TREATMENT OF FIBROTIC DISEASES

CROSS-REFERENCE TO RELATED APPLICATONS

[001] This application claims the priority benefit of U.S. Provisional Application No. 62/051,026, filed September 16, 2014, which is hereby incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

[002] The field of this invention generally relates to RSPO and LGR antagonists, including but not limited to, antibodies and other agents that bind RSPO, particularly human RSPOl, RSP02, and RSP03, as well as to methods of using the RSPO and/or LGR antagonists for the treatment of fibrotic diseases.

BACKGROUND OF THE INVENTION

[003] Fibrosis is estimated to contribute, either directly or indirectly, to nearly 45% of deaths in the developed world. Fibrotic diseases are characterized by an excessive accumulation of extracellular matrix components, which disrupts the physiological tissue architecture, leading to the dysfunction of the affected organ. In some instances, fibrosis is thought to be a consequence of chronic tissue irritation or chronic inflammation. In some instances, fibrosis is thought to be a consequence of autoimmune reactions within the body. The progressive replacement of parenchymal tissues with extracellular matrix components is observed in fibrotic diseases such as scleroderma, pulmonary fibrosis, and liver cirrhosis. However, the cellular and molecular factors that sustain the fibrotic cascade remain poorly understood.

[004] Wnt-signaling has been implicated in several human fibrotic diseases. Activated Wnt-signaling in some types of fibrotic diseases may be identified by accumulation of β-catenin, up-regulation of Wnt pathway ligands such as Wntl and Wnt 10B, and down-regulation of Wnt pathway negative regulators such as Dkkl (Akhmetshina et al., 2012, Nature Communications, 3:735; Guo et al., 2012, Physiol. Res., 61 :337-346). The Wnt signaling pathway is one of several critical regulators of embryonic pattern formation, post-embryonic tissue maintenance, and stem cell biology. Unregulated activation of the Wnt pathway is associated with many human diseases where pathogenesis may proceed by overtaking homeostatic mechanisms which control normal development and/or tissue repair.

[005] 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.

[006] The R-Spondin (RSPO) family of proteins is conserved among vertebrates and comprises four members, RSPOl, 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).

[007] 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). RSPOl 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 by endothelial cells and their cellular precursors in Xenopus and mouse. Furthermore, it has been suggested that RSP03 may act as an angiogenic factor in embryogenesis (Kazanskaya et al., 2008, Development, 135:3655-3664).

[008] Drug treatment options for patients diagnosed with a fibrotic disease are very limited. There is a need for new agents targeting fibrosis, and signaling pathways involved in fibrosis. Thus, biomolecules such as RSPO-binding agents that disrupt signaling pathways involved in fibrosis are a potential source of new therapeutic agents.

BRIEF SUMMARY OF THE INVENTION

[009] The present invention provides methods for treating a fibrotic disease in a subject, where the methods include administering to a subject a therapeutically effective amount of a RPSO and/or LGR antagonist, such as an anti-RSPO antibody, an anti-LGR antibody, or a soluble LGR receptor.

[010] In some embodiments, a RSPO or LGR antagonist includes, but is not limited to, antibodies and other polypeptides that bind to at least one RSPO or LGR protein, small molecules that bind at least one RSPO or LGR protein, and soluble LGR proteins. The RSPO protein (e.g., human protein) may be one of RSPOl, RSP02, RSP03, and RSP04. The LGR protein may be LGR4, LGR5, or LGR6.

[011] In some embodiments, the RSPO or LGR antagonist is a RSPO-binding agent. In some embodiments, the RSPO or LGR antagonist is a LGR-binding agent. In some embodiments, the RSPO or LGR antagonist is an antibody. In some embodiments, the RSPO or LGR antagonist is an anti-RSPO antibody. In some embodiments, the RSPO or LGR antagonist is an anti-LGR antibody. In some embodiments, the RSPO or LGR antagonist is a soluble receptor. In some embodiments, the RSPO or LGR antagonist is a LGR-Fc soluble receptor. In some embodiments, the RSPO or LGR antagonist is a LGR5-Fc soluble receptor.

[012] In some embodiments, the RSPO or LGR antagonist is an antibody that specifically binds at least one RSPO protein or portion thereof. In some embodiments, the antibody specifically binds at least one human RSPO protein selected from the group consisting of: RSPOl, RSP02, RSP03, and RSP04.

[013] The present invention provides methods of treating or preventing a fibrotic disease in a subject, comprising administering to the subject a therapeutically effective amount of a RSPO-binding agent, including any of those described herein. In some embodiments, the RSPO-binding agent is an antibody that specifically binds a human RSPO protein. In some embodiments, the RSPO-binding agent is an antibody that specifically binds human RSPO 1. In some embodiments, the RSPO-binding agent is an antibody that specifically binds human RSP02. In some embodiments, the RSPO-binding agent is an antibody that specifically binds human RSP03. In some embodiments, the RSPO-binding agent is an antibody that specifically binds human RSP04. In some embodiments, a method of treating or preventing a fibrotic disease in a subject comprises administering to the subject a therapeutically effective amount of an antibody that specifically binds human RSPOl . In some embodiments, a method of treating or preventing a fibrotic disease in a subject comprises administering to the subject a therapeutically effective amount of an antibody that specifically binds human RSP02. In some embodiments, a method of treating or preventing a fibrotic disease in a subject comprises administering to the subject a therapeutically effective amount of an antibody that specifically binds human RSP03. In some embodiments, a method of treating or preventing a fibrotic disease in a subject comprises administering to the subject a therapeutically effective amount of an antibody that specifically binds human RSPO 1 and a

therapeutically effective amount of an antibody that specifically binds human RSP02. In some embodiments, a method of treating or preventing a fibrotic disease in a subject comprises administering to the subject a therapeutically effective amount of an antibody that specifically binds human RSPOl and a therapeutically effective amount of an antibody that specifically binds human RSP03. In some embodiments, a method of treating or preventing a fibrotic disease in a subject comprises administering to the subject a therapeutically effective amount of an antibody that specifically binds human RSP02 and a therapeutically effective amount of an antibody that specifically binds human RSP03.

[014] In some embodiments, the RSPO-binding agent is a soluble receptor that specifically binds a human RSPO protein. In some embodiments, the RSPO-binding agent is a soluble receptor that specifically binds human RSPO 1. In some embodiments, the RSPO-binding agent a soluble receptor that specifically binds human RSP02. In some embodiments, the RSPO-binding agent a soluble receptor that specifically binds human RSP03. In some embodiments, the RSPO-binding agent is a soluble receptor that comprises the extracellular domain, or a fragment thereof, of LGR4, LGR5, or LGR6. In some embodiments, the RSPO-binding agent is a soluble receptor that comprises the extracellular domain, or a fragment thereof, of LGR5. In some embodiments, the RSPO-binding agent is a soluble receptor that comprises amino acids 22-564 LGR5. In some embodiments, the RSPO-binding agent is a soluble receptor that comprises a fragment of amino acids 22-264 of LGR5. In some embodiments, the soluble receptor comprises a non-LGR polypeptide. In some embodiments, the non-LGR polypeptide is directly linked to the extracellular domain of the LGR protein. In some embodiments, the non-LGR polypeptide is linked to the extracellular domain of the LGR protein by a linker. In some embodiments, the non-LGR polypeptide is a human Fc region. In some embodiments, the non-LGR polypeptide is a human Fc region, selected from the group consisting of SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, and SEQ ID NO: 80.

[015] The present invention also provides methods of treating or preventing a fibrotic disease in a subject, comprising administering to the subject a therapeutically effective amount of a LGR-binding agent, including any of those described herein. In some embodiments, the LGR-binding agent is an antibody that specifically binds a human LGR protein. In some embodiments, the LGR-binding agent is an antibody that specifically binds human LGR4. In some embodiments, the LGR-binding agent is an antibody that specifically binds human LGR5. In some embodiments, the LGR-binding agent is an antibody that specifically binds human LGR6. In some embodiments, a method of treating or preventing a fibrotic disease in a subject comprises administering to the subject a therapeutically effective amount of an antibody that specifically binds human LGR4. In some embodiments, a method of treating or preventing a fibrotic disease in a subject comprises administering to the subject a therapeutically effective amount of an antibody that specifically binds human LGR5. In some embodiments, a method of treating or preventing a fibrotic disease in a subject comprises administering to the subject a therapeutically effective amount of an antibody that specifically binds human LGR6.

[016] 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.

[017] In some embodiments, the method comprises administration of at least one additional therapeutic agent. In some embodiments, the additional therapeutic agent is a second antibody. In some

embodiments, the additional therapeutic agent is an anti-inflammatory agent.

[018] In certain embodiments of the methods described herein, the invention provides methods comprising a RSPO 1 -binding agent (e.g., an antibody) that specifically binds human RSPOl, wherein the RSPO 1 -binding agent (e.g., an antibody) comprises one, two, three, four, five, and/or six of the CDRs of antibody 89M5 (see Table 1 herein).

[019] In certain embodiments, the invention provides methods comprising a RSPOl -binding agent (e.g., an antibody) that specifically binds human RSPOl, wherein the RSPO 1 -binding agent comprises: (a) a heavy chain CDR1 comprising TGYTMH (SEQ ID NO:5), a heavy chain CDR2 comprising GINPNNGGTTYNQNFKG (SEQ ID NO: 6), and a heavy chain CDR3 comprising KEFSDGYYFFAY (SEQ ID NO:7); and (b) a light chain CDR1 comprising KASQDVIFAVA (SEQ ID NO:8), a light chain CDR2 comprising WASTRHT (SEQ ID NO:9), and a light chain CDR3 comprising QQHYSTPW (SEQ ID NO: 10).

[020] In certain embodiments, the invention provides methods comprising a RSPOl -binding agent (e.g., an antibody) that specifically binds RSPOl, wherein the RSPO 1 -binding agent comprises a heavy chain variable region having at least about 80%, at least about 85%>, at least about 90%>, at least about 95%, at least about 97%, or at least about 99% sequence identity to SEQ ID NO: 1 1 or SEQ ID NO:56. In certain embodiments, the RSPO 1 -binding agent comprises a light chain variable region having at least about 80%), at least about 85%>, at least about 90%>, at least about 95%>, at least about 97%>, or at least about 99% sequence identity to SEQ ID NO: 12 or SEQ ID NO:57. In certain embodiments, the RSPOl- binding agent comprises a heavy chain variable region comprising SEQ ID NO: 1 1 and/or a light chain variable region comprising SEQ ID NO: 12. In certain embodiments, the RSPO 1 -binding agent comprises a heavy chain variable region comprising SEQ ID NO:56 and/or a light chain variable region comprising SEQ ID NO:57.

