weeks. In certain embodiments, the RSPO antagonist 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 2 days, at least about 3 days, at least about 1 week, or at least about 2 weeks. Methods of increasing (or decreasing) the half-life of agents such as polypeptides and antibodies are known in the art. For example, known methods of increasing the circulating half-life of IgG antibodies include the introduction of mutations in the Fc region which increase the pH-dependent binding of the antibody to the neonatal Fc receptor (FcRn). Known methods of increasing the circulating half-life of antibody fragments lacking the Fc region include such techniques as PEGylation.

III. Methods of use and pharmaceutical compositions

[00190] The present invention provides methods of treating diseases such as cancer or a fibrotic disease with an RSPO antagonist (e.g., anti-RSP03 antibody), while screening for, monitoring, reducing, preventing, attenuating, and/or mitigating side effects and/or toxicities, including, but not limited to skeletal-related side effects and/or toxicities associated with the RSPO antagonist. Side effects and/or toxicities associated with cancer treatment may include, but are not limited to, fatigue, vomiting, nausea, diarrhea, pain, hair loss, neutropenia, anemia, thrombocytopenia, cardiovascular- related complications, skeletal-related complications, and any combination thereof. As used herein, "skeletal -related complications" (e.g., skeletal-related side effects and/or toxicities) include but are not limited to, osteopenia, osteoporosis, bone fractures (including silent fractures), and combinations thereof. Thus, in some aspects and/or embodiments of the methods described herein, the screening for, monitoring, reducing, preventing, attenuating, and/or mitigating skeletal-related side effects and/or toxicities is screening for, monitoring, reducing, preventing, attenuating, and/or mitigating bone density loss and/or fracture risk. Often bone density loss is asymptomatic and/or early signs of skeletal-related side effects are not evident with, for example, bone density scanning.

[00191] Bone metabolism is a continuous dual process of bone formation and bone destruction. Bone destruction is referred to as bone resorption and is carried out by osteoclasts, while bone formation is carried out by osteoblasts. In adults, the dual processes of bone formation and bone destruction are in balance, maintaining a constant, homeostatically controlled amount of bone. Bone metabolism may be assessed and/or monitored by measurement of biomarkers (e.g., enzymes, proteins, and/or degradation products) released during bone formation and bone resorption. These biomarkers are often referred to as "bone turnover markers", and include bone formation markers and bone resorption markers. Bone formation biomarkers include serum total alkaline phosphatase, serum bone-specific alkaline phosphatase, serum osteocalcin, serum procollagen type 1 amino-terminal propeptide (P1NP) and serum procollagen type 1 carboxy-terminal propeptide (PI CP). Bone resorption biomarkers include, urinary hydroxyproline, urinary total pyridinoline (PYD), urinary free deoxypryidinoline (DPD), urinary collagen type 1 cross-linked N-telopeptide (NTX), urinary or serum collagen type 1 cross-linked C-telopeptide (CTX), bone sialoprotein (BSP), and tartrate -resistant acid phosphatase 5b.

[00192] Approximately 90% of the organic matrix of bone is type 1 collagen, a helical protein that is cross-linked at the N- and C-terminal ends of the molecule. During bone resorption, osteoclasts secrete a mixture of acid and neutral proteases that degrade the collagen fibrils into molecular fragments including C-telopeptide (CTX). As bone ages, the alpha form of aspartic acid present in CTX converts to the beta form (β-CTX). β-CTX is released into the bloodstream during bone resorption and serves as a specific marker for the degradation of mature type 1 collagen.

[00193] Bone turnover markers have been used to monitor anti-resorptive therapies (e.g., hormone replacement therapies and bisphosphonate therapies) in post-menopausal women, as well as in individuals diagnosed with osteopenia. In addition, bone turnover markers may be used to assess drug-induced osteoporosis resulting from therapy with hormonal and non-hormonal drugs. These drugs may include, but are not limited to, glucocorticoids, thyroid hormone, aromatase inhibitors, ovarian suppressing agents, androgen deprivation therapy, thiazolidinediones, selective serotonin reuptake inhibitors, anticonvulsants, heparins, oral anticoagulants, loop diuretics, calcineurin inhibitors, anti -retroviral therapy, and proton pump inhibitors. Bone turnover markers have not previously been used to assess the effect of RSPO antagonists. Accordingly, in some embodiments, the present invention provides methods for using bone turnover markers to monitor skeletal-related side effects and/or toxicities in subjects being treated with an RSPO antagonist. In some embodiments, the methods use a bone formation biomarker to monitor and/or detect decreased levels of bone formation. In some embodiments, the methods use a bone resorption biomarker to monitor and/or detect increased levels of bone resorption. In some embodiments, monitoring the level of a bone formation biomarker gives an early indication of decreased levels of bone formation and/or increased risk of bone fracture, osteopenia, and/or osteoporosis. In some embodiments, monitoring the level of a bone resorption biomarker gives an early indication of increased levels of bone resorption and/or increased risk of bone fracture, osteopenia, and/or osteoporosis. In some embodiments, the methods detect skeletal-related side effects and/or toxicities prior to any evidence of skeletal dysfunction as evaluated by bone density scans.

[00194] In certain embodiments, the skeletal-related side effects and/or toxicities that are detected, identified, monitored, reduced, prevented, attenuated, and/or screened for are skeletal-related side effects and/or toxicities caused by, associated with, and/or related to administration of an RSPO antagonist (e.g., anti-RSP03 antibody) or treatment with an RSPO antagonist (e.g., anti-RSP03 antibody). In certain embodiments, the skeletal -related side effects and/or toxicities are related to the RSPO antagonist (e.g., anti-RSP03 antibody). In certain embodiments, the skeletal-related side effects and/or toxicities are related to the activity of the RSPO antagonist (e.g., anti-RSP03 antibody). In certain embodiments, the skeletal-related side effects and/or toxicities are related to an RSPO antagonist that is an anti-RSP03 antibody. In certain embodiments, the skeletal-related side effects and/or toxicities are related to an RSPO antagonist that is anti-RSP03 antibody OMP-131R10.

[00195] The invention provides methods for selecting a subject for treatment with an RSPO antagonist (e.g., anti-RSP03 antibody), comprising: determining the level of a biomarker in a sample, and selecting the subject for treatment with the RSPO antagonist if the level of the biomarker is below a predetermined level. In some embodiments, the methods for selecting a subject for treatment with an RSPO antagonist comprise: obtaining a biological sample from the subject, determining the level of a biomarker in the sample, and selecting the subject for treatment with the RSPO antagonist if the level of the biomarker is below a predetermined level. In some embodiments, the biomarker is a bone turnover marker. In some embodiments, the bone turnover marker is a bone resorption biomarker. In some embodiments, the bone resorption biomarker is β-CTX.

[00196] In some embodiments, the method of selecting a subject for treatment with an RSPO antagonist (e.g., anti-RSP03 antibody) comprises: obtaining a biological sample from the subject, determining the level of a bone turnover marker in the sample, and selecting the subject for treatment with the RSPO antagonist if the level of the bone turnover marker is below a predetermined level. In some embodiments, the biological sample is urine, blood, serum, or plasma. In some embodiments, the bone turnover marker is a bone resorptive biomarker. In some embodiments, the bone resorption biomarker is urinary hydroxyproline, urinary total pyridinoline (PYD), urinary free deoxypyridinoline (DPD), urinary collagen type 1 cross-linked N-telopeptide (NTX), urinary or serum collagen type 1 cross-linked C-telopeptide (CTX), bone sialoprotein (BSP), or tartrate-resistant acid phosphatase 5b. In some embodiments, the bone resorptive biomarker is CTX or β-CTX. Thus, in some embodiments, the methods of selecting a subject for treatment with an RSPO antagonist (e.g., anti-RSP03 antibody), comprising: obtaining a biological sample from the subject, determining the level of β-CTX in the sample, and selecting the subject for treatment with the RSPO antagonist if the level of β-CTX is below a predetermined level.

[00197] The invention provides methods of identifying a subject as eligible for treatment with an RSPO antagonist (e.g., anti-RSP03 antibody), comprising: determining the level of a biomarker in a sample, and identifying the subject as eligible for treatment with the RSPO antagonist if the level of the biomarker is below a predetermined level. In some embodiments, the methods of identifying a subject as eligible for treatment with an RSPO antagonist (e.g., anti-RSP03 antibody) comprise: obtaining a biological sample from the subject, determining the level of a biomarker in the sample, and identifying the subject as eligible for treatment with the RSPO antagonist if the level of the biomarker is below a predetermined level. In some embodiments, the biomarker is a bone turnover marker. In some embodiments, the biomarker is a bone resorption biomarker. In some embodiments, the bone resorption biomarker is urinary hydroxyproline, urinary total pyridinoline (PYD), urinary free deoxypyridinoline (DPD), urinary collagen type 1 cross-linked N-telopeptide (NTX), urinary or serum collagen type 1 cross-linked C-telopeptide (CTX), bone sialoprotein (BSP), or tartrate-resistant acid phosphatase 5b. In some embodiments, the bone resorption biomarker is CTX. In some embodiments, the bone resorption biomarker is β-CTX. In some embodiments, the methods of identifying a subject as eligible for treatment with an RSPO antagonist (e.g., anti-RSP03 antibody) comprise: obtaining a biological sample from the subject, determining the level of β-CTX in the sample, and identifying the subject as eligible for treatment with the RSPO antagonist if the level of β-CTX is below a predetermined level.

[00198] The invention also provides methods of monitoring a subject receiving treatment with an RSPO antagonist for the development of skeletal-related side effects and/or toxicity, comprising: determining the level of a biomarker in a sample, and comparing the level of the biomarker in the sample to a predetermined level of the biomarker, wherein an increase in the level of the biomarker indicates development of skeletal-related side effects and/or toxicity. In some embodiments, the methods of monitoring a subject receiving treatment with an RSPO antagonist for the development of skeletal -related side effects and/or toxicity comprise: obtaining a biological sample from the subject receiving treatment, determining the level of a biomarker in the sample, and comparing the level of the biomarker in the sample to a predetermined level of the biomarker, wherein an increase in the level of the biomarker indicates development of skeletal-related side effects and/or toxicity. In some embodiments, the skeletal -related side effect and/or toxicity is an increased risk of bone fracture. In some embodiments, the skeletal -related side effect and/or toxicity is osteopenia or osteoporosis. In some embodiments, the biomarker is a bone turnover marker. In some embodiments, the biomarker is a bone resorption biomarker. In some embodiments, the bone resorption biomarker is urinary hydroxyproline, urinary total pyridinoline (PYD), urinary free deoxypyridinoline (DPD), urinary collagen type 1 cross-linked N-telopeptide (NTX), urinary or serum collagen type 1 cross-linked C- telopeptide (CTX), bone sialoprotein (BSP), or tartrate-resistant acid phosphatase 5b. In some embodiments, the bone resorption biomarker is CTX. In some embodiments, the bone resorption biomarker is β-CTX. In some embodiments, a method of monitoring a subject receiving treatment with an RSPO antagonist for the development of skeletal-related side effects and/or toxicity, comprises: obtaining a biological sample from the subject receiving treatment, determining the level of β-CTX in the sample, and comparing the level of β-CTX in the sample to a predetermined level of β-CTX, wherein an increase in the level of β-CTX indicates development of skeletal-related side effects and/or toxicity.

[00199] The invention also provides methods of detecting the development of skeletal -related side effects and/or toxicity in a subject receiving treatment with an RSPO antagonist, comprising: determining the level of a biomarker in a sample, and comparing the level of a biomarker in the sample to a predetermined level of the biomarker, wherein an increase in the level of the biomarker indicates development of skeletal-related side effects and/or toxicity. In some embodiments, the methods of detecting the development of skeletal-related side effects and/or toxicity in a subject receiving treatment with an RSPO antagonist comprise: obtaining a biological sample from the subject receiving treatment, determining the level of a biomarker in the sample, and comparing the level of a biomarker in the sample to a predetermined level of the biomarker, wherein an increase in the level of the biomarker indicates development of skeletal-related side effects and/or toxicity. In some embodiments, the skeletal-related side effect and/or toxicity is an increased risk of bone fracture. In some embodiments, the skeletal -related side effect and/or toxicity is osteopenia or osteoporosis. In some embodiments, the biomarker is a bone turnover marker. In some embodiments, the biomarker is a bone resorption biomarker. In some embodiments, the bone resorption biomarker is urinary hydroxyproline, urinary total pyridinoline (PYD), urinary free deoxypyridinoline (DPD), urinary collagen type 1 cross-linked N-telopeptide (NTX), urinary or serum collagen type 1 cross- linked C-telopeptide (CTX), bone sialoprotein (BSP), or tartrate-resistant acid phosphatase 5b. In some embodiments, the bone resorption biomarker is CTX. In some embodiments, the bone resorption biomarker is β-CTX. In some embodiments, the methods of detecting the development of skeletal -related side effects and/or toxicity in a subject receiving treatment with an RSPO antagonist comprise: obtaining a biological sample from the subject receiving treatment, determining the level of β-CTX in the sample, and comparing the level of β-CTX in the sample to a predetermined level of β- CTX, wherein an increase in the level of β-CTX indicates development of skeletal-related side effects and/or toxicity.

[00200] The invention also provides methods for identifying skeletal-related side effects and/or toxicity in a subject receiving treatment with an RSPO antagonist, comprising: determining the level of a biomarker in a sample, and comparing the level of the biomarker in the sample to a predetermined level of the biomarker, wherein if the level of the biomarker in the sample is higher than the predetermined level of the biomarker then a skeletal-related side effect and/or toxicity is indicated. In some embodiments, the methods for identifying skeletal-related side effects and/or toxicity in a subject receiving treatment with an RSPO antagonist comprise: obtaining a biological sample from the subject receiving treatment, determining the level of a biomarker in the sample, and comparing the level of the biomarker in the sample to a predetermined level of the biomarker, wherein if the level of the biomarker in the sample is higher than the predetermined level of the biomarker then a skeletal-related side effect and/or toxicity is indicated. In some embodiments, the skeletal- related side effect and/or toxicity is an increased risk of bone fracture. In some embodiments, the skeletal-related side effect and/or toxicity is osteopenia or osteoporosis. In some embodiments, the biomarker is a bone turnover marker. In some embodiments, the biomarker is a bone resorption biomarker. In some embodiments, the bone resorption biomarker is urinary hydroxyproline, urinary total pyridinoline (PYD), urinary free deoxypyridinoline (DPD), urinary collagen type 1 cross-linked N-telopeptide (NTX), urinary or serum collagen type 1 cross-linked C-telopeptide (CTX), bone sialoprotein (BSP), or tartrate-resistant acid phosphatase 5b. In some embodiments, the bone resorption biomarker is CTX. In some embodiments, the bone resorption biomarker is β-CTX. In some embodiments, a method for identifying a skeletal-related side effect and/or toxicity in a subject receiving treatment with an RSPO antagonist comprises: obtaining a biological sample from the subject receiving treatment, determining the level of β-CTX in the sample, and comparing the level of β-CTX in the sample to a predetermined level of β-CTX, wherein if the level of β-CTX in the sample is higher than the predetermined level of β-CTX then a skeletal-related side effect and/or toxicity is indicated.

[00201] The invention also provides methods for monitoring skeletal-related side effects and/or toxicity in a subject receiving treatment with an RSPO antagonist (e.g., anti-RSP03 antibody), comprising: determining the level of a biomarker in a sample, and comparing the level of the biomarker in the sample to a predetermined level of the biomarker, wherein if the level of the biomarker in the sample is higher than the predetermined level of the biomarker then skeletal-related side effects and/or toxicity is indicated. In some embodiments, the methods for monitoring skeletal- related side effects and/or toxicity in a subject receiving treatment with an RSPO antagonist (e.g., anti-RSP03 antibody) comprise: obtaining a biological sample from the subject receiving treatment, determining the level of a biomarker in the sample, and comparing the level of the biomarker in the sample to a predetermined level of the biomarker, wherein if the level of the biomarker in the sample is higher than the predetermined level of the biomarker then skeletal-related side effects and/or toxicity is indicated. In some embodiments, the skeletal-related side effect and/or toxicity is an increased risk of bone fracture. In some embodiments, the skeletal -related side effect and/or toxicity is osteopenia or osteoporosis. In some embodiments, the biomarker is a bone turnover marker. In some embodiments, the biomarker is a bone resorption biomarker. In some embodiments, the bone resorption biomarker is urinary hydroxyproline, urinary total pyridinoline (PYD), urinary free deoxypyridinoline (DPD), urinary collagen type 1 cross-linked N-telopeptide (NTX), urinary or serum collagen type 1 cross-linked C-telopeptide (CTX), bone sialoprotein (BSP), or tartrate-resistant acid phosphatase 5b. In some embodiments, the bone resorption biomarker is CTX. In some embodiments, the bone resorption biomarker is β-CTX. In some embodiments, a method for monitoring skeletal -related side effects and/or toxicity in a subject receiving treatment with an RSPO antagonist (e.g., anti-RSP03 antibody) comprises: obtaining a biological sample from the subject receiving treatment, determining the level of β-CTX in the sample, and comparing the level of β-CTX in the sample to a predetermined level of β-CTX, wherein if the level of β-CTX in the sample is higher than the predetermined level of β-CTX then a skeletal-related side effect and/or toxicity is indicated.

[00202] The invention also provides methods of reducing skeletal-related side effects and/or toxicity in a subject receiving treatment with an RSPO antagonist (e.g., anti-RSP03 antibody), comprising: determining the level of a biomarker in a sample from the subject, comparing the level of the biomarker in the sample to a predetermined level of the biomarker, and administering to the subject a therapeutically effective amount of an anti-resorptive medication such as a bisphosphonate if the level of the biomarker in the sample is higher than the predetermined level of the biomarker. In some embodiments, the methods of reducing skeletal-related side effects and/or toxicity in a subject receiving treatment with an RSPO antagonist (e.g., anti-RSP03 antibody) comprise: obtaining a biological sample from the subject receiving treatment, determining the level of a biomarker in the sample, comparing the level of the biomarker in the sample to a predetermined level of the biomarker, and administering to the subject a therapeutically effective amount of an anti-resorptive medication such as a bisphosphonate if the level of the biomarker in the sample is higher than the predetermined level of the biomarker. In some embodiments, the skeletal-related side effect and/or toxicity is an increased risk of bone fracture. In some embodiments, the skeletal -related side effect and/or toxicity is osteopenia or osteoporosis. In some embodiments, the biomarker is a bone turnover marker. In some embodiments, the biomarker is a bone resorption biomarker. In some embodiments, the bone resorption biomarker is urinary hydroxyproline, urinary total pyridinoline (PYD), urinary free deoxypyridinoline (DPD), urinary collagen type 1 cross-linked N-telopeptide (NTX), urinary or serum collagen type 1 cross-linked C-telopeptide (CTX), bone sialoprotein (BSP), or tartrate-resistant acid phosphatase 5b. In some embodiments, the bone resorption biomarker is CTX. In some embodiments, the bone resorption biomarker is β-CTX. In some embodiments, a method for reducing skeletal -related side effects and/or toxicity in a subject receiving treatment with an RSPO antagonist (e.g., anti-RSP03 antibody) comprises: obtaining a biological sample from the subject receiving treatment, determining the level of β-CTX in the sample, and comparing the level of β-CTX in the sample to a predetermined level of β-CTX, and administering to the subject a therapeutically effective amount of an anti-resorptive medication if the level of β-CTX in the sample is higher than the predetermined level of β-CTX. In some embodiments, the anti-resorptive medication is a bisphosphonate.

[00203] The invention also provides methods of preventing or attenuating the development of skeletal- related side effects and/or toxicity in a subject receiving treatment with an RSPO antagonist (e.g., anti-RSP03 antibody), comprising: determining the level of a biomarker in a sample from the subject, comparing the level of the biomarker in the sample to a predetermined level of the biomarker; administering to the subject a therapeutically effective amount of an anti-resorptive medication, and administering to the subject the RSPO antagonist (e.g., anti-RSP03 antibody). In some embodiments, the methods of preventing or attenuating the development of skeletal-related side effects and/or toxicity in a subject receiving treatment with an RSPO antagonist (e.g., anti-RSP03 antibody) comprise: obtaining a biological sample from the subject prior to treatment with the RSPO antagonist (e.g., anti-RSP03 antibody), determining the level of a biomarker in the sample, comparing the level of the biomarker in the sample to a predetermined level of the biomarker; administering to the subject a therapeutically effective amount of an anti-resorptive medication, and administering to the subject the RSPO antagonist (e.g., anti-RSP03 antibody). In some embodiments, the skeletal-related side effect and/or toxicity is an increased risk of bone fracture. In some embodiments, the skeletal-related side effect and/or toxicity is osteopenia or osteoporosis. In some embodiments, the biomarker is a bone turnover marker. In some embodiments, the biomarker is a bone resorption biomarker. In some embodiments, the bone resorption biomarker is urinary hydroxyproline, urinary total pyridinoline (PYD), urinary free deoxypyridinoline (DPD), urinary collagen type 1 cross-linked N-telopeptide (NTX), urinary or serum collagen type 1 cross-linked C-telopeptide (CTX), bone sialoprotein (BSP), or tartrate-resistant acid phosphatase 5b. In some embodiments, the bone resorption biomarker is CTX. In some embodiments, the bone resorption biomarker is β-CTX. In some embodiments, a method of preventing or attenuating the development of a skeletal-related side effect and/or toxicity in a subject receiving treatment with an RSPO antagonist (e.g., anti-RSP03 antibody) comprises: obtaining a biological sample from the subject prior to treatment with the RSPO antagonist (e.g., anti- RSP03 antibody), determining the level of β-CTX in the sample, comparing the level of β-CTX in the sample to a predetermined level of β-CTX; administering to the subject a therapeutically effective amount of an anti-resorptive medication if the level of β-CTX in the sample is higher than the predetermined level of β-CTX; and administering to the subject the RSPO antagonist (e.g., anti- RSP03 antibody). [00204] In some embodiments of the methods described herein, the predetermined level is about 1500pg/ml or less in a blood, serum, or plasma sample. In some embodiments, the predetermined level is about 1200pg/ml or less in a blood, serum, or plasma sample. In some embodiments, the predetermined level is about lOOOpg/ml or less in a blood, serum, or plasma sample. In some embodiments, the predetermined level is about 800pg/ml or less in a blood, serum, or plasma sample. In some embodiments, the predetermined level is about 600pg/ml or less in a blood, serum, or plasma sample. In some embodiments, the predetermined level is about 400pg/ml or less in a blood, serum, or plasma sample. In the context of predetermined levels of β-CTX, the term "about" means the referenced amount plus or minus 10% of that referenced amount.

[00205] In some embodiments, the predetermined level of a biomarker (e.g., a bone resorption biomarker or β-CTX) is the amount of the biomarker in a sample obtained at an earlier date. In some embodiments, the predetermined level of a biomarker (e.g., a bone resorption biomarker or β-CTX) is the amount of the biomarker in a sample obtained at an initial screening. In some embodiments, the predetermined level of a biomarker (e.g., a bone resorption biomarker or β-CTX) is the amount of the biomarker in a sample obtained prior to treatment. In some embodiments, the predetermined level of a biomarker (e.g., a bone resorption biomarker or β-CTX) is the amount of the biomarker in a sample obtained at an initial screening. In some embodiments, the predetermined level of a biomarker (e.g., a bone resorption biomarker or β-CTX) is a normal reference level. In some embodiments, the predetermined level of a biomarker (e.g., a bone resorption biomarker or β-CTX) is a baseline level. In some embodiments, the baseline level is the amount of the biomarker determined at an initial screening. In some embodiments, the baseline level is the amount of the biomarker determined prior to treatment.

[00206] In some embodiments, if the β-CTX level in the sample is increased 2-fold or greater (i.e., a doubling or greater) as compared to a predetermined level, the subject is administered a therapeutically effective amount of an anti-resorptive medication. In some embodiments, if the β- CTX level in the sample is increased 2-fold or greater (i.e., a doubling or greater) as compared to a baseline level, the subject is administered a therapeutically effective amount of an anti-re sorptive medication.

[00207] The invention provides methods for selecting a subject for treatment with an RSPO antagonist (e.g., anti-RSP03 antibody), comprising: determining the level of a bone formation biomarker in a sample, and selecting the subject for treatment with the RSPO antagonist if the level of the bone formation biomarker is above a predetermined level. In some embodiments, the methods for selecting a subject for treatment with an RSPO antagonist comprise: obtaining a biological sample from the subject, determining the level of a bone formation biomarker in the sample, and selecting the subject for treatment with the RSPO antagonist if the level of the bone formation biomarker is above a predetermined level. In some embodiments, the bone formation biomarker is P1NP. [00208] In some embodiments, the method of selecting a subject for treatment with an RSPO antagonist (e.g., anti-RSP03 antibody) comprises: obtaining a biological sample from the subject, determining the level of a bone formation biomarker in the sample, and selecting the subject for treatment with the RSPO antagonist if the level of the bone formation biomarker is above a predetermined level. In some embodiments, the biological sample is urine, blood, serum, or plasma. In some embodiments, the bone formation biomarker is P1NP. Thus, in some embodiments, the methods of selecting a subject for treatment with an RSPO antagonist (e.g., anti-RSP03 antibody), comprising: obtaining a biological sample from the subject, determining the level of P1NP in the sample, and selecting the subject for treatment with the RSPO antagonist if the level of P1NP is above a predetermined level.

[00209] The invention provides methods of identifying a subject as eligible for treatment with an RSPO antagonist (e.g., anti-RSP03 antibody), comprising: determining the level of a bone formation biomarker in a sample, and identifying the subject as eligible for treatment with the RSPO antagonist if the level of the biomarker is above a predetermined level. In some embodiments, the methods of identifying a subject as eligible for treatment with an RSPO antagonist (e.g., anti-RSP03 antibody) comprise: obtaining a biological sample from the subject, determining the level of a bone formation biomarker in the sample, and identifying the subject as eligible for treatment with the RSPO antagonist if the level of the bone formation biomarker is above a predetermined level. In some embodiments, the biomarker is P1NP. In some embodiments, the methods of identifying a subject as eligible for treatment with an RSPO antagonist (e.g., anti-RSP03 antibody) comprise: obtaining a biological sample from the subject, determining the level of P1NP in the sample, and identifying the subject as eligible for treatment with the RSPO antagonist if the level of P1NP is above a predetermined level.

[00210] The invention also provides methods of monitoring a subject receiving treatment with an RSPO antagonist (e.g., anti-RSP03 antibody) for the development of skeletal-related side effects and/or toxicity, comprising: determining the level of a bone formation biomarker in a sample, and comparing the level of the bone formation biomarker in the sample to a predetermined level of the bone formation biomarker, wherein a decrease in the level of the bone formation biomarker indicates development of skeletal-related side effects and/or toxicity. In some embodiments, the methods of monitoring a subject receiving treatment with an RSPO antagonist (e.g., anti-RSP03 antibody) for the development of skeletal-related side effects and/or toxicity comprise: obtaining a biological sample from the subject receiving treatment, determining the level of a bone formation biomarker in the sample, and comparing the level of the bone formation biomarker in the sample to a predetermined level of the bone formation biomarker, wherein an decrease in the level of the biomarker indicates development of skeletal-related side effects and/or toxicity. In some embodiments, the skeletal- related side effect and/or toxicity is an increased risk of bone fracture. In some embodiments, the skeletal-related side effect and/or toxicity is osteopenia or osteoporosis. In some embodiments, the bone formation biomarker is P1NP. In some embodiments, a method of monitoring a subject receiving treatment with an RSPO antagonist (e.g., anti-RSP03 antibody) for the development of skeletal-related side effects and/or toxicity, comprises: obtaining a biological sample from the subject receiving treatment, determining the level of P1NP in the sample, and comparing the level of P1NP in the sample to a predetermined level of P1NP, wherein an increase in the level of P1NP indicates development of skeletal-related side effects and/or toxicity.

[00211] The invention also provides methods of detecting the development of skeletal -related side effects and/or toxicity in a subject receiving treatment with an RSPO antagonist (e.g., anti-RSP03 antibody), comprising: determining the level of a bone formation biomarker in a sample, and comparing the level of a bone formation biomarker in the sample to a predetermined level of the bone formation biomarker, wherein a decrease in the level of the bone formation biomarker indicates development of skeletal-related side effects and/or toxicity. In some embodiments, the methods of detecting the development of skeletal-related side effects and/or toxicity in a subject receiving treatment with an RSPO antagonist (e.g., anti-RSP03 antibody) comprise: obtaining a biological sample from the subject receiving treatment, determining the level of a bone formation biomarker in the sample, and comparing the level of a bone formation biomarker in the sample to a predetermined level of the bone formation biomarker, wherein a decrease in the level of the bone formation biomarker indicates development of skeletal-related side effects and/or toxicity. In some embodiments, the skeletal-related side effect and/or toxicity is an increased risk of bone fracture. In some embodiments, the skeletal -related side effect and/or toxicity is osteopenia or osteoporosis. In some embodiments, the bone formation biomarker is P1NP. In some embodiments, the methods of detecting the development of skeletal -related side effects and/or toxicity in a subject receiving treatment with an RSPO antagonist (e.g., anti-RSP03 antibody) comprise: obtaining a biological sample from the subject receiving treatment, determining the level of P1NP in the sample, and comparing the level of P1NP in the sample to a predetermined level of P1NP, wherein an increase in the level of P 1NP indicates development of skeletal -related side effects and/or toxicity.

[00212] The invention also provides methods for identifying skeletal-related side effects and/or toxicity in a subject receiving treatment with an RSPO antagonist (e.g., anti-RSP03 antibody), comprising: determining the level of a bone formation biomarker in a sample, and comparing the level of the bone formation biomarker in the sample to a predetermined level of the bone formation biomarker, wherein if the level of the bone formation biomarker in the sample is lower than the predetermined level of the bone formation biomarker then a skeletal-related side effect and/or toxicity is indicated. In some embodiments, the methods for identifying skeletal-related side effects and/or toxicity in a subject receiving treatment with an RSPO antagonist (e.g., anti-RSP03 antibody) comprise: obtaining a biological sample from the subject receiving treatment, determining the level of a bone formation biomarker in the sample, and comparing the level of the bone formation biomarker in the sample to a predetermined level of the bone formation biomarker, wherein if the level of the bone formation biomarker in the sample is higher than the predetermined level of the bone formation biomarker then a skeletal-related side effect and/or toxicity is indicated. In some embodiments, the skeletal -related side effect and/or toxicity is an increased risk of bone fracture. In some embodiments, the skeletal-related side effect and/or toxicity is osteopenia or osteoporosis. In some embodiments, the bone formation biomarker is P 1NP. In some embodiments, a method for identifying a skeletal- related side effect and/or toxicity in a subject receiving treatment with an RSPO antagonist (e.g., anti- RSP03 antibody) comprises: obtaining a biological sample from the subject receiving treatment, determining the level of P1NP in the sample, and comparing the level of P1NP in the sample to a predetermined level of P1NP, wherein if the level of P1NP in the sample is lower than the predetermined level of P1NP then a skeletal-related side effect and/or toxicity is indicated.