[021] In certain embodiments, the invention provides methods comprising a RSPOl -binding agent (e.g., an antibody) that specifically binds RSPOl, wherein the RSPO 1 -binding agent comprises a heavy chain having at least 90% sequence identity to SEQ ID NO: 13 or SEQ ID NO: 14, and/or a light chain having at least 90%> sequence identity to SEQ ID NO: 15 or SEQ ID NO: 16. In some embodiments, the RSPO 1 -binding agent comprises a heavy chain having at least 95% sequence identity to SEQ ID NO: 13 or SEQ ID NO: 14, and/or a light chain having at least 95% sequence identity to SEQ ID NO: 15 or SEQ ID NO: 16. In some embodiments, the RSPO 1 -binding agent comprises a heavy chain comprising SEQ ID NO: 14 and/or a light chain comprising SEQ ID NO: 16. In some embodiments, the RSPO 1 -binding agent comprises a heavy chain comprising SEQ ID NO: 14 and a light chain comprising SEQ ID NO: 16. In some embodiments, the anti-RSPOl antibody is h89M5-H8L5.

[022] In certain embodiments, the invention provides methods comprising a RSPOl -binding agent (e.g., an antibody) that specifically binds RSPOl, wherein the RSPO 1 -binding agent comprises a heavy chain having at least 90% sequence identity to SEQ ID NO:58 or SEQ ID NO:59, and/or a light chain having at least 90% sequence identity to SEQ ID NO:60 or SEQ ID NO:61. In some embodiments, the RSPO 1 -binding agent comprises a heavy chain having at least 95% sequence identity to SEQ ID NO:58 or SEQ ID NO:59, and/or a light chain having at least 95% sequence identity to SEQ ID NO:60 or SEQ ID NO:61. In some embodiments, the RSPO 1 -binding agent comprises a heavy chain comprising SEQ ID NO:59 and/or a light chain comprising SEQ ID NO:61. In some embodiments, the RSPO 1 -binding agent comprises a heavy chain comprising SEQ ID NO:59 and a light chain comprising SEQ ID NO:61. In some embodiments, the anti-RSPOl antibody is h89M5-H2L2.

[023] In certain embodiments of the methods described herein, the invention provides methods comprising a RSP02-binding agent (e.g., an antibody) that specifically binds human RSP02, wherein the RSP02-binding agent (e.g., an antibody) comprises one, two, three, four, five, and/or six of the CDRs of antibody 130M23 (see Table 1 herein).

[024] In certain embodiments, the invention provides methods comprising a RSP02-binding agent (e.g., an antibody) that specifically binds human RSP02, wherein the RSP02-binding agent comprises: (a) a heavy chain CDR1 comprising SSYAMS (SEQ ID NO:21), a heavy chain CDR2 comprising SISSGGSTYYPDSVKG (SEQ ID NO:22), and a heavy chain CDR3 comprising

RGGDPGVYNGDYEDAMDY (SEQ ID NO:23); and (b) a light chain CDR1 comprising

KASQDVSSAVA (SEQ ID NO:24), a light chain CDR2 comprising WASTRHT (SEQ ID NO:25), and a light chain CDR3 comprising QQHYSTP (SEQ ID NO:26). [025] In certain embodiments, the invention provides methods comprising a RSP02 -binding agent (e.g., an antibody) that specifically binds RSP02, wherein the RSP02-binding agent comprises a heavy chain variable region having at least about 80%, at least about 85%, at least about 90%, at least about 95%), at least about 97%, or at least about 99% sequence identity to SEQ ID NO:27. In certain embodiments, the RSP02-binding agent comprises a light chain variable region having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% sequence identity to SEQ ID NO:28 or SEQ ID NO:66. In certain embodiments, the RSP02-binding agent comprises a heavy chain variable region comprising SEQ ID NO:27 and/or a light chain variable region comprising SEQ ID NO:28. In certain embodiments, the RSP02-binding agent comprises a heavy chain variable region comprising SEQ ID NO:27 and/or a light chain variable region comprising SEQ ID NO:66.

[026] In certain embodiments, the invention provides methods comprising a RSP02 -binding agent (e.g., an antibody) that specifically binds RSP02, wherein the RSP02-binding agent comprises a heavy chain having at least 90% sequence identity to SEQ ID NO:29 or SEQ ID NO:30, and/or a light chain having at least 90% sequence identity to SEQ ID NO:31 or SEQ ID NO:32. In some embodiments, the RSP02-binding agent comprises a heavy chain having at least 95% sequence identity to SEQ ID NO:29 or SEQ ID NO:30, and/or a light chain having at least 95% sequence identity to SEQ ID NO:31 or SEQ ID NO:32. In some embodiments, the RSP02-binding agent comprises a heavy chain comprising SEQ ID NO:30 and/or a light chain comprising SEQ ID NO:32. In some embodiments, the RSP02-binding agent comprises a heavy chain comprising SEQ ID NO:30 and a light chain comprising SEQ ID NO:32. In some embodiments, the anti-RSP02 antibody is hl30M23-HlL6.

[027] In certain embodiments, the invention provides methods comprising a RSP02 -binding agent (e.g., an antibody) that specifically binds RSP02, wherein the RSP02-binding agent comprises a heavy chain having at least 90% sequence identity to SEQ ID NO:29 or SEQ ID NO:30, and/or a light chain having at least 90% sequence identity to SEQ ID NO:67 or SEQ ID NO:68. In some embodiments, the RSP02-binding agent comprises a heavy chain having at least 95% sequence identity to SEQ ID NO:29 or SEQ ID NO:30, and/or a light chain having at least 95% sequence identity to SEQ ID NO:67 or SEQ ID NO:68. In some embodiments, the RSP02-binding agent comprises a heavy chain comprising SEQ ID NO:30 and/or a light chain comprising SEQ ID NO:68. In some embodiments, the RSP02-binding agent comprises a heavy chain comprising SEQ ID NO:30 and a light chain comprising SEQ ID NO:68. In some embodiments, the anti-RSP02 antibody is hl30M23-HlL2.

[028] In certain embodiments of the methods described herein, the invention provides methods comprising a 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 131R01 1 (see Table 1 herein).

[029] In certain embodiments, the invention provides methods comprising a RSP03 -binding agent (e.g., an antibody) that specifically binds human RSP03, wherein the RSP03-binding agent comprises: (a) a heavy chain CDR1 comprising DYSIH (SEQ ID NO:37), a heavy chain CDR2 comprising

YIYPSNGDSGYNQKFK (SEQ ID NO:38), and a heavy chain CDR3 comprising TYFANNFD (SEQ ID NO:39) or ATYFAN FDY (SEQ ID NO:40); and (b) a light chain CDR1 comprising

KASQSVDYDGDSYMN (SEQ ID NO:41), a light chain CDR2 comprising AASNLES (SEQ ID NO:42) or AAS (SEQ ID NO:43), and a light chain CDR3 comprising QQSNEDPLT (SEQ ID NO:44)or QQSNEDPLTF (SEQ ID NO:45).

[030] In certain embodiments, the invention provides a 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%, 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:46. In certain embodiments, the RSP03-binding agent comprises a light chain variable region having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% sequence identity to SEQ ID NO:47. In certain embodiments, the RSP03 -binding agent comprises a heavy chain variable region comprising SEQ ID NO:46 and/or a light chain variable region comprising SEQ ID NO:47.

[031] In certain embodiments, the invention provides a RSP03-binding agent (e.g., an antibody) that specifically binds RSP03, wherein the RSP03 -binding agent comprises a heavy chain having at least 90% sequence identity to SEQ ID NO:48 or SEQ ID NO:49, and/or a light chain having at least 90% sequence identity to SEQ ID NO:50 or SEQ ID NO:51. In some embodiments, the RSP03-binding agent comprises a heavy chain having at least 95% sequence identity to SEQ ID NO:48 or SEQ ID NO:49, and/or a light chain having at least 95% sequence identity to SEQ ID NO:50 or SEQ ID NO:51. In some embodiments, the RSP03-binding agent comprises a heavy chain comprising SEQ ID NO:49 and/or a light chain comprising SEQ ID NO:51. In some embodiments, the RSP03-binding agent comprises a heavy chain comprising SEQ ID NO:49 and a light chain comprising SEQ ID NO:51. In some embodiments, the anti-RSP03 antibody is 131R010.

[032] In some embodiments, the RSPO-binding agent is a monoclonal antibody. In some

embodiments, the RSPO-binding agent is a recombinant antibody. In some embodiments, the RSPO- binding agent is a chimeric antibody. In some embodiments, the RSPO-binding agent is a humanized antibody. In some embodiments, the RSPO-binding agent is a human antibody. In some embodiments, the RSPO-binding agent is an IgGl antibody. In some embodiments, the RSPO-binding agent is an IgG2 antibody. In some embodiments, the RSPO-binding agent is a bispecific antibody. In some embodiments, the RSPO-binding agent is a monovalent antibody.

[033] In some embodiments of the methods described herein, a method comprises a RSPO 1 -binding agent that competes for specific binding to human RSPO 1 with an anti-RSPO 1 antibody of the invention. In some embodiments, the RSPO 1 -binding agent competes for specific binding to human RSPO 1 with an antibody that comprises a heavy chain variable region comprising SEQ ID NO: 1 1 and a light chain variable region comprising SEQ ID NO: 12. In some embodiments, the RSPO 1 -binding agent competes for specific binding to human RSPO 1 with an antibody that comprises a heavy chain variable region comprising SEQ ID NO:56 and a light chain variable region comprising SEQ ID NO:57. In some embodiments, the antibody with which the RSPO 1 -binding agent competes is an antibody comprising the CDRs of antibody 89M5. In some embodiments, the RSPO 1 -binding agent competes for specific binding to RSPO 1 with an anti-RSPO 1 antibody of the invention in an in vitro competitive binding assay.

[034] In certain embodiments, the RSPO 1 -binding agent is an antibody that binds the same epitope, or essentially the same epitope, on RSPOl as an anti-RSPO 1 antibody of the invention (e.g., 89M5, h89M5- H8L5, or h89M5-H2L2). In some embodiments, the RSPO 1 -binding agent is an antibody that binds an epitope on RSPO 1 that overlaps with the epitope on RSPO 1 bound by an anti-RSPO 1 antibody of the invention (e.g., 89M5, h89M5-H8L5, or h89M5-H2L2).

[035] In some embodiments of the methods described herein, a method comprises a RSP02-binding agent that competes for specific binding to human RSP02 with an anti-RSP02 antibody of the invention. In some embodiments, the RSP02-binding agent competes for specific binding to human RSP02 with an antibody that comprises a heavy chain variable region comprising SEQ ID NO:27 and a light chain variable region comprising SEQ ID NO:28. In some embodiments, the RSP02-binding agent competes for specific binding to human RSP02 with an antibody that comprises a heavy chain variable region comprising SEQ ID NO:27 and a light chain variable region comprising SEQ ID NO:66. In some embodiments, the antibody with which the RSP02-binding agent competes is an antibody comprising the CDRs of antibody 130M23. In some embodiments, the RSP02-binding agent competes for specific binding to RSP02 with an anti-RSP02 antibody of the invention in an in vitro competitive binding assay.

[036] In certain embodiments, the RSP02-binding agent is an antibody that binds the same epitope, or essentially the same epitope, on RSP02 as an anti-RSP02 antibody of the invention (e.g., 130M23, hl30M23-HlL6, or hl30M23-HlL2). In some embodiments, the RSP02-binding agent is an antibody that binds an epitope on RSP02 that overlaps with the epitope on RSP02 bound by an anti-RSP02 antibody of the invention (e.g., 130M23, hl30M23-HlL6, hl30M23-HlL2). [037] In some embodiments of the methods described herein, a method comprises a RSP03-binding agent that competes for specific binding to human RSP03 with an anti-RSP03 antibody of the invention. In some embodiments, the RSP03 -binding agent competes for specific binding to human RSP03 with an antibody that comprises a heavy chain variable region comprising SEQ ID NO:46 and a light chain variable region comprising SEQ ID NO:47. In some embodiments, the antibody with which the RSP03- binding agent competes is an antibody comprising the CDRs of antibody 131R010. In some

embodiments, the RSP03 -binding agent competes for specific binding to RSP03 with an anti-RSP03 antibody of the invention in an in vitro competitive binding assay.

[038] In certain embodiments, the RSP03-binding agent is an antibody that binds the same epitope, or essentially the same epitope, on RSP03 as an anti-RSP03 antibody of the invention (e.g., 131R010). In some embodiments, the RSP03-binding agent is an antibody that binds an epitope on RSP03 that overlaps with the epitope on RSP03 bound by an anti-RSP03 antibody of the invention (e.g., 131R010).

[039] In certain embodiments of any of the methods described herein, the RSPO-binding agent or antibody is isolated. In some embodiments, the RSPO-binding agent or antibody is substantially pure.

[040] In some embodiments, the RSPO or LGR antagonist is an antibody that specifically binds at least one human LGR protein or a portion thereof. In some embodiments, the antibody specifically binds at least one human LGR protein selected from the group consisting of LGR4, LGR5, and LGR6. In some embodiments, the antibody specifically binds human LGR5. In certain embodiments, the antibody comprises (a) the heavy chain CDRl, CDR2, and CDR3 sequences of the monoclonal antibody produced by the 88M1 hybridoma having the ATCC deposit number PTA-9342; and (b) the light chain CDRl, CDR2, and CDR3 sequences of the monoclonal antibody produced by the 88M1 hybridoma having the ATCC deposit number PTA-9342. In some embodiments, the antibody comprises the heavy chain variable region and light chain variable region of the monoclonal antibody produced by the 88M1 hybridoma having the ATCC deposit number PTA-9342.

[041] In some embodiments, the RSPO or LGR antagonist is a soluble receptor including an extracellular domain of a human LGR protein (e.g., LGR5) or a fragment thereof, where the extracellular domain is capable of binding a human RSPO protein. In some embodiments, the extracellular domain of a human LGR protein comprises amino acids 22-564 of human LGR5 (SEQ ID NO:74). In some embodiments, a soluble receptor comprises a non-LGR polypeptide. In some embodiments, the non-LGR polypeptide is directly linked to the extracellular domain of the human LGR protein or is connected to the extracellular domain of the human LGR protein by a linker. In some embodiments, the non-LGR polypeptide comprises a human Fc region (e.g., comprises SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, or SEQ ID NO:80, or an amino acid sequence at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, or SEQ ID NO:80).

[042] In another aspect, the invention provides a method of inhibiting Wnt signaling in a cell, comprising contacting the cell with an effective amount of a RSPO-binding agent or a LGR-binding agent, including each of those described herein. In some embodiments, the cell expresses elevated levels of at least one RSPO protein. In some embodiments, the cell expresses elevated levels of RSPO 1. In some embodiments, the cell expresses elevated levels of RSP02. In some embodiments, the cell expresses elevated levels of RSP03. In some embodiments, the cell expresses a high level of at least one RSPO protein. In some embodiments, the cell expresses a high level of RSPO 1. In some embodiments, the cell expresses a high level of RSP02. In some embodiments, the cell expresses a high level of RSP03. In certain embodiments, the RSPO-binding agent inhibits formation of fibrotic tissue. In certain embodiments, the LGR-binding agent inhibits formation of fibrotic tissue.

[043] In certain embodiments of any of the methods described herein, the method further comprises a step of determining the expression level of at least one RSPO protein in a cell. In some embodiments, the cell is from a fibrotic tissue.

[044] In certain embodiments of any of the methods described herein, the method comprises administering to the subject a RSPO-binding agent and at least one additional therapeutic agent. In certain embodiments of any of the methods described herein, the method comprises administering to the subject a LGR-binding agent and at least one additional therapeutic agent.

[045] Compositions comprising a RSPO-binding agent (e.g., antibody or soluble receptor) or a LGR- binding agent described herein are provided. Pharmaceutical compositions comprising a RSPO-binding agent (e.g., antibody or soluble receptor) or a LGR-binding agent described herein and a pharmaceutically acceptable carrier are provided. Methods of treating fibrotic diseases in a subject (e.g., a human) comprising administering to the subject an effective amount of a pharmaceutical composition comprising a RSPO-binding agent or LGR-binding agent are also provided. Use of a RSPO-binding agent and/or a LGR-binding agent for treatment of a fibrotic disease is also provided.

[046] 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

[047] Figure 1. RSPO expression in bleomycin-induced lung fibrosis model.

[048] Figure 2. RSPO expression in bleomycin-induced dermal fibrosis model.

[049] Figure 3. Inhibition of bleomycin-induced dermal lesions by RSPO antibodies.

[050] Figure 4. Gene expression in tissue from bleomycin-induced dermal lesions. Tissue samples were taken from Day 12.

[051] Figure 5. Gene expression of interferon gamma from bleomycin-induced dermal lesions. Tissue samples were taken 24 hours after administration of a single dose of bleomycin and a single dose of anti- RSPO antibodies.

DETAILED DESCRIPTION OF THE INVENTION

[052] The present invention provides novel methods from treating fibrotic diseases and for reducing scarring resulting from wound healing. The invention provides methods of treating or preventing a fibrotic disease in a subject, comprising administering to the subject a therapeutically effective amount of a human RSPO or LGR antagonist. The methods comprise administering RSPO-binding agents and/or LGR-binding agents, particularly anti-RSPO l antibodies, anti-RSP02 antibodies, anti-RSP03 antibodies, anti-LGR5 antibodies, or a LGR5-Fc soluble receptor, to a subject in need thereof. The RSPO-binding agents and LGR-binding agents include, but are not limited to, inhibitors of RSPO and LGR protein interactions.

I. Definitions

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

[054] 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.

[055] 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. 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., a RSPO protein or LGR protein). Suitable antagonist molecules specifically include, but are not limited to, antagonist antibodies or antibody fragments.

[056] The terms "modulation" and "modulate" as used herein refer to a change or an alteration in a biological activity. Modulation includes, but is not limited to, stimulating or inhibiting an activity.

Modulation may be an increase or a decrease in activity, a change in binding characteristics, or any other change in the biological, functional, or immunological properties associated with the activity of a protein, pathway, or other biological point of interest.

[057] 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(s) of the immunoglobulin molecule. As used herein, the term encompasses intact polyclonal antibodies, intact monoclonal antibodies, single chain antibodies, antibody fragments (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 recognition site (i.e., 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.

[058] The term "antibody fragment" refers to a portion of an intact antibody and refers to the antigenic determining variable regions of an intact antibody. Examples of 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 an antigen-binding site or epitope-binding site.

[059] The term "variable region" of an antibody refers to the variable region of an antibody light chain or the variable region of an antibody heavy chain, either alone or in combination. The variable region of each heavy and light chain consists of four framework regions (FR) connected by three complementarity determining regions (CDRs), also known as "hypervariable regions". The CDRs in each chain are held together in close proximity by the framework regions and, with the CDRs from the other chain, 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 may be used in the art to determine CDRs.

[060] The term "monoclonal antibody" as used herein refers to a homogeneous 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 a variety of different antigenic determinants. The term "monoclonal antibody" encompasses both intact and full-length monoclonal antibodies as well as antibody fragments (e.g., Fab, Fab', F(ab')2, Fv), single chain (scFv) antibodies, bispecific antibodies, fusion proteins comprising an antibody portion, and any other modified immunoglobulin molecule comprising an antigen recognition site (antigen-binding site). Furthermore, "monoclonal antibody" refers to such antibodies made by any number of techniques, including but not limited to, hybridoma production, phage selection, recombinant expression, and transgenic animals.

[061] The term "humanized antibody" as used herein refers to forms of 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. In some instances, the framework region amino acid residues of a human immunoglobulin are replaced with the corresponding amino acid residues in an antibody from a non-human species. The humanized antibody can be further modified by the substitution of additional amino acid residues either in the framework region and/or within the replaced non-human amino acid residues to refine and optimize antibody specificity, affinity, structural, and/or binding capability. In general, a humanized antibody will comprise substantially all of at least one, and typically two or three 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. A humanized antibody can also comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin.

[062] 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. A human antibody may be made using any of the techniques known in the art. [063] 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 the light chain and heavy chain corresponds to the variable region of an antibody derived from one species of mammal (e.g., mouse, rat, rabbit, etc.) with the desired specificity, affinity, and/or binding capability, while the constant region corresponds to sequences in antibodies derived from another species (usually human).

[064] The phrase "affinity-matured antibody" as used herein refers to an antibody with one or more alterations in one or more CDRs thereof that result in an improvement in the affinity of the antibody for an antigen, compared to a parent antibody that does not possess those alterations(s). The definition also includes alterations in non-CDR amino acid residues made in conjunction with alterations to CDR amino acid residues. Preferred affinity-matured antibodies generally have nanomolar or even picomolar affinities for the target antigen. Affinity-matured antibodies are produced by procedures known in the art including, but not limited to, heavy chain and light chain variable domain shuffling, random mutagenesis of CDR and/or framework amino acid residues, or site-directed mutagenesis of CDR and/or framework amino acid residues.

[065] 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

noncontiguous 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.

[066] The terms "selectively binds" or "specifically binds" 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 protein with a K _D of about 0. lmM or less, but more usually less than about 1 μΜ. In certain embodiments, "specifically binds" means that an antibody binds a target at times with a K _D of at least about 0.1 μΜ or less, at other times at least about 0.01 μΜ or less, and at other times 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 RSP03 and mouse RSP03). 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 RSP03 and human RSPO 1). It is understood that, in certain embodiments, an antibody or binding moiety that specifically binds a first target may or may not specifically bind a second target. As such, "specific binding" does not necessarily require (although it can include) exclusive binding, i.e. binding to a single target. Thus, an antibody may, in certain embodiments, specifically bind more than one target. In certain embodiments, multiple targets may be bound by the same antigen-binding site on the antibody. For example, an antibody may, in certain instances, comprise two identical antigen-binding sites, each of which specifically binds the same epitope on two or more proteins (e.g., RSP03 and RSPOl). In certain alternative embodiments, an antibody may be multispecific and comprise at least two antigen-binding sites with differing specificities. By way of non-limiting example, a bispecific antibody may comprise one antigen-binding site that recognizes an epitope on one protein (e.g., human RSP03) 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.