[00213] The invention also provides methods for monitoring skeletal-related side effects and/or toxicity in a subject receiving treatment with an RSPO antagonist (e.g., anti-RSP03 antibody), comprising: determining the level of a bone formation biomarker in a sample, and comparing the level of the bone formation biomarker in the sample to a predetermined level of the bone formation biomarker, wherein if the level of the bone formation biomarker in the sample is lower than the predetermined level of the bone formation biomarker then skeletal-related side effects and/or toxicity is indicated. In some embodiments, the methods for monitoring skeletal-related side effects and/or toxicity in a subject receiving treatment with an RSPO antagonist (e.g., anti-RSP03 antibody) comprise: obtaining a biological sample from the subject receiving treatment, determining the level of a bone formation biomarker in the sample, and comparing the level of the bone formation biomarker in the sample to a predetermined level of the bone formation biomarker, wherein if the level of the bone formation biomarker in the sample is lower than the predetermined level of the bone formation biomarker then skeletal-related side effects and/or toxicity is indicated. In some embodiments, the skeletal -related side effect and/or toxicity is an increased risk of bone fracture. In some embodiments, the skeletal-related side effect and/or toxicity is osteopenia or osteoporosis. In some embodiments, the bone formation biomarker is P 1NP. In some embodiments, a method for monitoring skeletal- related side effects and/or toxicity in a subject receiving treatment with an RSPO antagonist (e.g., anti-RSP03 antibody) comprises: obtaining a biological sample from the subject receiving treatment, determining the level of P1NP in the sample, and comparing the level of P1NP in the sample to a predetermined level of P1NP, wherein if the level of P1NP in the sample is lower than the predetermined level of PINP then a skeletal -related side effect and/or toxicity is indicated.

[00214] The invention also provides methods of reducing skeletal-related side effects and/or toxicity in a subject receiving treatment with an RSPO antagonist (e.g., anti-RSP03 antibody), comprising: determining the level of a bone formation biomarker in a sample from the subject, comparing the level of the bone formation biomarker in the sample to a predetermined level of the bone formation biomarker, and administering to the subject a therapeutically effective amount of an anti-re sorptive medication such as a bisphosphonate if the level of the bone formation biomarker in the sample is lower than the predetermined level of the bone formation biomarker. In some embodiments, the methods of reducing skeletal-related side effects and/or toxicity in a subject receiving treatment with an RSPO antagonist (e.g., anti-RSP03 antibody) comprise: obtaining a biological sample from the subject receiving treatment, determining the level of a bone formation biomarker in the sample, comparing the level of the bone formation biomarker in the sample to a predetermined level of the bone formation biomarker, and administering to the subject a therapeutically effective amount of an anti-resorptive medication such as a bisphosphonate if the level of the bone formation biomarker in the sample is lower than the predetermined level of the bone formation biomarker. In some embodiments, the skeletal -related side effect and/or toxicity is an increased risk of bone fracture. In some embodiments, the skeletal-related side effect and/or toxicity is osteopenia or osteoporosis. In some embodiments, the bone formation biomarker is P1NP. In some embodiments, a method for reducing skeletal-related side effects and/or toxicity in a subject receiving treatment with an RSPO antagonist (e.g., anti-RSP03 antibody) comprises: obtaining a biological sample from the subject receiving treatment, determining the level of P1NP in the sample, and comparing the level of P1NP in the sample to a predetermined level of P1NP, and administering to the subject a therapeutically effective amount of an anti-resorptive medication if the level of P1NP in the sample is lower than the predetermined level of P1NP. In some embodiments, the anti-resorptive medication is a bisphosphonate.

[00215] The invention also provides methods of preventing or attenuating the development of skeletal- related side effects and/or toxicity in a subject receiving treatment with an RSPO antagonist (e.g., anti-RSP03 antibody), comprising: determining the level of a bone formation biomarker in a sample from the subject, comparing the level of the bone formation biomarker in the sample to a predetermined level of the bone formation biomarker; administering to the subject a therapeutically effective amount of an anti-re sorptive medication, and administering to the subject the RSPO antagonist (e.g., anti-RSP03 antibody). In some embodiments, the methods of preventing or attenuating the development of skeletal -related side effects and/or toxicity in a subject receiving treatment with an RSPO antagonist (e.g., anti-RSP03 antibody) comprise: obtaining a biological sample from the subject prior to treatment with the RSPO antagonist (e.g., anti-RSP03 antibody), determining the level of a bone formation biomarker in the sample, comparing the level of the bone formation biomarker in the sample to a predetermined level of the bone formation biomarker; administering to the subject a therapeutically effective amount of an anti-resorptive medication, and administering to the subject the RSPO antagonist (e.g., anti-RSP03 antibody). In some embodiments, the skeletal-related side effect and/or toxicity is an increased risk of bone fracture. In some embodiments, the skeletal -related side effect and/or toxicity is osteopenia or osteoporosis. In some embodiments, the bone formation biomarker is P1NP. In some embodiments, a method of preventing or attenuating the development of a skeletal -related side effect and/or toxicity in a subject receiving treatment with an RSPO antagonist (e.g., anti-RSP03 antibody) comprises: obtaining a biological sample from the subject prior to treatment with the RSPO antagonist (e.g., anti-RSP03 antibody), determining the level of PINP in the sample, comparing the level of PINP in the sample to a predetermined level of PINP; administering to the subject a therapeutically effective amount of an anti-resorptive medication if the level of PINP in the sample is lower than the predetermined level of PINP; and administering to the subject the RSPO antagonist (e.g., anti-RSP03 antibody).

[00216] In some embodiments of the methods described herein, the predetermined level of PINP is at least about 16 microg/ml in a blood, serum, or plasma sample. In some embodiments, the predetermined level of PINP is at least about 16 microg/ml in a blood, serum, or plasma sample. In some embodiments, the predetermined level of PINP is at least about 19 microg/ml in a blood, serum, or plasma sample. In some embodiments, the predetermined level of PINP is at least about 22 microg/ml in a blood, serum, or plasma sample. In some embodiments, the predetermined level of PINP is at least about 25 microg/ml in a blood, serum, or plasma sample. In some embodiments, the predetermined level of PINP is about 30 microg/ml in a blood, serum, or plasma sample. In the context of predetermined levels of PINP, the term "about" means the referenced amount plus or minus 10% of that referenced amount.

[00217] In some embodiments, the predetermined level of a bone formation biomarker (e.g., PINP) is the amount of the bone formation biomarker in a sample obtained at an earlier date. In some embodiments, the predetermined level of a bone formation biomarker (e.g., PINP) is the amount of the bone formation biomarker in a sample obtained at an initial screening. In some embodiments, the predetermined level of a bone formation biomarker (e.g., PINP) is the amount of the bone formation biomarker in a sample obtained prior to treatment. In some embodiments, the predetermined level of a bone formation biomarker (e.g., PINP) is the amount of the bone formation biomarker in a sample obtained at an initial screening. In some embodiments, the predetermined level of a bone formation biomarker (e.g., PINP) is a normal reference level. In some embodiments, the predetermined level of a bone formation biomarker (e.g., PINP) is a baseline level. In some embodiments, the baseline level is the amount of the bone formation biomarker determined at an initial screening. In some embodiments, the baseline level is the amount of the bone formation biomarker determined prior to treatment.

[00218] In some embodiments, if the PINP level in the sample is decreased by 50% or more (i.e., a half or less) as compared to a predetermined level, the subject is administered a therapeutically effective amount of an anti-resorptive medication. In some embodiments, if the PINP level in the sample is decreased by 50% or more (i.e., a half or less) as compared to a baseline level, the subject is administered a therapeutically effective amount of an anti-resorptive medication.

[00219] In any of the methods described herein, a biological sample is obtained approximately every week, every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, or every 6 weeks. [00220] In any of the methods described herein, a biological sample is obtained once, twice or more, three times or more, four times or more, five times or more, six times or more, seven times or more, or ten times or more.

[00221] In any of the methods described herein, a biological sample is obtained no more than once, twice, three times, four times, five times, six times, seven times or ten times.

[00222] In any of the methods described herein, a biological sample is obtained between 1 and 5 times, between 1 and 10 times, or between 1 and 20 times.

[00223] In any of the methods described herein, a biological sample is obtained once, twice or more, three times or more, or four times or more every week, every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, or every 6 weeks for a total of 2 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, 12 weeks, 4 months, 6 months or 1 year.

[00224] In any of the methods described herein, a biological sample is obtained once, twice or more, three times or more, or four times or more every week, every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, or every 6 weeks for at least 2 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, 12 weeks, 4 months, 6 months or 1 year.

[00225] In any of the methods described herein, a biological sample is obtained once, twice or more, three times or more, or four times or more every week, every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, or every 6 weeks for no more than 2 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, 12 weeks, 4 months, 6 months or 1 year.

[00226] In any of the methods described herein, a biological sample is obtained once, twice or more, three times or more, or four times or more every week, every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, or every 6 weeks for the period of time during which the subject receives treatment with the RSPO antagonist.

[00227] In any of the methods described herein, the level of the biomarker is determined approximately every week, every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, or every 6 weeks.

[00228] In any of the methods described herein, the level of the biomarker is determined once, twice or more, three times or more, four times or more, five times or more, six times or more, seven times or more, or ten times or more.

[00229] In any of the methods described herein, the level of the biomarker is determined no more than once, twice, three times, four times, five times, six times, seven times or ten times.

[00230] In any of the methods described herein, the level of the biomarker is determined between 1 and 5 times, between 1 and 10 times, or between 1 and 20 times.

[00231] In any of the methods described herein, the level of the biomarker is determined once, twice or more, three times or more, or four times or more every week, every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, or every 6 weeks for a total of 2 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, 12 weeks, 4 months, 6 months or 1 year. [00232] In any of the methods described herein, the level of the biomarker is determined once, twice or more, three times or more, or four times or more every week, every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, or every 6 weeks for at least 2 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, 12 weeks, 4 months, 6 months or 1 year.

[00233] In any of the methods described herein, the level of the biomarker is determined once, twice or more, three times or more, or four times or more every week, every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, or every 6 weeks for no more than 2 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, 12 weeks, 4 months, 6 months or 1 year.

[00234] In any of the methods described herein, the level of the biomarker is determined once, twice or more, three times or more, or four times or more every week, every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, or every 6 weeks for the period of time during which the subject receives treatment with the RSPO antagonist.

[00235] In some embodiments of any of the methods described herein, the subjects are evaluated using a DEXA (dual energy X-ray absorptiometry) bone density scan. This technique is the most commonly used test for measuring bone mineral density (BMD). The DEXA output includes a T- score, which compares the subject's bone density to a 30-35 year old person, and a Z-score, which compares the subject's bone density to the average bone density of someone their age and gender. The T-score is used to determine if an individual has osteopenia or osteoporosis according to a standard scale. A T-score greater than -1 is considered normal bone density; a T-score between -1 and -2.5, is considered osteopenia; a T-score less than -2.5 is considered osteoporosis; and a T-score less than -2.5 and 1+ osteoporotic fractures is considered severe (established) osteoporosis. In some embodiments, a skeletal-related side effect and/or toxicity is indicated if the T-score declines to less than -2.5 in the total femur or vertebrae L1-L4. In some embodiments, a skeletal-related side effect and/or toxicity is indicated if the T-score declines to less than -2.0 in the total femur or vertebrae Ll- L4. In some embodiments, a skeletal-related side effect and/or toxicity is indicated if the T-score declines to less than -1.5 in the total femur or vertebrae L1-L4. In some embodiments, a skeletal- related side effect and/or toxicity is indicated if the T-score declines to less than -1.0 in the total femur or vertebrae L1-L4. Methods for measuring are known in the art. See, e.g., Fogelman &Blake, J. Nucl Med, 41: 2015-2025 (2000).

[00236] The invention also provides methods of ameliorating skeletal-related side effects and/or toxicity in a subject administered an RSPO antagonist (e.g., anti-RSP03 antibody), comprising: administering to the subject a therapeutically effective amount of an anti-re sorptive medication.

[00237] The invention also provides methods of screening a subject for the risk of skeletal-related side effects and/or toxicity from treatment with an RSPO antagonist (e.g., anti-RSP03 antibody), comprising: determining the level of a biomarker in a sample from the subject, and comparing the level of the biomarker in the sample to a predetermined level of the biomarker, wherein if the level of the biomarker in the sample is higher than the predetermined level of the biomarker then the subject is at risk for skeletal-related side effects and/or toxicity. In some embodiments, the methods of screening a subject for the risk of skeletal -related side effects and/or toxicity from treatment with an RSPO antagonist (e.g., anti-RSP03 antibody) comprise: obtaining a biological sample from the subject prior to treatment with the RSPO antagonist (e.g., anti-RSP03 antibody), determining the level of a biomarker in the sample, and comparing the level of the biomarker in the sample to a predetermined level of the biomarker, wherein if the level of the biomarker in the sample is higher than the predetermined level of the biomarker then the subject is at risk for skeletal-related side effects and/or toxicity. In some embodiments, the skeletal-related side effect and/or toxicity is an increased risk of bone fracture. In some embodiments, the skeletal-related side effect and/or toxicity is osteopenia or osteoporosis. In some embodiments, the biomarker is a bone turnover marker. In some embodiments, the biomarker is a bone resorption biomarker. In some embodiments, the bone resorption biomarker is urinary hydroxyproline, urinary total pyridinoline (PYD), urinary free deoxypyridinoline (DPD), urinary collagen type 1 cross-linked N-telopeptide (NTX), urinary or serum collagen type 1 cross-linked C-telopeptide (CTX), bone sialoprotein (BSP), or tartrate-resistant acid phosphatase 5b. In some embodiments, the bone resorption biomarker is CTX. In some embodiments, the bone resorption biomarker is β-CTX. In some embodiments, a method of screening a subject for the risk of a skeletal-related side effect and/or toxicity from treatment with an RSPO antagonist (e.g., anti-RSP03 antibody) comprises: obtaining a biological sample from the subject prior to treatment with the RSPO antagonist (e.g., anti-RSP03 antibody), determining the level of β- CTX in the sample, and comparing the level of β-CTX in the sample to a predetermined level of β- CTX, wherein if the level of β-CTX in the sample is higher than the predetermined level of β-CTX then the subject is at risk for a skeletal-related side effect and/or toxicity. In some embodiments, the predetermined level of β-CTX is a value determined at an initial screening. In some embodiments, the predetermined level of β-CTX is from about 400 to 1200pg/ml. In some embodiments, if the subject is at risk for a skeletal-related side effect and/or toxicity, the subject is administered a therapeutically effective amount of an anti-re sorptive medication prior to treatment with the RSPO antagonist (e.g., anti-RSP03 antibody).