[067] The terms "polypeptide" and "peptide" and "protein" are used interchangeably herein and refer to polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond 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 of this invention may be based upon antibodies, in certain embodiments, the polypeptides can occur as single chains or associated chains.

[068] 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.

[069] "Conditions of high stringency" may be identified by those that: (1) employ low ionic strength and high temperature for washing, for example 15mM NaCl/1.5mM sodium citrate/0.1% sodium dodecyl sulfate at 50°C; (2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0.1%> bovine serum albumin/0.1%) Ficoh70.1%> polyvinylpyrrolidone/50mM sodium phosphate buffer at pH 6.5 in 5x SSC (0.75M NaCl, 75mM sodium citrate) at 42°C; or (3) employ during hybridization 50% formamide in 5x SSC, 50mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5x Denhardt's solution, sonicated salmon sperm DNA (50μg/ml), 0.1 % SDS, and 10%> dextran sulfate at 42°C, with washes at 42°C in 0.2x SSC and 50%> formamide, followed by a high- stringency wash consisting of 0.1 x SSC containing EDTA at 55°C.

[070] 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 may be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that may be used to obtain alignments of amino acid or nucleotide sequences are well-known in the art. These include, but are not limited to, BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin Package, and variations thereof. In some embodiments, two nucleic acids or polypeptides of the invention 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 residues, at least about 60-80 residues in length or any integral value therebetween. In some embodiments, identity exists over a longer region than 60-80 residues, such as at least about 80-100 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.

[071] A "conservative amino acid substitution" is one 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), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For example, substitution of a phenylalanine for a tyrosine is a conservative substitution. Preferably, conservative substitutions in the sequences of the polypeptides and antibodies of the invention do not abrogate the binding of the polypeptide or antibody containing the amino acid sequence, to the antigen(s), i.e., the one or more RSPO protein(s) to which the polypeptide or antibody binds. Methods of identifying nucleotide and amino acid conservative substitutions which do not eliminate antigen binding are well-known in the art.

[072] 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.

[073] 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.

[074] 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.

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

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

[077] The terms "pharmaceutically acceptable excipient, carrier or adjuvant" or "acceptable pharmaceutical carrier" refer to an excipient, carrier or adjuvant that can be administered to a subject, together with at least one binding agent (e.g., an antibody) of the present disclosure, and which does not destroy the activity of the binding agent. The excipient, carrier, or adjuvant should be non-toxic when administered with a binding agent in doses sufficient to deliver a therapeutic effect. In general, those of skill in the art and the U.S. FDA consider a pharmaceutically acceptable excipient, carrier, or adjuvant to be an inactive ingredient of any formulation.

[078] The terms "effective amount" or "therapeutically effective amount" or "therapeutic effect" refer to an amount of a binding agent, an antibody, polypeptide, polynucleotide, small organic molecule, or other drug effective to "treat" a disease or disorder in a subject or mammal. 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.

[079] The terms "treating" or "treatment" or "to treat" or "alleviating" or "to alleviate" refer to both 1) therapeutic measures that cure, slow down, lessen symptoms of, and/or halt progression of a diagnosed pathologic condition or disorder and 2) prophylactic or preventative measures that prevent or slow the development of a targeted pathologic condition or disorder. Thus those in need of treatment include those already with the disorder; those prone to have the disorder; and those in whom the disorder is to be prevented. In some embodiments, a subject is successfully "treated" according to the methods of the 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.

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

[081] 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.

[082] 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. Methods of use and pharmaceutical compositions

[083] The invention provides methods for the prevention and/or treatment of fibrotic diseases. In some embodiments, a method comprises using RSPO-binding agents and/or LGR-binding agents (e.g., antibodies or soluble receptors) as described herein. In certain embodiments, a RSPO-binding agent is an antagonist of human RSPOl, RSP02, RSP03, and/or RSP04. In certain embodiments, a LGR-binding agent is an antagonist of human LGR4, LGR5, and/or LGR6. The methods may be in vitro, ex vivo, or in vivo methods. The invention also provides the use of a RSPO-binding agent or a LGR-binding agent (e.g., an antibody or a soluble receptor) described herein for the manufacture of a medicament for the treatment of a fibrotic disease.

[084] The present invention provides methods for treating or preventing a fibrotic disease using the RSPO-binding agents or LGR-binding agents (e.g., antibodies or soluble receptors) described herein. In certain embodiments, a method of treating a fibrotic disease comprises contacting a cell or a tissue with a RSPO-binding agent or a LGR-binding agent (e.g., an antibody or a soluble receptor) in vitro. For example, fibroblasts, either a cell line or primary cells from an organ of interest, are contacted with an agent of interest (e.g., a RSPO-binding agent or LGR-binding agent). Affects from the agent of the interest can be observed by evaluating myofibroblast/mesenchymal differentiation, including but not limited to up-regulation of alpha-smooth muscle actin, transforming growth factor best 1, and/or fibronectin; extracellular matrix deposition; and collagen/hydroxyproline content.

[085] In certain embodiments, a method of treating a fibrotic disease comprises contacting a cell or a tissue with a RSPO-binding agent or a LGR-binding agent (e.g., an antibody or a soluble receptor) in vivo. In certain embodiments, contacting a cell or a tissue with a RSPO-binding agent or LGR-binding agent is undertaken in an animal model. For example, a RSPO-binding agent may be administered to mice which have a fibrotic disease. In some embodiments, a RSPO-binding agent may be administered to a transgenic mouse. In some embodiments, a RSPO-binding agent is administered to the animal. In some embodiments, the RSPO-binding agent is administered at the same time or shortly after administration of a fibrosis-inducing agent in the animal to prevent development of fibrosis ("preventative model"). In some embodiments, the RSPO-binding agent is administered as a therapeutic after fibrosis has been induced ("therapeutic model"). In some embodiments, the RSPO-binding agent is an antibody. In some embodiments, the RSPO-binding agent is an anti-RSPO l antibody. In some embodiments, the RSPO- binding agent is an anti-RSP02 antibody. In some embodiments, the RSPO-binding agent is an anti- RSP03 antibody. In some embodiments, the RSPO-binding agent is a soluble receptor. In some embodiments, the RSPO-binding agent is a LGR5 soluble receptor. In some embodiments, the RSPO- binding is a LGR5-Fc fusion protein.

[086] Models for inducing fibrosis in animals are known to those of skill in the art. For example, pulmonary fibrosis can be induced in mice by intraperitoneal injection with bleomycin. Pulmonary fibrosis can also be induced by intratracheal instillation of bleomycin, fluorescein isothiocyanate or particulate matter, such as silica and asbestos. Liver fibrosis can be induced in mice by intraperitoneal injection with carbon tetrachloride. Dermal fibrosis can be induced in mice by local injection of bleomycin.

[087] In certain embodiments, a method of treating or preventing a fibrotic disease comprises administering to a subject a therapeutically effective amount of a RSPO-binding agent. In certain embodiments, a method of treating or preventing a fibrotic disease comprises administering to a subject a therapeutically effective amount of an anti-RSPO antibody. In certain embodiments, a method of treating or preventing a fibrotic disease comprises administering to a subject a therapeutically effective amount of a LGR soluble receptor. In certain embodiments, a method of treating or preventing a fibrotic disease comprises administering to a subject a therapeutically effective amount of a LGR-binding agent. In certain embodiments, a method of treating or preventing a fibrotic disease comprises administering to a subject a therapeutically effective amount of an anti-LGR antibody.

[088] In certain embodiments, a method of treating or preventing a fibrotic disease comprises administering to a subject a therapeutically effective amount of an anti-RSPO 1 antibody. In certain embodiments, a method of treating or preventing a fibrotic disease comprises administering to a subject a therapeutically effective amount of an anti-RSPO 1 antibody, wherein the anti-RSPO 1 antibody comprises a heavy chain CDR1 comprising TGYTMH (SEQ ID NO:5), a heavy chain CDR2 comprising

GINPNNGGTTYNQNFKG (SEQ ID NO: 6), and a heavy chain CDR3 comprising KEFSDGYYFFAY (SEQ ID NO:7); and a light chain CDR1 comprising KASQDVIFAVA (SEQ ID NO:8), a light chain CDR2 comprising WASTRHT (SEQ ID NO:9), and a light chain CDR3 comprising QQHYSTPW (SEQ ID NO: 10). In some embodiments, a method of treating or preventing a fibrotic disease comprises administering to a subject a therapeutically effective amount of an anti-RSPO 1 antibody, wherein the anti-RSPO 1 antibody comprises a heavy chain variable region having at least 90% sequence identity to SEQ ID NO: 1 1 and a light chain variable region having at least 90%> sequence identity to SEQ ID NO: 12. In some embodiments, a method of treating or preventing a fibrotic disease comprises administering to a subject a therapeutically effective amount of an anti-RSPO 1 antibody, wherein the anti-RSPO 1 antibody comprises a heavy chain variable region having at least 90%> sequence identity to SEQ ID NO:56 and a light chain variable region having at least 90%> sequence identity to SEQ ID NO:57. In some

embodiments, a method of treating or preventing a fibrotic disease comprises administering to a subject a therapeutically effective amount of an anti-RSPO 1 antibody, wherein the anti-RSPO 1 antibody comprises a heavy chain variable region of SEQ ID NO: 1 1 and a light chain variable region of SEQ ID NO: 12. In some embodiments, a method of treating or preventing a fibrotic disease comprises administering to a subject a therapeutically effective amount of an anti-RSPO 1 antibody, wherein the anti-RSPO 1 antibody comprises a heavy chain variable region of SEQ ID NO:56 and a light chain variable region of SEQ ID NO:57. In some embodiments, the anti-RSPOl antibody is a humanized version of antibody 89M5. In some embodiments, the anti-RSPOl antibody is antibody h89M5-H8L5. In some embodiments, the anti- RSPO l antibody is antibody h89M5-H2L2.

[089] In certain embodiments, a method of treating or preventing a fibrotic disease comprises administering to a subject a therapeutically effective amount of an anti-RSP02 antibody. In certain embodiments, a method of treating or preventing a fibrotic disease comprises administering to a subject a therapeutically effective amount of an anti-RSP02 antibody, wherein the anti-RSP02 antibody comprises a heavy chain CDRl comprising SSYAMS (SEQ ID NO:21), a heavy chain CDR2 comprising

SISSGGSTYYPDSVKG (SEQ ID NO:22), and a heavy chain CDR3 comprising

RGGDPGVYNGDYEDAMDY (SEQ ID NO:23); and a light chain CDRl comprising KASQDVSSAVA (SEQ ID NO:24), a light chain CDR2 comprising WASTRHT (SEQ ID NO:25), and a light chain CDR3 comprising QQHYSTP (SEQ ID NO:26). In some embodiments, a method of treating or preventing a fibrotic disease comprises administering to a subject a therapeutically effective amount of an anti-RSP02 antibody, wherein the anti-RSP02 antibody comprises a heavy chain variable region having at least 90% sequence identity to SEQ ID NO:27 and a light chain variable region having at least 90% sequence identity to SEQ ID NO:28 or SEQ ID NO:66. In some embodiments, a method of treating or preventing a fibrotic disease comprises administering to a subject a therapeutically effective amount of an anti-RSP02 antibody, wherein the anti-RSP02 antibody comprises a heavy chain variable region of SEQ ID NO:27 and a light chain variable region of SEQ ID NO:28 or SEQ ID NO:66. In some embodiments, the anti- RSP02 antibody is a humanized version of antibody 130M23. In some embodiments, the anti-RSP02 antibody is antibody hl30M23-HlL6. In some embodiments, the anti-RSP02 antibody is antibody hl30M23-HlL2.