[00238] The invention also provides methods of screening a subject for the risk of skeletal-related side effects and/or toxicity from treatment with an RSPO antagonist (e.g., anti-RSP03 antibody), comprising: determining the level of a bone formation biomarker in a sample from the subject, and comparing the level of the bone formation biomarker in the sample to a predetermined level of the bone formation biomarker, wherein if the level of the bone formation biomarker in the sample is lower than the predetermined level of the bone formation biomarker then the subject is at risk for skeletal-related side effects and/or toxicity. In some embodiments, the methods of screening a subject for the risk of skeletal-related side effects and/or toxicity from treatment with an RSPO antagonist (e.g., anti-RSP03 antibody) comprise: obtaining a biological sample from the subject prior to treatment with the RSPO antagonist (e.g., anti-RSP03 antibody), determining the level of a bone formation biomarker in the sample, and comparing the level of the bone formation biomarker in the sample to a predetermined level of the bone formation biomarker, wherein if the level of the bone formation biomarker in the sample is lower than the predetermined level of the bone formation biomarker then the subject is at risk for skeletal-related side effects and/or toxicity. In some embodiments, the skeletal -related side effect and/or toxicity is an increased risk of bone fracture. In some embodiments, the skeletal -related side effect and/or toxicity is osteopenia or osteoporosis. In some embodiments, the biomarker is PINP. In some embodiments, a method of screening a subject for the risk of a skeletal-related side effect and/or toxicity from treatment with an RSPO antagonist (e.g., anti-RSP03 antibody) comprises: obtaining a biological sample from the subject prior to treatment with the RSPO antagonist (e.g., anti-RSP03 antibody), determining the level of PINP in the sample, and comparing the level of PINP in the sample to a predetermined level of PINP, wherein if the level of PINP in the sample is lower than the predetermined level of PINP then the subject is at risk for a skeletal-related side effect and/or toxicity. In some embodiments, the predetermined level of PINP is a value determined at an initial screening. In some embodiments, the predetermined level of PINP is from about 16 to 30 microg/ml. In some embodiments, if the subject is at risk for a skeletal-related side effect and/or toxicity, the subject is administered a therapeutically effective amount of an anti-resorptive medication prior to treatment with the RSPO antagonist (e.g., anti- RSP03 antibody).

[00239] In some embodiments of the methods described herein, the anti-resorptive medication is a bisphosphonate. It is believed that bisphosphonates prevent loss of bone mass by "inducing" osteoclasts to undergo apoptosis and thereby inhibiting the digestion of bone. In some embodiments, the bisphosphonate is selected from the group consisting of: etidronate, clodronate, tiludronate, pamidronate, neridronate, olpadronate, alendronate (FOSAMAX), ibandronate (BONIVA), risedronate (ACTONEL), and zoledronic acid (RECLAST). In some embodiments, the bisphosphonate is zoledronic acid. In some embodiments, the anti-resorptive medication is anti- RANKL antibody denosumab (PROLIA).

[00240] In any of the methods described herein, the RSPO antagonist is an anti-RSPO antibody. In any of the methods described herein, the RSPO antagonist is an anti-RSP03 antibody.

[00241] In certain embodiments of any of the methods described herein, the RSPO antagonist is an anti-RSP03 antibody comprising: (a) a heavy chain CDR1 comprising DYSIH (SEQ ID NO:5), a heavy chain CDR2 comprising YIYPSNGDSGYNQKFK (SEQ ID NO:6), and a heavy chain CDR3 comprising TYFANNFD (SEQ ID NO: 7); and (b) a light chain CDR1 comprising KASQSVDYDGDSYMN (SEQ ID NO: 9), a light chain CDR2 comprising AASNLES (SEQ ID NO: 10), and a light chain CDR3 comprising QQSNEDPLT (SEQ ID NO: 12).

[00242] In certain embodiments of any of the methods described herein, the RSPO antagonist is an antibody comprising a heavy chain variable region comprising SEQ ID NO: 14 and a light chain variable region comprising SEQ ID NO: 15. [00243] In certain embodiments, the RSPO antagonist comprises the same heavy chain variable region and the same light chain variable region sequences as OMP-131R10. In some embodiments, the RSPO antagonist is antibody OMP-131R10. OMP-131R10 is an affinity matured humanized monoclonal IgGl antibody that binds human RSP03 and has been previously described in U.S. Patent No. 9, 181,333.

[00244] In certain embodiments, the RSPO antagonist comprises the same heavy and light chain amino acid sequences as an antibody encoded by a plasmid deposited with ATCC having deposit no. PTA-120420 and PTA-120421. In certain embodiments, the RSPO antagonist is encoded by the plasmid having ATCC deposit no. PTA-120420 and PTA-120421which was deposited with American Type Culture Collection (ATCC), at 10801 University Boulevard, Manassas, VA, 20110, under the conditions of the Budapest Treaty on Jun. 18, 2013. In certain embodiments, the RSPO antagonist competes for specific binding to a human FZD with an antibody encoded by the plasmid deposited with ATCC having deposit no. PTA-120420 and PTA-120421.

[00245] In some embodiments, the subject has cancer. In some embodiments, the cancer is selected from the group consisting of: lung cancer, breast cancer, colon cancer, colorectal cancer, melanoma, pancreatic cancer, gastrointestinal cancer, renal cancer, ovarian cancer, liver cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, neuroendocrine cancer, neuroblastoma, glioma, glioblastoma multiforme, cervical cancer, stomach cancer, bladder cancer, hepatoma, and head and neck cancer. As used herein, "lung cancer" refers to non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). In certain embodiments, the cancer is a hematological cancer, such as a lymphoma or leukemia. In certain embodiments, the cancer is NSCLC. In certain embodiments, the cancer is ovarian cancer. In certain embodiments, the cancer is pancreatic cancer. In another embodiment, the cancer is colorectal cancer. In some embodiments, Wnt signaling is activated in the colorectal cancer (e.g., by an inactivating mutation in the APC gene or an activating mutation in the β- catenin gene). In certain embodiments, the colorectal cancer is third-line colorectal cancer. In some embodiments, the colorectal cancer is resistant to treatment with chemotherapy comprising 5- fluorouracil, irinotecan, and/or oxaliplatin. In certain embodiments, the cancer is not a neuroendocrine cancer.

[00246] In some embodiments of the aforementioned methods, the cancer is colorectal cancer, breast cancer, ovarian cancer, lung cancer, or pancreatic cancer. In some embodiments, the cancer comprises an RSPO gene fusion. In some embodiments, the cancer comprises an RSP03 gene fusion. In one embodiment of the invention, the cancer is colorectal cancer. In another embodiment, the colorectal cancer comprises an inactivating mutation in the adenomatous polyposis coli (APC) gene. In another embodiment, the colorectal cancer does not comprise an inactivating mutation in the APC gene. In another embodiment, the colorectal cancer comprises a wild-type APC gene. In another embodiment, the colorectal cancer comprises an activating mutation in the β-catenin gene. In another embodiment, the colorectal cancer does not comprise an activating mutation in the β-catenin gene. [00247] In another embodiment, the colorectal cancer comprises an RSPO gene fusion. In another embodiment, the RSPO gene fusion is an RSP03 gene fusion. In some embodiments, the fusion is between Protein Tyrosine Phosphatase, Receptor Type, K (PTPRK) and RSP03. For example, Seshagiri et al. reported RSP03 translocations that result in a fusion between PTPRK and RSP03. Seshagiri S, et al., Nature 488(7413): 660-4 (2012); see also U.S. Publication No. 20130209473.

[00248] In some embodiments of the present invention, the cancer expresses high RSP03 levels compared to a pre-determined level of expression of RSPOl, RSP02, RSP03, and/or RSP04, respectively. In another embodiment, the pre-determined RSPO l, RSP02, RSP03, or RSP04 expression level is the expression level of RSPO l, RSP02, RSP03, or RSP04 in a tumor or a group of tumors of the same tissue type. In another embodiment, the pre-determined RSPOl, RSP02, RSP03, or RSP04 expression level is the expression level of RSPOl, RSP02, RSP03, or RSP04 in normal tissue of the same tissue type.

[00249] In some embodiments, the cancer has elevated expression level of RSPOl, RSP02, RSP03, and/or RSP04. In some embodiments, the cancer is colorectal cancer with an elevated expression level of RSP03. In some embodiments, the cancer does not have elevated expression level of an RSPO polypeptide. In some embodiments, the cancer does not have elevated expression level of RSPOl, RSP02, RSP03, and/or RSP04. In some embodiments, the cancer is colorectal cancer that does not have elevated expression level of RSP03. In some embodiments, the cancer has substantially the same expression level of an RSPO polypeptide as normal tissue of the same tissue type. In some embodiments, the cancer is colorectal cancer that has substantially the same expression level of RSP03 as normal tissue of the same tissue type.

[00250] The phrases "a cancer or tumor has elevated expression levels of," "a cancer or tumor has substantially the same expression level as normal tissue of the same tissue type," "a cancer or tumor has low RSP03 expression," or "a cancer or tumor has high RSP03 expression" may refer to expression levels of a protein or expression levels of a nucleic acid. In general, the phrase "a cancer or tumor has elevated expression levels of," "a cancer or tumor has high expression levels of," "a cancer or tumor has low expression levels of," or "a cancer or tumor has substantially the same expression levels of a protein or a gene (or similar phrases) refers to expression levels of a protein or a gene in a cancer or tumor as compared to expression levels of the same protein or the same gene in a reference sample or to a pre-determined expression level. In some embodiments, the reference sample is normal tissue of the same tissue type. In some embodiments, the reference sample is normal tissue of a group of tissue types. In some embodiments, the reference sample is a cancer or tumor or a group of cancers or tumors of the same tissue type. In some embodiments, the reference sample is a cancer or tumor or group of cancers or tumors of a different tissue type. Thus in some embodiments, the expression levels of a protein or a gene in a cancer or tumor are "elevated," "high," "low," or "substantially the same" as compared to the average expression level of the protein or the gene within a group of tissue types. In some embodiments, the expression levels of a protein or a gene in a cancer or tumor are "elevated," "high," "low," or "substantially the same" as compared to the expression level of the protein or the gene in other cancers or tumors of the same tissue type or a different tissue type. In some embodiments, the cancer or tumor expresses "elevated," "high," "low," or "substantially the same" levels of RSPOl, RSP02, RSP03, and/or RSP04 as compared to the RSPO levels expressed in normal tissue of the same tissue type. In some embodiments, the cancer or tumor expresses "elevated," "high," or "substantially the same" levels of RSPOl, RSP02, RSP03, and/or RSP04 as compared to a pre-determined level.

[00251] Thus, the invention also provides methods of treating cancer. In some embodiments, the methods comprise a method of treating cancer in a subject in need thereof, comprising: (a) administering to the subject a therapeutically effective amount of an RSPO antagonist (e.g., anti- RSP03 antibody); and (b) determining the level of a bone resorption biomarker in a sample from the subject. In some embodiments, a method of treating cancer comprises (a) administering to the subject a therapeutically effective amount of an RSPO antagonist (e.g., anti-RSP03 antibody); (b) determining the level of a bone resorption biomarker in a sample from the subject; and (c) comparing the level of the bone resorption biomarker in the sample to a predetermined level of the bone resorption biomarker. In some embodiments, a method of treating cancer comprises (a) administering to the subject a therapeutically effective amount of an RSPO antagonist (e.g., anti-RSP03 antibody); (b) determining the level of a bone resorption biomarker in a sample from the subject; and (c) comparing the level of the bone resorption biomarker in the sample to a predetermined level of the bone resorption biomarker; wherein if the level of the bone resorption biomarker in the sample is higher than the predetermined level of the bone resorption biomarker then the subject is at risk for a skeletal-related side effect and/or toxicity. In some embodiments, a method of treating cancer comprises (a) administering to the subject a therapeutically effective amount of an RSPO antagonist (e.g., anti-RSP03 antibody); (b) determining the level of a bone resorption biomarker in a sample from the subject; and (c) comparing the level of the bone resorption biomarker in the sample to a predetermined level of the bone resorption biomarker; wherein if the level of the bone resorption biomarker in the sample is higher than the predetermined level of the bone resorption biomarker then the subject is administered a therapeutically effective amount of an anti-resorptive medication. In some embodiments, the bone formation biomarker is β-CTX.

[00252] In other embodiments, the methods comprise a method of treating cancer in a subject in need thereof, comprising: (a) administering to the subject a therapeutically effective amount of an RSPO antagonist (e.g., anti-RSP03 antibody); and (b) determining the level of a bone formation biomarker in a sample from the subject. In some embodiments, a method of treating cancer comprises (a) administering to the subject a therapeutically effective amount of an RSPO antagonist (e.g., anti- RSP03 antibody); (b) determining the level of a bone formation biomarker in a sample from the subject; and (c) comparing the level of the bone formation biomarker in the sample to a predetermined level of the bone formation biomarker. In some embodiments, a method of treating cancer comprises (a) administering to the subject a therapeutically effective amount of an RSPO antagonist (e.g., anti- RSP03 antibody); (b) determining the level of a bone formation biomarker in a sample from the subject; and (c) comparing the level of the bone formation biomarker in the sample to a predetermined level of the bone formation biomarker; wherein if the level of the bone formation biomarker in the sample is lower than the predetermined level of the bone formation biomarker then the subject is at risk for a skeletal-related side effect and/or toxicity. In some embodiments, a method of treating cancer comprises (a) administering to the subject a therapeutically effective amount of an RSPO antagonist (e.g., anti-RSP03 antibody); (b) determining the level of a bone formation biomarker in a sample from the subject; and (c) comparing the level of the bone formation biomarker in the sample to a predetermined level of the bone formation biomarker; wherein if the level of the bone formation biomarker in the sample is lower than the predetermined level of the bone formation biomarker then the subject is administered a therapeutically effective amount of an anti-resorptive medication. In some embodiments, the bone resorption biomarker is P1NP.

[00253] The invention also provides methods of inhibiting tumor growth. In some embodiments, the methods comprise a method of inhibiting tumor growth in a subject in need thereof, comprising: (a) administering to the subject a therapeutically effective amount of an RSPO antagonist (e.g., anti- RSP03 antibody); and (b) determining the level of a bone resorption biomarker in a sample from the subject. In some embodiments, a method of inhibiting tumor growth comprises (a) administering to the subject a therapeutically effective amount of an RSPO antagonist (e.g., anti-RSP03 antibody); (b) determining the level of a bone resorption biomarker in a sample from the subject; and (c) comparing the level of the bone resorption biomarker in the sample to a predetermined level of the bone resorption biomarker. In some embodiments, a method of inhibiting tumor growth comprises (a) administering to the subject a therapeutically effective amount of an RSPO antagonist (e.g., anti- RSP03 antibody); (b) determining the level of a bone resorption biomarker in a sample from the subject; and (c) comparing the level of the bone resorption biomarker in the sample to a predetermined level of the bone resorption biomarker; wherein if the level of the bone resorption biomarker in the sample is higher than the predetermined level of the bone resorption biomarker then the subject is at risk for a skeletal-related side effect and/or toxicity. In some embodiments, a method of inhibiting tumor growth comprises (a) administering to the subject a therapeutically effective amount of an RSPO antagonist (e.g., anti-RSP03 antibody); (b) determining the level of a bone resorption biomarker in a sample from the subject; and (c) comparing the level of the bone resorption biomarker in the sample to a predetermined level of the bone resorption biomarker; wherein if the level of the bone resorption biomarker in the sample is higher than the predetermined level of the bone resorption biomarker then the subject is administered a therapeutically effective amount of an anti-resorptive medication. In some embodiments, the bone resorption biomarker is β-CTX.