[090] In certain embodiments, a method of treating or preventing a fibrotic disease comprises administering to a subject a therapeutically effective amount of an anti-RSP03 antibody. In certain embodiments, a method of treating or preventing a fibrotic disease comprises administering to a subject a therapeutically effective amount of an anti-RSP03 antibody, wherein the anti-RSP03 antibody comprises a heavy chain CDRl comprising DYSIH (SEQ ID NO:37), a heavy chain CDR2 comprising

YIYPSNGDSGYNQKFK (SQ ID NO:38), and a heavy chain CDR3 comprising TYFANNFD (SEQ ID NO:39) or ATYFANNFDY (SEQ ID NO:40); and a light chain CDRl comprising

KASQSVDYDGDSYMN (SEQ ID NO:41), a light chain CDR2 comprising AASNLES (SEQ ID NO:42) or AAS (SEQ ID NO:43), and a light chain CDR3 comprising QQSNEDPLT (SEQ ID NO:44) or QQSNEDPLTF (SEQ ID NO:45). In some embodiments, a method of treating or preventing a fibrotic disease comprises administering to a subject a therapeutically effective amount of an anti-RSP03 antibody, wherein the anti-RSP03 antibody comprises a heavy chain variable region having at least 90% sequence identity to SEQ ID NO:46 and a light chain variable region having at least 90% sequence identity to SEQ ID NO:47. In some embodiments, a method of treating or preventing a fibrotic disease comprises administering to a subject a therapeutically effective amount of an anti-RSP03 antibody, wherein the anti-RSP03 antibody comprises a heavy chain variable region of SEQ ID NO:46 and a light chain variable region of SEQ ID NO:47. In some embodiments, the anti-RSP03 antibody is antibody 131R010.

[091] In certain embodiments, a method of treating or preventing a fibrotic disease comprises administering to a subject a therapeutically effective amount of an anti-LGR antibody. In certain embodiments, a method of treating or preventing a fibrotic disease comprises administering to a subject a therapeutically effective amount of an anti-LGR5 antibody, wherein the anti-LGR5 antibody comprises the heavy chain CDR1, CDR2, and CDR3 sequences of the monoclonal antibody produced by the 88M1 hybridoma having the ATCC deposit number PTA-9342, and the light chain CDR1, CDR2, and CDR3 sequences of the monoclonal antibody produced by the 88M1 hybridoma having the ATCC deposit number PTA-9342. In certain embodiments, a method of treating or preventing a fibrotic disease comprises administering to a subject a therapeutically effective amount of an anti-LGR5 antibody, wherein the anti-LGR5 antibody comprises the heavy chain variable region and light chain variable region of the monoclonal antibody produced by the 88M1 hybridoma having the ATCC deposit number PTA-9342.

[092] In certain embodiments, a method of treating or preventing a fibrotic disease comprises administering to a subject a therapeutically effective amount of a LGR soluble receptor. In certain embodiments, a method of treating or preventing a fibrotic disease comprises administering to a subject a therapeutically effective amount of a LGR5 soluble receptor, wherein the LGR5 soluble receptor comprises the extracellular domain, or a fragment thereof, of LGR5. In some embodiments, a method of treating or preventing a fibrotic disease comprises administering to a subject a therapeutically effective amount of a LGR5 soluble receptor, wherein the LGR5 soluble receptor comprises amino acids 22-564 of human LGR5. In certain embodiments, a method of treating or preventing a fibrotic disease comprises administering to a subject a therapeutically effective amount of a LGR5 soluble receptor, wherein the LGR5 soluble receptor comprises SEQ ID NO:74. In certain embodiments, a method of treating or preventing a fibrotic disease comprises administering to a subject a therapeutically effective amount of a LGR5 soluble receptor, wherein the LGR5 soluble receptor comprises SEQ ID NO: 75.

[093] In some embodiments of the methods described herein, the RSPO-binding agent binds RSPOl and prevents the development of a fibrotic disease. In some embodiments, the RSPO-binding agent binds RSPO l and slows down the development of a fibrotic disease. In some embodiments, the RSPO-binding agent binds RSPOl and slows down the progression of a fibrotic disease. In some embodiments, the RSPO-binding agent binds RSPOl and reduces the amount of fibrosis in a disease. In some

embodiments, the RSPO-binding agent binds RSPO 1 and reduces pathological deposits of fibrotic material in an organ. In some embodiments, the RSPO-binding agent binds RSPO 1 and reduces the pathological deposits of connective tissue and/or extracellular matrix in an organ. In some embodiments, the RSPO-binding agent is an anti-RSPOl antibody. In some embodiments, the anti-RSPOl antibody is a humanized version of antibody 89M5. In some embodiments, the anti-RSPOl antibody is h89M5-H8L5. In some embodiments, the anti-RSPOl antibody is h89M5-H2L2.

[094] In some embodiments of the methods described herein, the RSPO-binding agent binds RSP02 and prevents the development of a fibrotic disease. In some embodiments, the RSPO-binding agent binds RSP02 and slows down the development of a fibrotic disease. In some embodiments, the RSPO-binding agent binds RSP02 and slows down the progression of a fibrotic disease. In some embodiments, the RSPO-binding agent binds RSP02 and reduces the amount of fibrosis in a disease. In some

embodiments, the RSPO-binding agent binds RSP02 and reduces pathological deposits of fibrotic material in an organ. In some embodiments, the RSPO-binding agent binds RSP02 and reduces the pathological deposits of connective tissue and/or extracellular matrix in an organ. In some embodiments, the RSPO-binding agent is an anti-RSP02 antibody. In some embodiments, the anti-RSP02 antibody is a humanized version of antibody 130M23. In some embodiments, the anti-RSP02 antibody is hl30M23- H1L6. In some embodiments, the anti-RSP02 antibody is hl30M23-HlL2.

[095] In some embodiments of the methods described herein, the RSPO-binding agent binds RSP03 and prevents the development of a fibrotic disease. In some embodiments, the RSPO-binding agent binds RSP03 and slows down the development of a fibrotic disease. In some embodiments, the RSPO-binding agent binds RSP03 and slows down the progression of a fibrotic disease. In some embodiments, the RSPO-binding agent binds RSP03 and reduces the amount of fibrosis in a disease. In some

embodiments, the RSPO-binding agent binds RSP03 and reduces pathological deposits of fibrotic material in an organ. In some embodiments, the RSPO-binding agent binds RSP03 and reduces the pathological deposits of connective tissue and/or extracellular matrix in an organ. In some embodiments, the RSPO-binding agent is an anti-RSP03 antibody. In some embodiments, the anti-RSP03 antibody is 131R010.

[096] In some embodiments of the methods described herein, the RSPO-binding agent binds RSPOl, RSP02, RSP03, and/or RSP04 and prevents the development of a fibrotic disease. In some

embodiments, the RSPO-binding agent binds RSPOl, RSP02, RSP03, and/or RSP04 and slows down the development of a fibrotic disease. In some embodiments, the RSPO-binding agent binds RSPOl, RSP02, RSP03, and/or RSP04 and slows down the progression of a fibrotic disease. In some embodiments, the RSPO-binding agent binds RSPOl, RSP02, RSP03, and/or RSP04 and reduces the amount of fibrosis in a disease. In some embodiments, the RSPO-binding agent binds RSPOl, RSP02, RSP03, and/or RSP04 and reduces pathological deposits of fibrotic material in an organ. In some embodiments, the RSPO-binding agent binds RSPOl, RSP02, RSP03, and/or RSP04 and reduces the pathological deposits of connective tissue and/or extracellular matrix in an organ. In some embodiments, the RSPO-binding agent is a LGR soluble receptor. In some embodiments, the LGR soluble receptor is a LGR5 soluble receptor. In some embodiments, the LGR5 soluble receptor comprises a Fc region. In some embodiments, the LGR soluble receptor is the LGR5-Fc fusion protein comprising SEQ ID NO:72.

[097] 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.

[098] In certain embodiments, a method further comprises a step of determining the expression level of at least one RSPO (i.e., protein or nucleic acid) in a tissue. In some embodiments, the step of determining the expression level of a RSPO in a tissue comprises determining the expression level of RSPO 1 , RSP02, RSP03, and/or RSP04. In some embodiments, the expression level of RSPOl, RSP02, RSP03, and/or RSP04 in a tissue sample (e.g., fibrotic tissue) is compared to the expression level of RSPO l, RSP02, RSP03, and/or RSP04 in a reference sample. In some embodiments, the expression level of RSPOl, RSP02, RSP03, and/or RSP04 in a tissue sample is compared to the expression level of RSPOl, RSP02, RSP03, and/or RSP04 in normal tissue. In some embodiments, the level of expression of RSPO 1, RSP02, RSP03, and/or RSP04 in a tissue sample is compared to a pre-determined level of expression of RSPO l, RSP02, RSP03, and/or RSP04. In some embodiments, the level of expression of RSPO 1, RSP02, RSP03, and/or RSP04 in a tissue sample is compared to a pre-determined level of expression of RSPO l, RSP02, RSP03, and/or RSP04 in normal tissue. In some embodiments, the tissue has a high expression level of RSPOl . In some embodiments, the tissue has a high expression level of RSP02. In some embodiments, the tissue has a high expression level of RSP03. In general, the expression level of a RSPO (i.e., protein or nucleic acid) in a tissue sample is compared to the expression level of the RSPO (i.e., protein or nucleic acid) in normal tissue of the same tissue type. However, in some embodiments, the expression level of a RSPO (i.e., protein or nucleic acid) is compared to the average expression level of the RSPO (i.e., protein or nucleic acid) within a group of tissue types. In some embodiments, the expression level of a RSPO (i.e., protein or nucleic acid) in a tissue sample is compared to the expression level of the RSPO (i.e., protein or nucleic acid) in other sample of tissue of the same tissue type or a different tissue type.

[099] In some embodiments, determining the level of RSPO expression is done prior to treatment. In some embodiments, the subject is administered a RSPO-binding agent or LGR-binding agent (e.g., an antibody or soluble receptor) describe herein if the tissue sample has an elevated expression level of RSPO as compared to the expression level of the same RSPO in a reference sample (e.g., normal tissue) or a pre-determined level. For example, in some embodiments, the subject is administered a RSP03- binding agent (e.g., anti-RSP03 antibody) if the tissue sample has an elevated expression level of RSP03 (i.e., protein or nucleic acid) as compared to the expression level of RSP03 in normal or control tissue.

[0100] In addition, the present invention provides methods of identifying a human subject for treatment with a RSPO-binding agent or LGR-binding agent, comprising determining if the subject has tissue sample (e.g., fibrotic tissue) that has an elevated expression level of RSPO (i.e., protein or nucleic acid) as compared to expression of the same RSPO (i.e., protein or nucleic acid) in normal tissue, in a reference sample, or to a pre-determined level of the RSPO protein.

[0101] In some embodiments, a method of identifying a human subject for treatment with a RSPO 1 - binding agent comprises determining if the subject has fibrotic tissue that has an elevated expression level of RSPO 1 as compared to a reference sample or a pre-determined level of RSPO 1. In some

embodiments, a method of identifying a human subject for treatment with a RSPO 1 -binding agent comprises obtaining a sample of fibrotic tissue from the subject, and determining if the fibrotic tissue has an elevated expression level of RSPOl as compared to a reference sample or a pre-determined level of RSPO l. In some embodiments, if the fibrotic tissue has an elevated expression level of RSPOl, the subj ect is selected for treatment with an antibody that specifically binds RSPO 1. In some embodiments, if selected for treatment, the subject is administered an anti-RSPOl antibody described herein. In some embodiments, if the fibrotic tissue has an elevated expression level of RSPO 1, the subject is selected for treatment with a LGR soluble receptor that specifically binds RSPO 1. In some embodiments, if selected for treatment, the subject is administered a LGR soluble receptor described herein.

[0102] In some embodiments, a method of identifying a human subject for treatment with a RSP02- binding agent comprises determining if the subject has fibrotic tissue that has an elevated expression level of RSP02 as compared to a reference sample or a pre-determined level of RSP02. In some

embodiments, a method of identifying a human subject for treatment with a RSP02-binding agent comprises obtaining a sample of fibrotic tissue from the subject, and determining if the fibrotic tissue has an elevated expression level of RSP02 as compared to a reference sample or a pre-determined level of RSP02. In some embodiments, if the fibrotic tissue has an elevated expression level of RSP02, the subject is selected for treatment with an antibody that specifically binds RSP02. In some embodiments, if selected for treatment, the subject is administered an anti-RSP02 antibody described herein. In some embodiments, if the fibrotic tissue has an elevated expression level of RSP02, the subject is selected for treatment with a LGR soluble receptor that specifically binds RSP02. In some embodiments, if selected for treatment, the subject is administered a LGR soluble receptor described herein.

[0103] In some embodiments, a method of identifying a human subject for treatment with a RSP03- binding agent comprises determining if the subject has fibrotic tissue that has an elevated expression level of RSP03 as compared to a reference sample or a pre-determined level of RSP03. In some

embodiments, a method of identifying a human subject for treatment with a RSP03 -binding agent comprises obtaining a sample of fibrotic tissue from the subject, and determining if the fibrotic tissue has an elevated expression level of RSP03 as compared to a reference sample or a pre-determined level of RSP03. In some embodiments, if the fibrotic tissue has an elevated expression level of RSP03, the subject is selected for treatment with an antibody that specifically binds RSP03. In some embodiments, if selected for treatment, the subject is administered an anti-RSP03 antibody described herein. In some embodiments, if the fibrotic tissue has an elevated expression level of RSP03, the subject is selected for treatment with a LGR soluble receptor that specifically binds RSP03. In some embodiments, if selected for treatment, the subject is administered a LGR soluble receptor described herein.

[0104] In some embodiments, if the fibrotic tissue has an elevated expression level of more than one RSPO (i.e., protein or nucleic acid), the subject is administered a RSPO-binding agent that binds the RSPO with the highest level of expression. In some embodiments, the expression level of RSPO 1, RSP02, RSP03, and/or RSP04 in the fibrotic tissue is determined, if the fibrotic tissue has an elevated level of RSPOl expression as compared to the level of RSPO l in normal tissue, the subject is selected for treatment with an antibody that specifically binds RSPOl . If selected for treatment, the subject is administered an anti-RSPOl antibody describe herein. In some embodiments, the RSPO 1 -binding agent is a humanized version of antibody 89M5. In some embodiments, the RSPO 1 -binding agent is antibody h89M5-H8L5. In some embodiments, the RSPO l-binding agent is antibody h89M5-H2L3. In some embodiments, the expression level of RSPOl, RSP02, RSP03, and/or RSP04 in fibrotic tissue is determined, if the fibrotic tissue has an elevated level of RSP02 expression as compared to the level of RSP02 in normal tissue, the subject is selected for treatment with an antibody that specifically binds RSP02. If selected for treatment, the subject is administered an anti-RSP02 antibody describe herein. In some embodiments, the RSP02-binding agent is a humanized version of antibody 130M23. In some embodiments, the RSP02-binding agent is antibody hl30M23-HlL6. In some embodiments, the RSP02- binding agent is antibody hl30M23-HlL2. In some embodiments, the expression level of RSPO l, RSP02, RSP03, and/or RSP04 in fibrotic tissue is determined, if the fibrotic tissue has an elevated level of RSP03 expression as compared to the level of RSP03 in normal tissue, the subject is selected for treatment with an antibody that specifically binds RSP03. If selected for treatment, the subject is administered an anti-RSP03 antibody describe herein. In some embodiments, the RSP03-binding agent is antibody 131R01 1.

[0105] The present invention also provides methods of treating a fibrotic disease in a human subject, comprising: (a) selecting a subject for treatment based, at least in part, on the subject having a tissue sample that has an elevated level of RSPOl, and (b) administering to the subject a therapeutically effective amount of a RSPOl-binding agent described herein. In some embodiments, the RSPOl-binding agent is a humanized version of antibody 89M5. In some embodiments, the RSPOl-binding agent is antibody h89M5-H8L5. In some embodiments, the RSPOl -binding agent is antibody h89M5-H2L2. In some embodiments, the RSPO 1 -binding agent is a LGR soluble receptor.

[0106] The present invention also provides methods of treating a fibrotic disease in a human subject, comprising: (a) selecting a subject for treatment based, at least in part, on the subject having a tissue sample that has an elevated level of RSP02, and (b) administering to the subject a therapeutically effective amount of a RSP02-binding agent described herein. In some embodiments, the RSP02-binding agent is a humanized version of antibody 130M23. In some embodiments, the RSP02-binding agent is antibody hl30M23-HlL6. In some embodiments, the RSP02-binding agent is antibody hl30M23-HlL2. In some embodiments, the RSP02-binding agent is a LGR soluble receptor.

[0107] The present invention also provides methods of treating a fibrotic disease in a human subject, comprising: (a) selecting a subject for treatment based, at least in part, on the subject having a tissue sample that has an elevated level of RSP03, and (b) administering to the subject a therapeutically effective amount of a RSP03-binding agent described herein. In some embodiments, the RSP03-binding agent is antibody 131R010. In some embodiments, the RSP03 -binding agent is a LGR soluble receptor.

[0108] Methods for determining the level of RSPO expression in a cell or tissue are known by those of skill in the art. For nucleic acid expression these methods include, but are not limited to, PCR-based assays, microarray analyses and nucleotide sequencing (e.g., NextGen sequencing). For protein expression these methods include, but are not limited to, Western blot analyses, protein arrays, ELISAs, immunohistochemistry (IHC) assays, and FACS.

[0109] Methods for determining whether fibrotic tissue has an elevated level of RSPO expression can use a variety of samples. In some embodiments, the sample is taken from a subject having a fibrotic disease. In some embodiments, the sample is a fresh sample. In some embodiments, the sample is a frozen sample. In some embodiments, the sample is a formalin-fixed paraffin-embedded sample. In some embodiments, the sample is processed to a cell lysate. In some embodiments, the sample is processed to DNA or RNA.

[0110] The present invention further provides compositions comprising the RSPO-binding agents or LGR-binding agents described herein. In certain embodiments, the compositions are pharmaceutical compositions which comprise a pharmaceutically acceptable vehicle. In some embodiments, the pharmaceutical compositions find use in treating or preventing a fibrotic disease in a subject (e.g., a human patient).

[0111] Formulations are prepared for storage and use by combining a purified antibody or agent of the present invention with a pharmaceutically acceptable vehicle (e.g., a carrier or excipient). Those of skill in the art generally consider pharmaceutically acceptable carriers, excipients, and/or stabilizers to be inactive ingredients of a formulation or pharmaceutical composition.

[0112] Suitable pharmaceutically acceptable vehicles include, but are not limited to, nontoxic buffers such as phosphate, citrate, and other organic acids; salts such as sodium chloride; antioxidants including ascorbic acid and methionine; preservatives such as octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens, such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol; low molecular weight polypeptides (e.g., less than about 10 amino acid residues); proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; carbohydrates such as monosaccharides, disaccharides, glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes such as Zn-protein complexes; and non-ionic surfactants such as TWEEN or polyethylene glycol (PEG). (Remington: The Science and Practice of Pharmacy, 22 ^nd Edition, 2012, Pharmaceutical Press, London.)

[0113] The pharmaceutical compositions of the present invention can be administered in any number of ways for either local or systemic treatment. Administration can be topical by epidermal or transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders; pulmonary by inhalation or insufflation of powders or aerosols, including by nebulizer, intratracheal, and intranasal; oral; or parenteral including intravenous, intraarterial, subcutaneous, intraperitoneal, intramuscular (e.g., injection or infusion), or intracranial (e.g., intrathecal or intraventricular). In some embodiments of the methods described herein, the RSPO-binding agent or LGR-binding agent is administered locally. In some embodiments, the RSPO-binding agent or LGR-binding agent is administered subcutaneously. In some embodiments, the RSPO-binding agent or LGR-binding agent is administered intramuscularly. In some embodiments, the RSPO-binding agent or LGR-binding agent is administered topically. In some embodiments, the RSPO-binding agent or LGR-binding agent is administered intravenously. In some embodiments, the RSPO-binding agent or LGR-binding agent is administered by inhalation.

[0114] The therapeutic formulation can be in unit dosage form. Such formulations include tablets, pills, capsules, powders, granules, solutions or suspensions in water or non-aqueous media, or suppositories. In solid compositions such as tablets the principal active ingredient is mixed with a pharmaceutical carrier. Conventional tableting ingredients include corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, and diluents (e.g., water). These can be used to form a solid pre-formulation composition containing a homogeneous mixture of a compound of the present invention, or a non-toxic pharmaceutically acceptable salt thereof. The solid pre-formulation composition is then subdivided into unit dosage forms of a type described above. The tablets, pills, etc. of the formulation or composition can be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action. For example, the tablet or pill can comprise an inner composition covered by an outer component. Furthermore, the two components can be separated by an enteric layer that serves to resist disintegration and permits the inner component to pass intact through the stomach or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials include a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol and cellulose acetate.

[0115] The RSPO-binding agents or LGR-binding agents (e.g., antibodies or soluble receptors) described herein can also be entrapped in microcapsules. Such microcapsules are prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin- microcapsules and poly-(methylmethacrylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and

nanocapsules) or in macroemulsions as described in Remington: The Science and Practice of Pharmacy, 22 ^nd Edition, 2012, Pharmaceutical Press, London.