[00254] In further embodiments, the methods comprise a method of inhibiting tumor growth in a subject in need thereof, comprising: (a) administering to the subject a therapeutically effective amount of an RSPO antagonist (e.g., anti-RSP03 antibody); and (b) determining the level of a bone formation biomarker in a sample from the subject. In some embodiments, a method of inhibiting tumor growth comprises (a) administering to the subject a therapeutically effective amount of an RSPO antagonist (e.g., anti-RSP03 antibody); (b) determining the level of a bone formation biomarker in a sample from the subject; and (c) comparing the level of the bone formation biomarker in the sample to a predetermined level of the bone formation biomarker. In some embodiments, a method of inhibiting tumor growth comprises (a) administering to the subject a therapeutically effective amount of an RSPO antagonist (e.g., anti-RSP03 antibody); (b) determining the level of a bone formation biomarker in a sample from the subject; and (c) comparing the level of the bone formation biomarker in the sample to a predetermined level of the bone formation biomarker; wherein if the level of the bone formation biomarker in the sample is lower than the predetermined level of the bone formation biomarker then the subject is at risk for a skeletal-related side effect and/or toxicity. In some embodiments, a method of inhibiting tumor growth comprises (a) administering to the subject a therapeutically effective amount of an RSPO antagonist (e.g., anti-RSP03 antibody); (b) determining the level of a bone formation biomarker in a sample from the subject; and (c) comparing the level of the bone formation biomarker in the sample to a predetermined level of the bone formation biomarker; wherein if the level of the bone formation biomarker in the sample is lower than the predetermined level of the bone formation biomarker then the subject is administered a therapeutically effective amount of an anti-resorptive medication. In some embodiments, the bone formation biomarker is P1NP.

[00255] In some embodiments, the subject has a fibrotic disease. In further embodiments, the fibrotic disease is selected from the group consisting of pulmonary fibrosis, renal fibrosis, liver fibrosis, dermal fibrosis, cardiac fibrosis, and adhesion formation. In further embodiments, dermal fibrosis is selected from the group consisting of scleroderma, systemic sclerosis, scleroderma-like disease, sine scleroderma, keloid formation, and hypertrophic scarring. In further embodiments, renal fibrosis is chronic kidney disease. In further embodiments, pulmonary fibrosis is selected from the group consisting of idiopathic pulmonary fibrosis, interstitial pulmonary fibrosis, lung fibrosis, mediastinal fibrosis, secondary pulmonary fibrosis, and pleural fibrosis. In further embodiments, liver fibrosis is cirrhosis of the liver. In further embodiments, cardiac fibrosis is selected from the group consisting of myocardial fibrosis, cardiac valve fibrosis, endomyocardial fibrosis, and atherosclerosis.

[00256] The invention also provides methods of treating cancer. Any methods of treating a fibrotic disease known to a skilled artisan can be used together with the methods described herein. For example, methods of treating a fibrotic disease that can be used together with the methods described herein have been described in U.S. Patent Publication No. 2016/0166684, which is hereby incorporated by reference herein in its entirety for all purposes.

[00257] In some embodiments, the methods comprise a method of treating a fibrotic disease in a subject in need thereof, comprising: (a) administering to the subject a therapeutically effective amount of an RSPO antagonist (e.g., anti-RSP03 antibody); and (b) determining the level of a bone resorption biomarker in a sample from the subject. In some embodiments, a method of treating a fibrotic disease comprises (a) administering to the subject a therapeutically effective amount of an RSPO antagonist (e.g., anti-RSP03 antibody); (b) determining the level of a bone resorption biomarker in a sample from the subject; and (c) comparing the level of the bone resorption biomarker in the sample to a predetermined level of the bone resorption biomarker. In some embodiments, a method of treating a fibrotic disease comprises (a) administering to the subject a therapeutically effective amount of an RSPO antagonist (e.g., anti-RSP03 antibody); (b) determining the level of a bone resorption biomarker in a sample from the subject; and (c) comparing the level of the bone resorption biomarker in the sample to a predetermined level of the bone resorption biomarker; wherein if the level of the bone resorption biomarker in the sample is higher than the predetermined level of the bone resorption biomarker then the subject is at risk for a skeletal-related side effect and/or toxicity. In some embodiments, a method of treating a fibrotic disease comprises (a) administering to the subject a therapeutically effective amount of an RSPO antagonist (e.g., anti-RSP03 antibody); (b) determining the level of a bone resorption biomarker in a sample from the subject; and (c) comparing the level of the bone resorption biomarker in the sample to a predetermined level of the bone resorption biomarker; wherein if the level of the bone resorption biomarker in the sample is higher than the predetermined level of the bone resorption biomarker then the subject is administered a therapeutically effective amount of an anti-resorptive medication. In some embodiments, the bone formation biomarker is β-CTX.

[00258] In other embodiments, the methods comprise a method of treating a fibrotic disease in a subject in need thereof, comprising: (a) administering to the subject a therapeutically effective amount of an RSPO antagonist (e.g., anti-RSP03 antibody); and (b) determining the level of a bone formation biomarker in a sample from the subject. In some embodiments, a method of treating a fibrotic disease comprises (a) administering to the subject a therapeutically effective amount of an RSPO antagonist (e.g., anti-RSP03 antibody); (b) determining the level of a bone formation biomarker in a sample from the subject; and (c) comparing the level of the bone formation biomarker in the sample to a predetermined level of the bone formation biomarker. In some embodiments, a method of treating a fibrotic disease comprises (a) administering to the subject a therapeutically effective amount of an RSPO antagonist (e.g., anti-RSP03 antibody); (b) determining the level of a bone formation biomarker in a sample from the subject; and (c) comparing the level of the bone formation biomarker in the sample to a predetermined level of the bone formation biomarker; wherein if the level of the bone formation biomarker in the sample is lower than the predetermined level of the bone formation biomarker then the subject is at risk for a skeletal-related side effect and/or toxicity. In some embodiments, a method of treating a fibrotic disease comprises (a) administering to the subject a therapeutically effective amount of an RSPO antagonist (e.g., anti-RSP03 antibody); (b) determining the level of a bone formation biomarker in a sample from the subject; and (c) comparing the level of the bone formation biomarker in the sample to a predetermined level of the bone formation biomarker; wherein if the level of the bone formation biomarker in the sample is lower than the predetermined level of the bone formation biomarker then the subject is administered a therapeutically effective amount of an anti-resorptive medication. In some embodiments, the bone resorption biomarker is P1NP.

[00259] In some embodiments, the biological sample is a body fluid. In some embodiments, the biological sample is blood, plasma, serum, or urine. In some embodiments, the biological sample is a venous whole blood specimen. In some embodiments, the biological sample is a venous whole blood specimen using EDTA or heparin as an anticoagulant. In some embodiments, the biological sample is a plasma specimen. In some embodiments, the biological sample is a plasma specimen using EDTA or heparin as an anticoagulant. Samples of body fluids may be obtained by any method known in the art. In some embodiments, the biological sample is a frozen tissue sample or is fresh tissue sample.

[00260] Assays for measuring or determining the level of a bone resorption biomarker (e.g., β-CTX) in a sample are known to those of skilled in the art. For example, in some embodiments an immunoassay that quantitatively measures β-CTX levels in whole blood or plasma specimens is used. In some embodiments, the sample contains EDTA as an anticoagulant. In some embodiments, the sample contains heparin as an anticoagulant. In some embodiments, the immunoassay comprises two highly specific monoclonal antibodies against the amino acid sequence of ΕΚΑΗΟ-β-GGR of β-CTX, wherein the aspartic acid residue is β-isomerized. In order to obtain a specific signal in the immunoassay, two chains of ΕΚΑΗΟ-β-GGR must be cross-linked. In some embodiments, a sample and appropriate controls are placed into streptavidin-coated microtiter wells, followed by a solution containing biotinylated monoclonal antibodies against the amino acid sequence of ΕΚΑΗΟ-β-GGR of β-CTX. After incubation and washing, a chromogenic substrate solution is added to microtiter wells. After incubation, the reaction is stopped. Absorbance of the microtiter wells is read and the β-CTX concentration is determined.

[00261] Assays for measuring or determining the level of a bone formation biomarker (e.g., P1NP) in a sample are known to those of skilled in the art. For example, Gareno et al , Clinical Chemistry 54(1): 188-196 (2008) discloses a fully automated assay for determining P1NP in serum samples. Additional assays are discussed in Marcius et al, Biochemia Medica 16(2): 178-190 (2005).

[00262] In some embodiments of the methods described herein, a method of treating cancer or a fibrotic disease comprises administering a therapeutically effective amount of OMP-131R10 to a subject in need thereof at a dosage of 2.5 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, or 20 mg/kg. In some embodiments of the methods described herein, a method of treating cancer or a fibrotic disease comprises administering a therapeutically effective amount of OMP-131R10 to a subject in need thereof at a dosage of (a) at least about 2.5 mg/kg about every one to two weeks or (b) at least about 5.0 mg/kg about every three weeks. In some embodiments, a method of treating cancer or a fibrotic disease comprises administering a therapeutically effective amount of OMP-131R10 to a subject in need thereof at a dosage of about 2.5 mg/kg to about 20.0 mg/kg about every one to two weeks. In some embodiments, a method of treating cancer or a fibrotic disease comprises administering a therapeutically effective amount of OMP-131R10 to a subject in need thereof at a dosage of about 2.5 mg/kg to about 20.0 mg/kg about every three weeks. In some embodiments, a method of treating cancer or a fibrotic disease comprises administering a therapeutically effective amount of OMP- 131R10 to a subject in need thereof at a dosage of about 2.5 mg/kg to about 20.0 mg/kg about every four weeks.

[00263] The choice of delivery method for the initial and subsequent doses is made according to the ability of the subject to tolerate introduction of the RSPO antagonist (e.g., anti-RSP03 antibody) into the body. Thus, in any of the aspects and/or embodiments described herein, the administration of the RSPO antagonist (e.g., anti-RSP03 antibody) may be by intravenous injection or intravenously. In some embodiments, the administration is by intravenous infusion. In some embodiments, the administration is by subcutaneous injection. In any of the aspects and/or embodiments described herein, the administration of the RSPO antagonist (e.g., anti-RSP03 antibody) may be by a non- intravenous route.

[00264] In certain embodiments, in addition to administering an RSPO antagonist (e.g., anti-RSP03 antibody), the method or treatment further comprises administering at least one additional therapeutic agent. An additional therapeutic agent can be administered prior to, concurrently with, and/or subsequently to, administration of the RSP03 antagonist. Pharmaceutical compositions comprising the additional therapeutic agent(s) are also provided. In some embodiments, the at least one additional therapeutic agent comprises 1, 2, 3, or more additional therapeutic agents.

[00265] Combination therapy 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).

[00266] In some embodiments, the combination of an RSPO antagonist (e.g., anti-RSP03 antibody) and at least one additional therapeutic agent results in additive or synergistic results. In some embodiments, the combination therapy results in an increase in the therapeutic index of the RSPO antagonist. In some embodiments, the combination therapy results in an increase in the therapeutic index of the additional agent(s). In some embodiments, the combination therapy results in a decrease in the toxicity and/or side effects of the RSPO antagonist. In some embodiments, the combination therapy results in a decrease in the toxicity and/or side effects of the additional agent(s).

[00267] Therapeutic agents useful for the treatment of a fibrotic disease have been described in U.S. Patent Publication No. 2016/0166684, which is hereby incorporated by reference herein in its entirety for all purposes. [00268] Useful classes of therapeutic agents for the treatment of cancer 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 complexes and carboplatin), anthracyclines, antibiotics, antifolates, antimetabolites, chemotherapy sensitizers, duocarmycins, etoposides, fluorinated pyrimidines, ionophores, lexitropsins, nitrosoureas, platinols, 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 angiogenesis inhibitor.

[00269] Therapeutic agents that may be administered in combination with the RSPO (e.g., anti- RSP03 antibody) antagonists include chemotherapeutic agents. Thus, in some embodiments, the method or treatment involves the administration of an RSPO antagonist in combination with a chemotherapeutic agent or cocktail of multiple different chemotherapeutic agents. Treatment with an RSPO antagonist (e.g., anti-RSP03 antibody) can occur prior to, concurrently with, or subsequent to administration of chemotherapies. Combined administration can include co-administration, either in a single pharmaceutical 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. Preparation and dosing schedules for such chemotherapeutic 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 Book, 4th Edition, 2008, M. C. Perry, Editor, Lippincott, Williams & Wilkins, Philadelphia, PA.

[00270] Chemotherapeutic agents useful in the instant invention include, but are not limited to, alkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide and trimethylolomelamime; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, calicheamicin, carabicin, caminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytosine arabinoside, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenishers such as folinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK; razoxane; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (Ara-C); taxoids, e.g. paclitaxel (TAXOL) and docetaxel (TAXOTERE); chlorambucil; gemcitabine; 6- thioguanine; mercaptopurine; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; ibandronate; CPT11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (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 inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY 117018, onapristone, and toremifene (FARESTON); and anti-androgens 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 therapeutic agent is paclitaxel (taxol).

[00271] In certain embodiments, the chemotherapeutic agent is a topoisomerase inhibitor. Topoisomerase inhibitors are chemotherapy agents that interfere with the action of a topoisomerase enzyme (e.g., topoisomerase I or II). Topoisomerase inhibitors include, but are not limited to, doxorubicin HC1, daunorubicin citrate, mitoxantrone HC1, actinomycin D, etoposide, topotecan HC1, teniposide (VM-26), and irinotecan, as well as pharmaceutically acceptable salts, acids, or derivatives of any of these.

[00272] In certain embodiments, 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 division. Anti-metabolites include, but are not limited to, gemcitabine, fluorouracil, capecitabine, methotrexate sodium, ralitrexed, pemetrexed, tegafur, cytosine arabinoside, thioguanine, 5-azacytidine, 6- mercaptopurine, azathioprine, 6-thioguanine, pentostatin, fludarabine phosphate, and cladribine, as well as pharmaceutically acceptable salts, acids, or derivatives of any of these. [00273] In certain 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 docetaxel, or a pharmaceutically acceptable salt, acid, or derivative of paclitaxel or docetaxel. In certain embodiments, the agent is paclitaxel (TAXOL), docetaxel (TAXOTERE), albumin-bound paclitaxel (nab-paclitaxel; ABRAXANE), 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 an RSPO antagonist (e.g., anti- RSP03 antibody) is an anti-mitotic agent, the cancer or tumor being treated is breast cancer or a breast tumor. In certain embodiments, where the chemotherapeutic agent administered in combination with an RSPO antagonist (e.g., anti-RSP03 antibody) is an anti-mitotic agent, the cancer or tumor being treated is colorectal cancer or a colorectal tumor. In certain embodiments, the additional therapeutic agent is paclitaxel (taxol) or albumin-bound paclitaxel.