[0116] In certain embodiments, pharmaceutical formulations include a RSPO-binding agent or LGR- binding agent (e.g., an antibody or a soluble receptor) of the present invention complexed with liposomes. Methods to produce liposomes are known to those of skill in the art. For example, some liposomes can be generated by reverse phase evaporation with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes can be extruded through filters of defined pore size to yield liposomes with the desired diameter.

[0117] In certain embodiments, sustained-release preparations comprising the RSPO-binding agents or LGR-binding agents described herein can be produced. Suitable examples of sustained-release preparations include semi-permeable matrices of solid hydrophobic polymers containing a RSPO-binding agent or LGR-binding agent (e.g., an antibody or a soluble receptor), where the matrices are in the form of shaped articles (e.g., films or microcapsules). Examples of sustained-release matrices include polyesters, hydrogels such as poly(2-hydroxyethyl-methacrylate) or poly( vinyl alcohol), polylactides, copolymers of L-glutamic acid and 7 ethyl-L-glutamate, non-degradable ethylene -vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose acetate isobutyrate, and poly-D-(-)-3-hydroxybutyric acid.

[0118] In certain embodiments, in addition to administering a RSPO-binding agent or LGR-binding agent (e.g., an antibody or a soluble receptor), 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 RSPO-binding agent or the LGR-binding agent. 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.

[0119] 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). [0120] In some embodiments, the combination of a RSPO-binding agent or LGR-binding agent 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-binding agent or the LGR-binding agent. 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-binding agent or the LGR-binding agent. In some embodiments, the combination therapy results in a decrease in the toxicity and/or side effects of the additional agent(s).

[0121] In some embodiments, an additional therapeutic agent is an anti-inflammatory agent. In some embodiments, an anti-inflammatory agent is a steroid, a glucocorticosteroid, or a corticosteroid. Steroids may include, but are not limited to, beclometasone, budesonide, flunisolide, fluticasone propionate, triamcinolone, methylprednisolone, prednisolone, and prednisone.

[0122] In some embodiments, an anti-inflammatory agent is a non-steroid anti-inflammatory (NSAID). NSAIDs may include, but are not limited to, naproxen sodium, celecoxib, sulindac, oxaprozin, salsalate, diflunisal, piroxicam, indomethacin, etodolac, meloxicam, naproxen, nabumetone, ketorolac

tromethamine, naproxen/exomeprazole, diclofenac, and aspirin.

[0123] In some embodiments, an anti-inflammatory agent is a small molecule. In some embodiments, an anti-inflammatory agent is a tyrosine kinase inhibitor, such as imatinib (GLEEVEC).

[0124] In some embodiments, an anti-inflammatory agent is an inhibitor of pro-inflammatory cytokines, interleukins, lymphokines, and/or chemokines. In some embodiments, an anti-inflammatory agent is an antibody that inhibits the activity of a pro-inflammatory cytokine. In some embodiments, an antiinflammatory agent is antibody which binds and/or inhibits IL-1, IL-18, IL-33, IL36, IL-37, IL-6, IL-1 1, IL-31, IL-17, IL-25, tumor necrosis factor (TNF), interferon alpha, interferon gamma, and/or interferon beta.

[0125] 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 a RSPO-binding agent in combination with an anti-FZD antibody. In some embodiments, a method comprises administering a RSPO-binding agent in combination with the anti-FZD antibody 18R5. In some embodiments, the Wnt pathway inhibitors are Wnt protein binding 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 201 1/088127 and WO 201 1/088123. 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 a RSPO-binding agent or a LGR- binding agent in combination with a FZD-Fc soluble receptor. In some embodiments, a method comprises administering a RSPO-binding agent or a LGR-binding agent in combination with a FZD8-Fc soluble receptor. In some embodiments, a method comprises administering a RSPO-binding agent or a LGR-binding agent in combination with an anti-FZD antibody.

[0126] In some embodiments, the methods described herein comprise administering a therapeutically effective amount of a RSPO-binding agent or a LGR-binding agent in combination with more than one additional therapeutic agent.

[0127] 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.

[0128] It will be appreciated that the combination of a RSPO-binding agent or a LGR-binding agent and at least one additional therapeutic agent may be administered in any order or concurrently. In some embodiments, the RSPO-binding agent or the LGR-binding agent will be administered to patients that have previously undergone treatment with a second therapeutic agent. In certain other embodiments, the RSPO-binding agent or the LGR-binding agent and a second therapeutic agent will be administered substantially simultaneously or concurrently. For example, a subject may be given a RSPO-binding agent (e.g., an antibody) while undergoing a course of treatment with a second therapeutic agent (e.g., chemotherapy). In certain embodiments, a RSPO-binding agent or a LGR-binding agent will be administered within 1 year of the treatment with a second therapeutic agent. In certain alternative embodiments, a RSPO-binding agent or a LGR-binding agent will be administered within 10, 8, 6, 4, or 2 months of any treatment with a second therapeutic agent. In certain other embodiments, a RSPO-binding agent or a LGR-binding agent will be administered within 4, 3, 2, or 1 weeks of any treatment with a second therapeutic agent. In some embodiments, a RSPO-binding agent or a LGR-binding agent 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).

[0129] For the treatment of a disease, the appropriate dosage of an RSPO-binding agent or a LGR- binding agent (e.g., an antibody or a soluble receptor) of the present invention depends on the type of disease to be treated, the severity and course of the disease, the responsiveness of the disease, whether the RSPO-binding agent or LGR-binding agent 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-binding agent or LGR-binding agent 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 RSPO-binding agent 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-binding agent or LGR-binding agent is given once every week, once every two weeks or once every three weeks.

[0130] 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.

[0131] Thus, the present invention provides methods of treating fibrotic diseases in a subject comprising using an intermittent dosing strategy for administering one or more agents, which may reduce side effects and/or toxicities associated with administration of a RSPO-binding agent, a LGR-binding agent, a second agent, etc.

III. RSPO and LGR antagonists

[0132] The present invention provides methods comprising agents that specifically bind human RSPO proteins or human LGR proteins. These agents are referred to herein as "RSPO-binding agents" and "LGR-binding agents", respectively. Non-limiting examples of RSPO-binding agents and LGR-binding agents can be found in U.S. Patent Publication Nos. 2006/0275870, 2009/0074782, 2009/0191205, 2009/0220495, 2010/0071078, 2012/0039912, 2012/0263730, 2013/00951 16, 2013/0336885,

2013/0337533, 2014/0017253, 2014/0302054, 2015/0147333, and International Publication No. WO 2010/016766, WO 2014/012007, WO 2014/192974. 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 RSPO l ("RSPO 1 -binding agents"). In certain embodiments, the RSPO- binding agent binds RSP02 ("RSP02-binding agents"). 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 RSPOl, RSP02, RSP03, and RSP04 are known in the art and are provided herein as SEQ ID NO: l (RSPOl), SEQ ID NO:2 (RSP02), SEQ ID NO:3 (RSP03), and SEQ ID NO:4 (RSP04). In some embodiments, the RSPO-binding agent is a soluble receptor. In some embodiments, the RSPO-binding agent is a soluble receptor comprising the extracellular domain, or a fragment thereof, of a human LGR protein. In some embodiments, the human LGR protein is LGR4, LGR5, or LGR6. The full-length amino acid (aa) sequences for human LGR4, LGR5, and LGR6 are known in the art and are provided herein as SEQ ID NO:71 (LGR4), SEQ ID NO:72 (LGR5), and SEQ ID NO:73 (LGR6).

[0133] In some embodiments, the LGR-binding agent is an antibody. In some embodiments, the LGR- binding agent is a polypeptide. In certain embodiments, the LGR-binding agent binds LGR4 ("LGR4- binding agents"). In certain embodiments, the LGR-binding agent binds LGR5 ("LGR5-binding agents"). In certain embodiments, the LGR-binding agent binds LGR6 ("LGR6-binding agents"). In certain embodiments, the LGR-binding agent specifically binds one or more human LGR proteins.

[0134] In certain embodiments, the antigen-binding site of a RSPO-binding agent (e.g., an antibody or a bispecific antibody) described herein is capable of binding (or binds) one, two, three, or four RSPOs. In certain embodiments, the antigen-binding site of a RSPO-binding agent (e.g., an antibody or a bispecific antibody) described herein is capable of binding (or binds) a first RSPO protein (e.g., RSPOl) as well as one, two, or three other RSPOs (e.g., RSP02, RSP03, and/or RSP04). In some embodiments, the RSPO- binding agent (e.g., antibody) specifically binds both human RSPO and mouse RSPO.

[0135] In certain embodiments of the methods described herein, the RSPO-binding agent is an antibody that specifically binds within amino acids 21-263 of human RSPOl (SEQ ID NO: l). In certain embodiments, the RSPO-binding agent is an antibody that specifically binds within amino acids 31-263 of human RSPOl (SEQ ID NO: 1). In certain embodiments, the RSPO-binding agent is an antibody that specifically binds within amino acids 34-135 of human RSPOl (SEQ ID NO: 1). In certain embodiments, the RSPO-binding agent is an antibody that specifically binds within amino acids 34-85 of human RSPOl (SEQ ID NO: 1). In certain embodiments, the RSPO-binding agent is an antibody that specifically binds within amino acids 91-135 of human RSPOl (SEQ ID NO: l). In certain embodiments, the RSPO- binding agent is an antibody that specifically binds within amino acids 147-207 of human RSPOl (SEQ ID NO: l). In certain embodiments, the RSPO-binding agent binds a furin-like cysteine -rich domain of RSPO l. In some embodiments, the RSPO-binding agent binds at least one amino acid within a furin-like cysteine-rich domain of RSPOl . In some embodiments, the RSPO-binding agent binds the thrombospondin domain of RSPOl . In some embodiments, the RSPO-binding agent binds at least one amino acid within the thrombospondin domain of RSPO 1.

[0136] In certain embodiments, the RSPO-binding agent is an antibody that specifically binds within amino acids 22-243 of human RSP02 (SEQ ID NO:2). In certain embodiments, the RSPO-binding agent is an antibody that specifically binds within amino acids 22-205 of human RSP02 (SEQ ID NO:2). In certain embodiments, the RSPO-binding agent is an antibody that specifically binds within amino acids 35-134 of human RSP02 (SEQ ID NO:2). In certain embodiments, the RSPO-binding agent is an antibody that specifically binds within amino acids 34-84 of human RSP02 (SEQ ID NO:2). In certain embodiments, the RSPO-binding agent is an antibody that specifically binds within amino acids 90-134 of human RSP02 (SEQ ID NO:2). In certain embodiments, the RSPO-binding agent binds a furin-like cysteine-rich domain of RSP02. In some embodiments, the RSPO-binding agent binds at least one amino acid within a furin-like cysteine-rich domain of RSP02. In some embodiments, the RSPO-binding agent binds the thrombospondin domain of RSP02. In some embodiments, the RSPO-binding agent binds at least one amino acid within the thrombospondin domain of RSP02.