[00274] In some embodiments, an additional therapeutic agent comprises an agent such as a small molecule. For example, treatment can involve the combined administration of an RSPO antagonist (e.g., anti-RSP03 antibody) with a small molecule that acts as an inhibitor against additional tumor- associated antigens including, 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. In some embodiments, the additional therapeutic agent is an inhibitor of the BMP pathway.

[00275] In some embodiments, an additional therapeutic agent is a Wnt pathway inhibitor. In some embodiments, the Wnt pathway inhibitors are frizzled (FZD) protein binding agents, "FZD-binding agents". Non-limiting examples of FZD-binding agents can be found in U.S. Patent No. 7,982,013. FZD-binding agents may include, but are not limited to, anti-FZD antibodies. In some embodiments, a method comprises administering an RSPO antagonist (e.g., anti-RSP03 antibody) in combination with an anti-FZD antibody. In some embodiments, a method comprises administering an RSP03 antagonist in combination with the anti-FZD antibody 18R5. In some embodiments, the Wnt pathway inhibitors are Wnt protein binding agents, "Wnt-binding agents". Non-limiting examples of Wnt- binding agents can be found in U.S. Patent Nos. 7,723,477 and 7,947,277; and International Publications WO 2011/088127 and WO 2011/088123. 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 an RSPO antagonist (e.g., anti-RSP03 antibody) in combination with a FZD- Fc soluble receptor. In some embodiments, a method comprises administering an RSP03 antagonist in combination with a FZD8-Fc soluble receptor. In some embodiments, a method comprises administering an RSPO antagonist (e.g., anti-RSP03 antibody) in combination with an anti-FZD antibody.

[00276] In some embodiments, the methods described herein comprise administering a therapeutically effective amount of an RSPO antagonist (e.g., anti-RSP03 antibody) in combination with more than one additional therapeutic agent.

[00277] Combined administration can include co-administration, either in a single pharmaceutical 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.

[00278] It will be appreciated that the combination of an RSPO antagonist (e.g., anti-RSP03 antibody) and at least one additional therapeutic agent may be administered in any order or concurrently. In some embodiments, the RSPO antagonist will be administered to patients that have previously undergone treatment with a second therapeutic agent. In certain other embodiments, the RSPO antagonist and a second therapeutic agent will be administered substantially simultaneously or concurrently. For example, a subject may be given an RSPO antagonist (e.g., anti-RSP03 antibody) while undergoing a course of treatment with a second therapeutic agent (e.g., chemotherapy). In certain embodiments, an RSPO antagonist (e.g., anti-RSP03 antibody) will be administered within 1 year of the treatment with a second therapeutic agent. In certain alternative embodiments, an RSPO antagonist (e.g., anti-RSP03 antibody) will be administered within 10, 8, 6, 4, or 2 months of any treatment with a second therapeutic agent. In certain other embodiments, an RSPO antagonist (e.g., anti-RSP03 antibody) will be administered within 4, 3, 2, or 1 weeks of any treatment with a second therapeutic agent. In some embodiments, an RSPO antagonist (e.g., anti-RSP03 antibody) will be administered within 5, 4, 3, 2, or 1 days of any treatment with a second therapeutic agent. It will further be appreciated that the two (or more) agents or treatments may be administered to the subject within a matter of hours or minutes (i.e., substantially simultaneously).

[00279] For the treatment of a disease, the appropriate dosage of an RSPO antagonist (e.g., anti- RSP03 antibody) depends on the type of disease to be treated, the severity and course of the disease, the responsiveness of the disease, whether the RSPO antagonist is administered for therapeutic or preventative purposes, previous therapy, the patient's clinical history, and so on, all at the discretion of the treating physician. The RSPO antagonist can be administered one time or over a series of treatments lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation 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 repetition rates. In certain embodiments, dosage is from 0.0 ^g to lOOmg/kg of body weight, from O. ^g to lOOmg/kg of body weight, from ^g to lOOmg/kg of body weight, from lmg to lOOmg/kg of body weight, lmg to 80mg/kg of body weight from lOmg to lOOmg/kg of body weight, from lOmg to 75mg/kg of body weight, or from lOmg to 50mg/kg of body weight. In certain embodiments, the dosage of the RSP03 antagonist is from about O. lmg to about 20mg/kg of body weight. In certain embodiments, dosage can be given once or more daily, weekly, monthly, or yearly. In certain embodiments, the RSPO antagonist is given once every week, once every two weeks or once every three weeks.

[00280] 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 administration of the particular agent at a therapeutically effective dose. In some cases, drug 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 effects associated with the therapeutic agent.

[00281] Thus, the present invention provides methods of treating a fibrotic disease, treating cancer or inhibiting tumor growth 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 an RSPO antagonist (e.g., anti-RSP03 antibody), a second agent, etc.

[00282] In certain embodiments, the treatment involves the administration of an RSPO antagonist (e.g., anti-RSP03 antibody) in combination with radiation therapy. Treatment with an RSPO antagonist (e.g., anti-RSP03 antibody) can occur prior to, concurrently with, or subsequent to administration of radiation therapy. Dosing schedules for such radiation therapy can be determined by the skilled medical practitioner.

[00283] Embodiments of the present disclosure can be further defined by reference to the following non-limiting examples, which describe the use of an RSPO antagonist (e.g., anti-RSP03 antibody) for treatment of cancer. 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

Phase la/b Study of OMP-131R10 in patients with advanced solid tumors or metastatic colorectal cancer

[00284] The study was an open-label Phase la/b dose-escalation of OMP-131R10 as a single agent for advanced solid tumors and in combination with FOLFIRI (5-FU) for patients with previously treated metastatic colorectal cancer. The primary objectives of the study were to determine the safety and the maximum tolerated dose of OMP-131R10. The secondary objectives were to determine the rate of immunogenicity, the preliminary efficacy, and the pharmacokinetics of OMP-131R10.

[00285] The patients in the initial la portion of the trial were treated with a dosing regimen of OMP- 131R10 of 2.5 mg/kg (n = 3), 5 mg/kg (n = 3), 10 mg/kg (n = 4), or 15 mg/kg (n = 5) on the first day of each two-week cycle (Figures 1 and 2). The patients in the lb portion of the trial were treated with a dosing regimen of OMP-131R10 of 5 mg/kg (n = 3) on the first day of each two-week cycle. No dose limiting toxicities had been observed.

[00286] β-CTX and P1NP levels were monitored during the trial (Figures 3 A and B). A summary of bone biomarker changes observed during the trial and shown in Figure 3A are summarized in Figure 4. β-CTX and P1NP changes were determined relative to baseline results. Of the lowest 2.5 mg/kg cohort, only one out of three patients experienced any significant change in the bone biomarkers: a 50% increase in β-CTX level without a significant drop in P1NP. In contrast, 3 out of 4 patients in the highest 15 mg/kg cohort experienced at least a 50% increase in β-CTX level, with one of them showing a doubling of β-CTX level accompanied by a halving of P1NP levels. Three patients (101, 105, and 204) were administered zoledronic acid based on the changes in β-CTX and/or P1NP levels. None of the patients suffered a fracture. The correlation of increased OMP-131R10 doses with the increased frequency and magnitude of β-CTX and P1NP level changes indicated that OMP-131R10 treatment is associated with a change in bone homeostasis in some patients. β-CTX appeared to be an early and sensitive biomarker of the effect of OMP-131R10 on bone homeostasis.

[00287] Bone densities of the patients were also determined by DEXA (Figure 5). Several patients had a bone density T-score between -1 and -2.5 at the beginning of the trial indicating that they suffered from osteopenia. One patient had a T-score of less than -2.5 at the beginning of the trial indicating that he suffered from osteoporosis. None of the patients enrolled in the study had a significant change in their bone mineral density (BMD) as assessed by DEXA scans (T-scores) while on treatment with OMP-131R10 (Figure 5). None of the patients suffered a fracture. These data suggest that osteopenic patients can be treated with OMP-131R10 without a significant risk of developing a further decline in their bone mineral density. Furthermore, the data confirms that β-CTX appears to be an early and sensitive biomarker of skeletal-related side effects and/or toxicities resulting from treatment with an anti-RSP03 antibody. Finally, the study demonstrated that the skeletal-related side effects tied to treatment with OMP-131R10 appear to be manageable.

[00288] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application.

[00289] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, or patent application were specifically and individually indicated to be so incorporated by reference.

[00290] Following are the sequences disclosed in the application:

Human RSPOl amino acid sequence with signal sequence (SEQ ID NO: 1)

MRLGLCVVALVLSWTHLTISSRGIKGKRQRRISAEGSQACAKGCELCSEVNGCLKCSPKL FILLERNDIRQVGVCLPSCPPGYFDARNPDMNKCIKCKIEHCEACFSHNFCTKCKEGLYL HKGRCYPACPEGSSAA GTMECSSPAQCEMSEWSPWGPCSKKQQLCGFRRGSEERTRRVL HAPVGDHAACSDTKETRRCTVRRVPCPEGQKRRKGGQGRRENANRNLARKESKEAGAGSR RRKGQQQQQQQGTVGPL SAGPA Human RSP02 amino acid sequence with signal sequence (SEQ ID NO: 2)

MQFRLFSFALI ILNCMDYSHCQGNRWRRSKRASYVSNPICKGCLSCSKDNGCSRCQQKLF FFLRREGMRQYGECLHSCPSGYYGHRAPDMNRCARCRIENCDSCFSKDFCTKCKVGFYLH RGRCFDECPDGFAPLEETMECVEGCEVGHWSEWGTCSRNNRTCGFKWGLETRTRQIVKKP VKD IPCPTIAESRRCKMTMRHCPGGKRTPKAKEKRNKKKKRKLIERAQEQHSVFLATDR ANQ

Human RSP03 amino acid sequence with signal sequence (SEQ ID NO:3)

MHLRLISWLFI IL FMEYIGSQNASRGRRQRRMHPNVSQGCQGGCATCSDYNGCLSCKPR LFFALERIGMKQIGVCLSSCPSGYYGTRYPDINKCTKCKADCDTCFNKNFCTKCKSGFYL HLGKCLDNCPEGLEANNHTMECVSIVHCEVSEWNPWSPCTKKGKTCGFKRGTETRVREII QHPSAKGNLCPPTNETRKCTVQRKKCQKGERGKKGRERKRKKPNKGESKEAIPDSKSLES SKEIPEQRENKQQQKKRKVQDKQKSVSVSTVH

Human RSP04 amino acid sequence with signal sequence (SEQ ID NO: 4)

MRAPLCLLLLVAHAVDMLALNRRKKQVGTGLGGNCTGCIICSEENGCSTCQQRLFLFIRR EGIRQYGKCLHDCPPGYFGIRGQEVNRCKKCGATCESCFSQDFCIRCKRQFYLYKGKCLP TCPPGTLAHQNTRECQGECELGPWGGWSPCTHNGKTCGSAWGLESRVREAGRAGHEEAAT CQVLSESRKCPIQRPCPGERSPGQKKGRKDRRPRKDRKLDRRLDVRPRQPGLQP

131R010 Heavy chain CDR1 (SEQ ID NO:5)

DYSIH

131R010 Heavy chain CDR2 (SEQ ID NO:6)

YIYPSNGDSGYNQKFK

131R010 Heavy chain CDR3 (SEQ ID NO:7)

TYFANNFD

131R010 Alternative Heavy chain CDR3 (SEQ ID NO: 8)

A YFANNFDY

131R010 Light chain CDR1 (SEQ ID NO:9)

KASQSVDYDGDSYMN

131R010 Light chain CDR2 (SEQ ID NO: 10)

AASNLES

131R010 Alternative Light chain CDR2 (SEQ ID NO: 11)

AAS

131R010 Light chain CDR3 (SEQ ID NO: 12)

QQSNEDPLT

131R010 Alternative Light chain CDR3 (SEQ ID NO: 13)

QQSNEDPLTF

131R010 Heavy chain variable region amino acid sequence (SEQ ID NO: 14)

QVQLVQSGAEVKKPGASVKVSCKASGY F DYSIHWVRQAPGQGLEWIGYIYPSNGDSGY NQKFKNRVTMTRDTSTSTAYMELSRLRSEDTAVYYCATYFA NFDYWGQGTTLTVSS

131R010 Light chain variable region amino acid sequence (SEQ ID NO: 15)

DIQMTQSPSSLSASVGDRVTITCKASQSVDYDGDSYMNWYQQKPGKAPKLLIYAASNLES GVPSRFSGSGSGTDFTL ISPVQAEDFATYYCQQSNEDPLTFGAGTKLELKR 131R010 Heavy chain amino acid sequence with predicted signal sequence underlined (SEQ ID NO: 16)

MKHLWFFLLLVAAPRWVLSQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYSIHWVRQAP GQGLEWIGYIYPSNGDSGYNQKFKNRVTMTRDTSTSTAYMELSRLRSEDTAVYYCATYFA NNFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

131R010 Heavy chain amino acid sequence without predicted signal sequence (SEQ ID NO: 17)

QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYSIHWVRQAPGQGLEWIGYIYPSNGDSGY NQKFKNRVTM RD S S AYMELSRLRSEDTAVYYCATYFA NFDYWGQGTTLTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY SLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT PPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK

131R010 Light chain amino acid sequence with predicted signal sequence underlined (SEQ ID NO: 18)

MKHLWFFLLLVAAPRWVLSDIQMTQSPSSLSASVGDRVTITCKASQSVDYDGDSYMNWYQ QKPGKAPKLLIYAASNLESGVPSRFSGSGSGTDFTL ISPVQAEDFATYYCQQSNEDPLT FGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

131R010 Light chain amino acid sequence without predicted signal sequence (SEQ ID NO: 19)

DIQMTQSPSSLSASVGDRVTITCKASQSVDYDGDSYMNWYQQKPGKAPKLLIYAASNLES GVPSRFSGSGSGTDFTL ISPVQAEDFATYYCQQSNEDPLTFGAGTKLELKRTVAAPSVF I FPPSDEQLKSGTASVVCLLN FYPREAKVQWKVDNALQSGNSQESV EQDSKDSTYSLS STLTLSKADYEKHKVYACEVTHQGLSSPV KSFNRGEC

Human LGR4 protein sequence (NM_018490; SEQ ID NO:20)

MPGPLGLLCFLALGLLGSAGPSGAAPPLCAAPCSCDGDRRVDCSGKGLTAVPEGLSAFTQ ALDISMNNITQLPEDAFKNFPFLEELQLAGNDLSFIHPKALSGLKELKVLTLQNNQLKTV PSEAIRGLSALQSLRLDANHITSVPEDSFEGLVQLRHLWLDDNSLTEVPVHPLSNLPTLQ ALTLALNKISSIPDFAFTNLSSLVVLHLHNNKIRSLSQHCFDGLDNLETLDLNYNNLGEF PQAIKALPSLKELGFHSNSISVIPDGAFDGNPLLRTIHLYDNPLSFVGNSAFHNLSDLHS LVIRGASMVQQFPNLTGTVHLESLTLTGTKISSIPNNLCQEQKMLRTLDLSYNNIRDLPS FNGCHALEEISLQRNQIYQIKEG FQGLISLRILDLSRNLIHEIHSRAFATLGPITNLDV SFNELTSFPTEGLNGLNQLKLVGNFKLKEALAAKDFVNLRSLSVPYAYQCCAFWGCDSYA NLNTEDNSLQDHSVAQEKGTADAANVTSTLENEEHSQI IIHCTPSTGAFKPCEYLLGSWM IRLTVWFI FLVALFFNLLVILT FASC SLPSSKLFIGLISVSNLFMGIYTGIL FLDAV SWGRFAEFGIWWETGSGCKVAGFLAVFSSESAI FLLMLATVERSLSAKDIMKNGKSNHLK QFRVAALLAFLGATVAGCFPLFHRGEYSASPLCLPFPTGETPSLGFTVTLVLLNSLAFLL MAVIYTKLYCNLEKEDLSENSQSSMIKHVAWLI FTNCI FFCPVAFFSFAPLI AISISPE IMKSVTLI FFPLPACLNPVLYVFFNPKFKEDWKLLKRRV KKSGSVSVSISSQGGCLEQD FYYDCGMYSHLQGNLTVCDCCESFLLTKPVSCKHLIKSHSCPALAVASCQRPEGYWSDCG TQSAHSDYADEEDSFVSDSSDQVQACGRACFYQSRGFPLVRYAYNLPRVKD

Human LGR5 protein sequence (SEQ ID NO: 21)

MDTSRLGVLLSLPVLLQLATGGSSPRSGVLLRGCPTHCHCEPDGRMLLRVDCSDLGLSEL PSNLSVFTSYLDLSMNNISQLLPNPLPSLRFLEELRLAGNALTYIPKGAFTGLYSLKVLM LQNNQLRHVPTEALQNLRSLQSLRLDA HISYVPPSCFSGLHSLRHLWLDDNALTEIPVQ AFRSLSALQAMTLALNKIHHIPDYAFGNLSSLVVLHLHNNRIHSLGKKCFDGLHSLETLD LNYNNLDEFPTAIRTLSNLKELGFHSNNIRSIPEKAFVGNPSLITIHFYDNPIQFVGRSA FQHLPELRTLTLNGASQITEFPDLTGTANLESLTLTGAQISSLPQTVCNQLPNLQVLDLS YNLLEDLPSFSVCQKLQKIDLRHNEIYEIKVD FQQLLSLRSLNLAWNKIAI IHPNAFST LPSLIKLDLSSNLLSSFPITGLHGLTHLKLTGNHALQSLISSENFPELKVIEMPYAYQCC AFGVCENAYKISNQWNKGDNSSMDDLHKKDAGMFQAQDERDLEDFLLDFEEDLKALHSVQ CSPSPGPFKPCEHLLDGWLIRIGVWTIAVLALTCNALVTSTVFRSPLYISPIKLLIGVIA AVNMLTGVSSAVLAGVDAFTFGSFARHGAWWENGVGCHVIGFLSIFASESSVFLLTLAAL ERGFSVKYSAKFE KAPFSSLKVI ILLCALLALTMAAVPLLGGSKYGASPLCLPLPFGEP STMGYMVALILLNSLCFLMMTIAYTKLYCNLDKGDLENIWDCSMVKHIALLLFTNCILNC PVAFLSFSSLINLTFISPEVIKFILLVVVPLPACLNPLLYILFNPHFKEDLVSLRKQTYV WTRSKHPSLMSINSDDVEKQSCDSTQALVTFTSSSITYDLPPSSVPSPAYPVTESCHLSS VAFVPCL

Human LGR6 protein sequence (BC047905; SEQ ID NO:22)

MGRPRLTLVCQVSI I ISARDLSMNNLTELQPGLFHHLRFLEELRLSGNHLSHIPGQAFSG LYSLKILMLQNNQLGGIPAEALWELPSLQSLRLDA LISLVPERSFEGLSSLRHLWLDDN ALTEIPVRALNNLPALQAMTLALNRISHIPDYAFQNL SLVVLHLHNNRIQHLGTHSFEG LHNLETLDLNYNKLQEFPVAIRTLGRLQELGFHNNNIKAIPEKAFMGNPLLQTIHFYDNP IQFVGRSAFQYLPKLHTLSLNGAMDIQEFPDLKGTTSLEILTLTRAGIRLLPSGMCQQLP RLRVLELSHNQIEELPSLHRCQKLEEIGLQHNRIWEIGADTFSQLSSLQALDLSWNAIRS IHPEAFSTLHSLVKLDLTDNQLTTLPLAGLGGLMHLKLKGNLALSQAFSKDSFPKLRILE VPYAYQCCPYGMCASFFKASGQWEAEDLHLDDEESSKRPLGLLARQAENHYDQDLDELQL EMEDSKPHPSVQCSPTPGPFKPCEYLFESWGIRLAVWAIVLLSVLCNGLVLLTVFAGGPV PLPPVKFVVGAIAGANTLTGISCGLLASVDALTFGQFSEYGARWETGLGCRATGFLAVLG SEASVLLLTLAAVQCSVSVSCVRAYGKSPSLGSVRAGVLGCLALAGLAAALPLASVGEYG ASPLCLPYAPPEGQPAALGFTVALVMMNSFCFLVVAGAYIKLYCDLPRGDFEAVWDCAMV RHVAWLIFADGLLYCPVAFLSFASMLGLFPVTPEAVKSVLLVVLPLPACLNPLLYLLFNP HFRDDLRRLRPRAGDSGPLAYAAAGELEKSSCDSTQALVAFSDVDLILEASEAGRPPGLE TYGFPSVTLISCQQPGAPRLEGSHCVEPEGNHFGNPQPSMDGELLLRAEGS PAGGGLSG GGGFQPSGLAFASHV

Claims

WHAT IS CLAIMED IS:

1. A method of selecting a subject for treatment with an RSPO antagonist, comprising:

(a) obtaining a biological sample from the subject;

(b) determining the level of a bone formation biomarker and/or bone resorption

biomarker in the sample; and

(c) selecting the subject for treatment with the RSPO antagonist if

(i) the level of the bone formation biomarker is above a predetermined level; and/or

(ii) the level of bone resorption biomarker is below a predetermined level.

2. A method of identifying a subject as eligible for treatment with a RSPO antagonist,

comprising:

(a) obtaining a biological sample from the subject;

(b) determining the level of a bone formation biomarker and/or bone resorption

biomarker in the sample; and

(c) identifying the subject as eligible for treatment with the RSPO antagonist if

(ii) the level of bone resorption biomarker is below a predetermined level.

3. A method of selecting a subject for treatment with a RSPO antagonist, comprising:

(a) determining the level of a bone formation biomarker and/or bone resorption

biomarker in a sample from the subject; and

(b) selecting the subject for treatment with the RSPO antagonist if

(ii) the level of bone resorption biomarker is below a predetermined level.

4. A method of identifying a subject as eligible for treatment with a RSPO antagonist,

comprising:

(a) determining the level of a bone formation biomarker and/or bone resorption

biomarker in a sample from the subject; and

(b) identifying the subject as eligible for treatment with the RSPO antagonist if

(ii) the level of bone resorption biomarker is below a predetermined level.

5. A method of screening for a subject at risk of a skeletal -related side effect and/or toxicity from treatment with a RSPO antagonist, comprising:

(a) obtaining a biological sample from the subject prior to treatment with the RSPO antagonist; (b) determining the level of a bone formation biomarker and/or bone resorption biomarker in the sample; and

(c) comparing the level of the bone formation biomarker and/or bone resorption

biomarker in the sample to a predetermined level of the bone formation biomarker and/or bone resorption biomarker,

wherein the subject is at risk for a skeletal-related side effect and/or toxicity if

(i) the level of the bone formation biomarker in the sample is lower than the predetermined level of the bone formation biomarker; and/or

(ii) the level of the bone resorption biomarker in the sample is higher than the predetermined level of the bone resorption biomarker.

6. A method of screening for a subject at risk of a skeletal -related side effect and/or toxicity from treatment with a RSPO antagonist, comprising:

(a) determining the level of a bone formation biomarker and/or bone resorption

biomarker in a sample from the subject; and

(b) comparing the level of the bone formation biomarker and/or bone resorption

biomarker in the sample to a predetermined level of the bone formation biomarker and/or bone resorption biomarker;

7. The method of claim 5 or claim 6, wherein a subject at risk for a skeletal -related side effect and/or toxicity is administered a therapeutically effective amount of an anti-re sorptive medication prior to treatment with the RSPO antagonist.

8. The method of any one of claims 1 to 7, wherein the biological sample is blood, serum, or plasma.

9. The method of any one of claims 1 to 8, wherein the bone formation biomarker is serum procollagen type 1 amino-terminal propeptide (P1NP).

10. The method of claim 9, wherein the predetermined level of P1NP in blood, serum, or plasma is a level of P1NP determined at an earlier timepoint or at an initial screening.

1 1. The method of claim 9, wherein the predetermined level of P1NP in blood, serum, or plasma is about 16 microg/ml or more, about 19 microg/ml or more, about 22 microg/ml or more, about 25 microg/ml or more, or about 30 microg/ml or more.

12. The method of any one of claims 8 to 1 1, which further comprises administering the RSPO antagonist to the subject if the level of the bone formation biomarker is above the predetermined level.

13. The method of any one of claims 9 to 11, wherein if the P1NP level is 70% or less, 60% or less, 50% or less, 40% or less, or 30% or less than the predetermined level, then the subject is administered a therapeutically effective amount of an anti-resorptive medication prior to administering the RSPO antagonist to the subject.

14. The method of any one of claims 1 to 8, wherein the bone resorption biomarker is collagen type 1 cross-linked beta C-telopeptide (β-CTX).

15. The method of claim 14, wherein the predetermined level of β-CTX in blood, serum, or plasma is a level of β-CTX determined at an earlier timepoint or at an initial screening.

16. The method of claim 14, wherein the predetermined level of β-CTX in blood, serum, or plasma is about 1500 pg/ml or less, about 1200 pg/ml or less, about 1000 pg/ml or less, about 800 pg/ml or less, about 600 pg/ml or less, or about 400 pg/ml or less.

17. The method of any one of claims 8 and 14 to 16, which further comprises administering the RSPO antagonist to the subject if the level of the bone resorption biomarker is below the predetermined level.

18. The method of any one of claims 14 to 16, wherein if the β-CTX level is at least about 1.5- fold, at least about 2-fold, at least about 2.5-fold, or at least about 3-fold greater than the predetermined level, then the subject is administered a therapeutically effective amount of an anti-resorptive medication prior to administering the RSPO antagonist.

19. A method of selecting a subject for treatment with a RSPO antagonist, comprising:

(a) determining bone density in the subject; and

(b) selecting the subject for treatment with the RSPO antagonist if the bone density is at or above a predetermined level.

20. A method of identifying a subject as eligible for treatment with a RSPO antagonist,

comprising:

(a) determining bone density in the subject; and

21. A method of screening a subject for the risk of a skeletal-related side effect and/or toxicity from treatment with a RSPO antagonist, comprising:

(a) determining bone density in the subject; and

(b) comparing the bone density of the subject to a predetermined level of bone density; wherein the subject is at risk for a skeletal-related side effect and/or toxicity if the bone density in the subject is lower than the predetermined level of bone density.

22. The method of claim 21, wherein a subject at risk for a skeletal-related side effect and/or toxicity is administered a therapeutically effective amount of an anti-re sorptive medication prior to treatment with the RSPO antagonist.

23. The method of any one of claim 19 to 22, wherein bone density is determined by dual -energy x-ray absorptiometry (DEXA).

24. The method of claim 23, wherein bone density is expressed as a T-score.

25. The method of claim 24, wherein the predetermined level of bone density T-score is -1, -1.5, -2, -2.5, or -3.

26. A method of monitoring a subject receiving treatment with a RSPO antagonist for the

development of a skeletal -related side effect and/or toxicity, comprising:

(a) obtaining a biological sample from the subject receiving treatment;

(b) determining the level of a bone formation biomarker and/or bone resorption

biomarker in the sample; and

(c) comparing the level of the bone formation biomarker and/or bone resorption

wherein a decrease in the level of the bone formation biomarker and/or an increase in the level of the bone resorption biomarker indicates development of a skeletal-related side effect and/or toxicity.

27. A method of detecting the development of a skeletal-related side effect and/or toxicity in a subject receiving treatment with a RSPO antagonist, comprising:

(a) obtaining a biological sample from the subject receiving treatment;

(b) determining the level of a bone formation biomarker and/or bone resorption

biomarker in the sample; and

(c) comparing the level of the bone formation biomarker and/or bone resorption

28. A method for identifying a skeletal-related side effect and/or toxicity in a subject receiving treatment with a RSPO antagonist, comprising:

(a) obtaining a biological sample from the subject receiving treatment;

(b) determining the level of a bone formation biomarker and/or bone resorption

biomarker in the sample; and

(c) comparing the level of the bone formation biomarker and/or bone resorption

wherein a skeletal-related side effect and/or toxicity is indicated when (i) the level of the bone formation biomarker is below a predetermined level; and/or

(ii) the level of bone resorption biomarker is above a predetermined level.

29. A method for monitoring a skeletal -related side effect and/or toxicity in a subject receiving treatment with a RSPO antagonist, comprising:

(a) obtaining a biological sample from the subject receiving treatment;

(b) determining the level of a bone formation biomarker and/or bone resorption

biomarker in the sample; and

(c) comparing the level of the bone formation biomarker and/or bone resorption

wherein a skeletal-related side effect and/or toxicity is indicated when

(i) the level of the bone formation biomarker is below a predetermined level; and/or

(ii) the level of bone resorption biomarker is above a predetermined level.

30. A method of monitoring a subject receiving treatment with a RSPO antagonist for the

development of a skeletal-related side effect and/or toxicity, comprising:

(a) determining the level of a bone formation biomarker and/or bone resorption

biomarker in a sample from the subject; and

(b) comparing the level of the bone formation biomarker and/or bone resorption

31. A method of detecting the development of a skeletal-related side effect and/or toxicity in a subject receiving treatment with a RSPO antagonist, comprising:

(a) determining the level of a bone formation biomarker and/or bone resorption

biomarker in a sample from the subject; and

(b) comparing the level of the bone formation biomarker and/or bone resorption

wherein a skeletal-related side effect and/or toxicity is indicated when

(ii) the level of bone resorption biomarker is above a predetermined level. A method for identifying a skeletal-related side effect and/or toxicity in a subject receiving treatment with a RSPO antagonist, comprising:

(a) determining the level of a bone formation biomarker and/or bone resorption

biomarker in a sample from the subject; and

(b) comparing the level of the bone formation biomarker and/or bone resorption

wherein a skeletal-related side effect and/or toxicity is indicated when

(ii) the level of bone resorption biomarker is above a predetermined level.

A method for monitoring a skeletal -related side effect and/or toxicity in a subject receiving treatment with a RSPO antagonist, comprising:

(a) determining the level of a bone formation biomarker and/or bone resorption

biomarker in a sample from the subject; and

(b) comparing the level of the bone formation biomarker and/or bone resorption

wherein a skeletal-related side effect and/or toxicity is indicated when

(ii) the level of bone resorption biomarker is above a predetermined level. A method for reducing a skeletal -related side effect and/or toxicity in a subject receiving treatment with a RSPO antagonist, comprising:

(a) obtaining a biological sample from the subject receiving treatment;

(b) determining the level of a bone formation biomarker and/or bone resorption

biomarker in the sample;

(c) comparing the level of the bone formation biomarker and/or bone resorption

biomarker in the sample to a predetermined level of the bone formation biomarker and/or bone resorption biomarker; and

(d) administering to the subject a therapeutically effective amount of an anti-resorptive medication if

(ii) the level of the bone resorption biomarker in the sample is higher than the predetermined level of the bone resorption biomarker. A method of preventing or attenuating the development of a skeletal-related side effect and/or toxicity in a subject receiving treatment with a RSPO antagonist, comprising:

(a) obtaining a biological sample from the subject prior to treatment with the RSPO antagonist;

(b) determining the level of a bone formation biomarker and/or bone resorption

biomarker in the sample;

(c) comparing the level of the bone formation biomarker and/or bone resorption

(d) administering to the subject a therapeutically effective amount of an anti-re sorptive medication; and

(e) administering to the subject the RSPO antagonist.

A method for reducing a skeletal -related side effect and/or toxicity in a subject receiving treatment with a RSPO antagonist, comprising:

(a) determining the level of a bone formation biomarker and/or bone resorption

biomarker in a sample from the subject;

(b) comparing the level of the bone formation biomarker and/or bone resorption

(c) administering to the subject a therapeutically effective amount of an anti-resorptive medication if

A method of preventing or attenuating the development of a skeletal-related side effect and/or toxicity in a subject receiving treatment with a RSPO antagonist, comprising:

(a) determining the level of a bone formation biomarker and/or bone resorption

biomarker in a sample from the subject prior to treatment with the RSPO antagonist;

(b) comparing the level of the bone formation biomarker and/or bone resorption

(c) administering to the subject a therapeutically effective amount of an anti-resorptive medication; and

(d) administering to the subject the RSPO antagonist.

A method of treating cancer or a fibrotic disease in a subject in need thereof, comprising: (a) administering to the subject a therapeutically effective amount of a RSPO antagonist; and

(b) determining the level of a bone formation biomarker and/or bone resorption

biomarker in a sample from the subject.

39. The method of claim 38, further comprising:

(c) comparing the level of the bone formation biomarker and/or bone resorption

40. The method of claim 38, further comprising:

(c) comparing the level of the bone formation biomarker and/or bone resorption

wherein the subject is administered a therapeutically effective amount of an anti-resorptive medication if

41. The method of any one of claims 26 to 40, wherein the sample is blood, serum, or plasma.

42. The method of any one of claims 26 to 41, wherein the predetermined level of the bone

formation biomarker and/or bone resorption biomarker is the amount of bone formation biomarker and/or bone resorption biomarker in a sample obtained from the subject at an earlier date.

43. The method of any one of claims 26 to 42, wherein the predetermined level of the bone

formation biomarker and/or bone resorption biomarker is the amount of bone formation biomarker and/or bone resorption biomarker in a sample obtained from the subject prior to treatment.

44. The method of any one of claims 26 to 43, wherein the predetermined level of the bone

formation biomarker and/or bone resorption biomarker is a baseline level.

45. The method of any one of claims 26 to 44, wherein the subject is administered a

therapeutically effective amount of an anti-resorptive medication if (i) the bone formation biomarker level is below a predetermined level for any one sample; and/or

(ii) the bone resorption biomarker level is above a predetermined level for any one sample.

46. The method of any one of claims 26 to 44, wherein the subject is administered a

therapeutically effective amount of an anti-resorptive medication if

(i) the bone formation biomarker level is 70% or less, 60% or less, 50% or less, 40% or less, or 30% or less than a predetermined level; and/or

(ii) the bone resorption biomarker level is at least about 1.5-fold, at least about 2-fold, at least about 2.5 -fold, or at least about 3 -fold greater than a predetermined level.

47. The method of claims 26 to 46, wherein the bone formation biomarker is serum procollagen type 1 amino-terminal propeptide (P1NP).

48. The method of claims 26 to 46, wherein the bone resorption biomarker is collagen type 1 cross-linked beta C-telopeptide (β-CTX).

49. The method of claim 44 or claim 45, wherein the bone formation biomarker is serum

procollagen type 1 amino-terminal propeptide (P 1NP), and the predetermined level is about 16 microg/ml, about 19 microg/ml, about 22 microg/ml, about 25 microg/ml, or about 30 microg/ml.

50. The method of claim 44 or claim 45, wherein the bone resorption biomarker is collagen type 1 cross-linked beta C-telopeptide (β-CTX), and the predetermined level is about 1500 pg/ml, about 1200 pg/ml, about 1000 pg/ml, about 800 pg/ml, about 600 pg/ml, or about 400 pg/ml.

51. A method of monitoring a subject receiving treatment with a RSPO antagonist for the

development of a skeletal-related side effect and/or toxicity, comprising:

(a) determining bone density in the subject; and

(b) comparing the bone density in the subject to a predetermined level of bone density; wherein a decrease in bone density in the subject indicates development of a skeletal-related side effect and/or toxicity.

52. A method of detecting the development of a skeletal-related side effect and/or toxicity in a subject receiving treatment with a RSPO antagonist, comprising:

(a) determining bone density in the subject; and

(b) comparing the bone density in the subject to a predetermined level of bone density; wherein a skeletal-related side effect and/or toxicity is indicated when bone density in the subject is below the predetermined level.

53. A method for identifying a skeletal-related side effect and/or toxicity in a subject receiving treatment with a RSPO antagonist, comprising:

(a) determining bone density in the subject; and

54. A method for monitoring a skeletal-related side effect and/or toxicity in a subject receiving treatment with a RSPO antagonist, comprising:

(a) determining bone density in the subject; and

55. A method for reducing a skeletal -related side effect and/or toxicity in a subject receiving treatment with a RSPO antagonist, comprising:

(a) determining bone density in the subject;

(b) comparing the bone density in the subject to a predetermined level of bone density; and

(c) administering to the subject a therapeutically effective amount of an anti-resorptive medication if bone density in the subject is below the predetermined level.

56. A method of preventing or attenuating the development of a skeletal -related side effect and/or toxicity in a subject receiving treatment with a RSPO antagonist, comprising:

(a) determining bone density in the subject;

(b) comparing the bone density in the subject to a predetermined level of bone density;

(d) administering to the subject the RSPO antagonist.

57. A method of treating cancer or a fibrotic disease in a subject in need thereof, comprising:

(a) administering to the subject a therapeutically effective amount of a RSPO antagonist; and

(b) determining bone density in the subject.

58. The method of claim 38, further comprising:

(a) comparing the bone density in the subject to a predetermined level of bone density; wherein the subject is at risk for a skeletal-related side effect and/or toxicity if bone density in the subject is below the predetermined level.

59. The method of claim 38, further comprising:

(a) comparing the bone density in the subject to a predetermined level of bone density; wherein the subject is administered a therapeutically effective amount of an anti-resorptive medication if bone density in the subject is below the predetermined level.

60. The method of any one of claims 51 to 59, wherein the predetermined level of bone density is the bone density in the subject at an earlier date.

61. The method of any one of claims 51 to 60, wherein the predetermined level of bone density is the bone density in the subject prior to treatment.

62. The method of any one of claims 51 to 61, wherein the predetermined level of bone density is a baseline level.

63. The method of any one of claims 51 to 62, wherein the subject is administered a

therapeutically effective amount of an anti-resorptive medication if bone density in the subject is below the predetermined level.

64. The method of any one of claims 51 to 63, wherein bone density is determined by dual -energy x-ray absorptiometry (DEXA).

65. The method of claim 64, wherein bone density is expressed as a T-score.

66. The method of claim 65, wherein the predetermined level of bone density T-score is -1, -1.5, -2, -2.5, or -3.

67. The method of any one of claims 26 to 66, wherein

(a) the biological sample is obtained and the bone formation biomarker and/or bone resorption biomarker is determined,

(b) the bone formation biomarker and/or bone resorption biomarker is determined, or

(c) the bone density is determined

once, two or more times, three or more time, four or more times, five or more times, six or more times, seven or more times, or ten or more times.

68. The method of any one of claims 26 to 66, wherein

(c) the bone density is determined

no more than once, two or more times, three or more time, four or more times, five or more times, six or more times, seven or more times, or ten or more times.

69. The method of any one of claims 26 to 66, wherein

(c) the bone density is determined

between 1 and 5 times, between 1 and 10 times, or between 1 and 20 times.

70. The method of any one of claims 26 to 66, wherein

(c) the bone density is determined once, twice or more, three times or more, or four times or more every week, every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, or every 6 weeks for a total of 2 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, 12 weeks, 4 months, 6 months or 1 year.

71. The method of any one of claims 26 to 66, wherein

(c) the bone density is determined

once, twice or more, three times or more, or four times or more every week, every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, or every 6 weeks for at least 2 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, 12 weeks, 4 months, 6 months or 1 year.

72. The method of any one of claims 26 to 66, wherein

(c) the bone density is determined

once, twice or more, three times or more, or four times or more every week, every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, or every 6 weeks for no more than 2 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, 12 weeks, 4 months, 6 months or 1 year.

73. The method of any one of claims 26 to 66, wherein

(c) the bone density is determined

once, twice or more, three times or more, or four times or more every week, every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, or every 6 weeks for the period of time during which the subject receives treatment with the RSPO antagonist.

74. A method for reducing a skeletal-related side effect and/or toxicity in a subject receiving treatment with a RSPO antagonist, comprising administering to the subject a therapeutically effective amount of an anti-resorptive medication.

75. A method of preventing or attenuating the development of a skeletal -related side effect and/or toxicity in a subject receiving treatment with a RSPO antagonist, comprising administering to the subject a therapeutically effective amount of an anti-resorptive medication.

76. The method of any one of claims 1 to 75, wherein the skeletal-related side effect and/or toxicity is related to treatment with the RSPO antagonist.

77. The method of any one of claims 1 to 76, wherein the skeletal-related side effect and/or toxicity is an increased risk of bone fracture, osteopenia, or osteoporosis.

78. The method of any one of claims 1 to 77, wherein the RSPO antagonist is an antibody that specifically binds at least one RSPO protein or portion thereof.

79. The method of claim 78, wherein the antibody specifically binds at least one RSPO protein selected from the group consisting of human RSPOl, human RSP02, human RSP03, or human RSP04.

80. The method of claim 78 or claim 79, wherein the antibody specifically binds RSP03.

81. The method of any one of claims 78 to 80, wherein the antibody specifically binds RSP03 and comprises:

(a) a heavy chain CDR1 comprising DYSIH (SEQ ID NO:5), a heavy chain CDR2

comprising YIYPSNGDSGYNQKFK (SEQ ID NO:6), and a heavy chain CDR3 comprising TYFANNFD (SEQ ID NO:7); and

(b) a light chain CDR1 comprising KASQSVDYDGDSYMN (SEQ ID NO:9), a light chain CDR2 comprising AASNLES (SEQ ID NO: 10), and a light chain CDR3 comprising QQSNEDPLT (SEQ ID NO: 12).

82. The method of claim 81, wherein the antibody comprises a heavy chain variable region

having at least 90% sequence identity to SEQ ID NO: 14 and a light chain variable region having at least 90% sequence identity to SEQ ID NO: 15.

83. The method of claim 81, wherein the antibody comprises the heavy chain variable region of SEQ ID NO: 14 and the light chain variable region of SEQ ID NO: 15.

84. The method of any one of claims 78 to 83, wherein the antibody is a recombinant antibody, a monoclonal antibody, a chimeric antibody, a monospecific antibody, or a bispecific antibody.

85. The method of any one of claims 78 to 81, wherein the antibody is a humanized antibody.

86. The method of any one of claims 78 to 81, wherein the antibody is a human antibody.

87. The method of any one of claims 78 to 86, wherein the antibody is an IgGl antibody.

88. The method of any one of claims 78 to 86, wherein the antibody is an IgG2 antibody.

89. The method of any one of claims 78 to 86, wherein the antibody is an antibody fragment comprising an antigen binding site.

90. The method of any one of claims 78 to 83, wherein the antibody is OMP-131R10.

91. The method of claim 90, wherein OMP-131R10 is administered intravenously to the subject in need thereof at a dosage of 2.5 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, or 20 mg/kg.

92. The method according to any one of claims 45 to 35, 36, or 40 to 91, wherein the anti- resorptive medication is a bisphosphonate or denosumab.

93. The method of claim 92, wherein the bisphosphonate is selected from the group consisting of: etidronate, clodronate, tiludronate, pamidronate, neridronate, olpadronate, alendronate, ibandronate, risedronate, and zoledronic acid.

94. The method of claim 92 or claim 93, wherein the bisphosphonate is zoledronic acid.

95. The method of any one of claims 1 to 94, wherein the subject has cancer.

96. The method of claim 80, wherein the cancer is selected from the group consisting of: lung cancer, breast cancer, colon cancer, colorectal cancer, melanoma, pancreatic cancer, gastrointestinal cancer, renal cancer, ovarian cancer, neuroendocrine cancer, liver cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, neuroblastoma, glioma, glioblastoma multiforme, cervical cancer, stomach cancer, bladder cancer, hepatoma, and head and neck cancer.

97. The method of claim 96, wherein the cancer is colorectal cancer, breast cancer, ovarian

cancer, lung cancer, or pancreatic cancer.

98. The method of claim 97, wherein the cancer is colorectal cancer.

99. The method of claim 98, wherein the colorectal cancer comprises an inactivating mutation in the adenomatous polyposis coli (APC) gene.

100. The method of claim 98, wherein the colorectal cancer does not comprise an inactivating mutation in the APC gene.

101. The method of claim 98, wherein the colorectal cancer comprises a wild-type APC gene.

102. The method of claim 98, wherein the colorectal cancer does not comprise an activating

mutation in the β-catenin gene.

103. The method of any one of claims 95 to 102, wherein the cancer has elevated expression of one or more of RSPOl, RSP02, RSP03, and RSP04.

104. The method of any one of claims 95 to 102, wherein the cancer has elevated expression of RSP03.

105. The method of any one of claims 95 to 104, wherein the cancer comprises a RSPO gene

fusion.

106. The method of any one of claims 95 to 104, wherein the cancer comprises an RSP03 gene fusion.

107. The method of claim 106, wherein the cancer comprises a fusion between PTPRK and

RSP03.

108. The method of any one of claims 1 to 107, wherein the subject is treated with the RSPO

antagonist in combination with one or more additional anti-cancer agents.

109. The method of any one of claims 1 to 94, wherein the subject has a fibrotic disease.

110. The method of claim 109, wherein the fibrotic disease is selected from the group consisting of pulmonary fibrosis, renal fibrosis, liver fibrosis, dermal fibrosis, cardiac fibrosis, and adhesion formation.

111. The method of claim 110, wherein the dermal fibrosis is selected from the group consisting of scleroderma, systemic sclerosis, scleroderma-like disease, sine scleroderma, keloid formation, and hypertrophic scarring.

112. The method of claim 110, wherein the renal fibrosis is chronic kidney disease.

13. The method of claim 1 10, wherein the pulmonary fibrosis is selected from the group consisting of idiopathic pulmonary fibrosis, interstitial pulmonary fibrosis, lung fibrosis, mediastinal fibrosis, secondary pulmonary fibrosis, and pleural fibrosis.

14. The method of claim 1 10, wherein the liver fibrosis is cirrhosis of the liver.

15. The method of claim 1 10, wherein the cardiac fibrosis is selected from the group consisting of myocardial fibrosis, cardiac valve fibrosis, endomyocardial fibrosis, and atherosclerosis.