[0137] 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.

[0138] In certain embodiments, the RSPO-binding agent (e.g., an antibody or a soluble receptor) binds at least one RSPO protein with a dissociation constant (K _D ) of about ΙμΜ or less, about lOOnM or less, about 40nM or less, about 20nM or less, about lOnM or less, about lnM or less, or about O. lnM or less. In certain embodiments, a RSPO-binding agent (e.g., an antibody or a soluble receptor) binds at least one RSPO protein with a dissociation constant (K _D ) of about l uM or less, about lOOnM or less, about 40nM or less, about 20nM or less, about lOnM or less, about lnM or less, or about O. lnM or less. In some embodiments, a RSPO-binding agent (e.g., an antibody or a soluble receptor) binds at least one RSPO protein with a K _D of about 20nM or less. In some embodiments, a RSPO-binding agent (e.g., an antibody or a soluble receptor) binds at least one RSPO protein with a K _D of about 1 OnM or less. In some embodiments, a RSPO-binding agent (e.g., an antibody or a soluble receptor) binds at least one RSPO protein with a K _D of about InM or less. In some embodiments, a RSPO-binding agent (e.g., an antibody or a soluble receptor) binds at least one RSPO protein with a K _D of about 0.5nM or less. In some embodiments, a RSPO-binding agent (e.g., an antibody or a soluble receptor) binds at least one RSPO protein with a K _D of about 0. InM or less. In certain embodiments, a RSPO-binding agent (e.g., an antibody or a soluble receptor) 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, a RSPO-binding agent binds both human RSPO and mouse RSPO with a K _D of about InM or less. In some embodiments, a 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 or a soluble receptor) to a RSPO protein is the dissociation constant determined using a 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 or a soluble receptor) to a RSPO protein is the dissociation constant determined using the binding agent captured by an anti- human IgG antibody on a Biacore chip and a RSPO protein.

[0139] In certain embodiments, the RSPO-binding agent (e.g., an antibody or a soluble receptor) binds to at least one human RSPO protein with a half maximal effective concentration (EC ₅ o) of about 1 μΜ or less, about lOOnM 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, a RSPO-binding agent (e.g., an antibody or a soluble receptor) binds to at least one human RSPO with a half maximal effective concentration (EC ₅ o) of about Ι μΜ or less, about lOOnM or less, about 40nM or less, about 20nM or less, about lOnM or less, about InM or less, or about O. lnM or less.

[0140] In certain embodiments, the LGR-binding agent (e.g., an antibody) binds at least one LGR protein with a dissociation constant (K _D ) of about 1 μΜ or less, about 1 OOnM 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, a LGR-binding agent (e.g., an antibody) binds at least one LGR protein with a dissociation constant (K _D ) of about ΙμΜ or less, about lOOnM 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 some embodiments, a LGR-binding agent (e.g., an antibody) binds at least one LGR protein with a K _D of about 20nM or less. In some embodiments, a LGR-binding agent (e.g., an antibody) binds at least one LGR protein with a K _D of about lOnM or less. In some embodiments, a LGR-binding agent (e.g., an antibody) binds at least one LGR protein with a K _D of about InM or less. In some embodiments, a LGR-binding agent (e.g., an antibody) binds at least one LGR protein with a K _D of about 0.5nM or less. In some embodiments, a LGR-binding agent (e.g., an antibody) binds at least one LGR protein with a K _D of about 0. InM or less. In certain embodiments, a LGR-binding agent (e.g., an antibody) described herein binds at least two LGR proteins. In some embodiments, the dissociation constant of a binding agent (e.g., an antibody) to a LGR protein is the dissociation constant determined using a LGR fusion protein comprising at least a portion of the LGR protein immobilized on a Biacore chip. In some embodiments, the dissociation constant of a binding agent (e.g., an antibody) to a LGR protein is the dissociation constant determined using the binding agent captured by an anti-human IgG antibody on a Biacore chip and a LGR protein.

[0141] In certain embodiments, the LGR-binding agent (e.g., an antibody) binds to at least one human LGR protein with a half maximal effective concentration (EC ₅₀ ) of about 1 μΜ or less, about 1 OOnM or less, about 40nM or less, about 20nM or less, about lOnM or less, about InM or less, or about 0. InM or less. In certain embodiments, a LGR-binding agent (e.g., an antibody) binds to at least one human LGR with a half maximal effective concentration (EC ₅ o) of about 1 μΜ or less, about 1 OOnM or less, about 40nM or less, about 20nM or less, about l OnM or less, about InM or less, or about O. lnM or less.

[0142] In certain embodiments, the RSPO-binding agent or LGR-binding agent 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.

[0143] The RSPO-binding agents (e.g., antibodies) of the present invention 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).

[0144] For example, the specific binding of an agent to a human RSPO protein may 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 be knowledgeable as to the parameters that can be modified to increase the signal detected as well as other variations of ELISAs known in the art.

[0145] In another example, the specific binding of an agent to a human RSPO protein may be determined using FACS. A FACS screening assay may comprise generating a cDNA construct that expresses an antigen as a fusion protein (e.g., RSPO-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 may 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 be knowledgeable as to the parameters that can be modified to optimize the signal detected as well as other variations of FACS that may enhance screening (e.g., screening for blocking antibodies).

[0146] The binding affinity of an agent 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 ³ H or ¹²⁵ I), 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.

[0147] In certain embodiments of the methods described herein, the invention provides methods comprising a RSPO 1 -binding agent (e.g., an antibody) that specifically binds human RSPOl, wherein the RSPO 1 -binding agent (e.g., an antibody) comprises one, two, three, four, five, and/or six of the CDRs of antibody 89M5 (see Table 1).

Table 1

[0148] In certain embodiments, the invention provides methods comprising a RSPOl -binding agent (e.g., an antibody) that specifically binds human RSPOl, wherein the RSPO 1 -binding agent comprises a heavy chain CDRl comprising TGYTMH (SEQ ID NO:5), a heavy chain CDR2 comprising

GINPNNGGTTYNQNFKG (SEQ ID NO: 6), and a heavy chain CDR3 comprising KEFSDGYYFFAY (SEQ ID NO:7). In some embodiments, the RSPO 1 -binding agent further comprises a light chain CDRl comprising KASQDVIFAVA (SEQ ID NO:8), a light chain CDR2 comprising WASTRHT (SEQ ID NO:9), and a light chain CDR3 comprising QQHYSTPW (SEQ ID NO: 10). In some embodiments, the RSPO 1 -binding agent comprises a light chain CDR1 comprising KASQDVIFAVA (SEQ ID NO: 8), a light chain CDR2 comprising WASTRHT (SEQ ID NO:9), and a light chain CDR3 comprising

QQHYSTPW (SEQ ID NO: 10). In certain embodiments, the RSPO 1 -binding agent comprises: (a) a heavy chain CDR1 comprising TGYTMH (SEQ ID NO:5), a heavy chain CDR2 comprising

GINPNNGGTTYNQNFKG (SEQ ID NO: 6), and a heavy chain CDR3 comprising KEFSDGYYFFAY (SEQ ID NO:7); and (b) a light chain CDR1 comprising KASQDVIFAVA (SEQ ID NO:8), a light chain CDR2 comprising WASTRHT (SEQ ID NO:9), and a light chain CDR3 comprising QQHYSTPW (SEQ ID NO: 10).

[0149] In certain embodiments, the invention provides methods comprising a RSPO 1 -binding agent (e.g., an antibody or bispecific antibody) that specifically binds human RSPOl, wherein the RSPOl- binding agent comprises: (a) a heavy chain CDR1 comprising TGYTMH (SEQ ID NO: 5) or a variant thereof comprising 1, 2, 3, or 4 amino acid substitutions; (b) a heavy chain CDR2 comprising

GINPNNGGTTYNQNFKG (SEQ ID NO:6) or a variant thereof comprising 1, 2, 3, or 4 amino acid substitutions; (c) a heavy chain CDR3 comprising KEFSDGYYFFAY (SEQ ID NO:7) or a variant thereof comprising 1, 2, 3, or 4 amino acid substitutions; (d) a light chain CDR1 comprising

KASQDVIFAVA (SEQ ID NO:8) or a variant thereof comprising 1, 2, 3, or 4 amino acid substitutions; (e) a light chain CDR2 comprising WASTRHT (SEQ ID NO: 9) or a variant thereof comprising 1, 2, 3, or 4 amino acid substitutions; and (f) a light chain CDR3 comprising QQHYSTPW (SEQ ID NO: 10) 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.

[0150] In certain embodiments, the invention provides methods comprising a RSPO 1 -binding agent (e.g., an antibody) that specifically binds RSPOl, wherein the RSPO 1 -binding agent comprises a heavy chain variable region having at least about 80% sequence identity to SEQ ID NO: l 1 and/or a light chain variable region having at least 80% sequence identity to SEQ ID NO: 12. In certain embodiments, the RSPO 1 -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: 1 1. In certain embodiments, the RSPO 1 -binding agent comprises a light chain variable region having at least about 85%o, at least about 90%>, at least about 95%>, at least about 97%>, or at least about 99%> sequence identity to SEQ ID NO: 12. In certain embodiments, the RSPO 1 -binding agent comprises a heavy chain variable region having at least about 95%> sequence identity to SEQ ID NO: 1 1 and/or a light chain variable region having at least about 95%> sequence identity to SEQ ID NO: 12. In certain embodiments, the RSPO l-binding agent comprises a heavy chain variable region comprising SEQ ID NO: l 1, and/or a light chain variable region comprising SEQ ID NO: 12. In certain embodiments, the RSPO 1 -binding agent comprises a heavy chain variable region comprising SEQ ID NO: 1 1 and a light chain variable region comprising SEQ ID NO: 12. In certain embodiments, the RSPO 1 -binding agent comprises a heavy chain variable region consisting of SEQ ID NO: 1 1 and a light chain variable region consisting of SEQ ID NO: 12.

[0151] In certain embodiments, the invention provides methods comprising a RSPOl-binding agent (e.g., an antibody) that specifically binds RSPOl, wherein the RSPO l-binding agent comprises a heavy chain variable region having at least about 80% sequence identity to SEQ ID NO:56 and/or a light chain variable region having at least 80%> sequence identity to SEQ ID NO:57. In certain embodiments, the RSPO l-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:56. In certain embodiments, the RSPO 1 -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:57. In certain embodiments, the RSPOl-binding agent comprises a heavy chain variable region having at least about 95% sequence identity to SEQ ID NO:56 and/or a light chain variable region having at least about 95% sequence identity to SEQ ID NO: 57. In certain embodiments, the RSPO l-binding agent comprises a heavy chain variable region comprising SEQ ID NO:56 and/or a light chain variable region comprising SEQ ID NO:57. In certain embodiments, the RSPOl-binding agent comprises a heavy chain variable region comprising SEQ ID NO:56 and a light chain variable region comprising SEQ ID NO: 57. In certain embodiments, the RSPOl-binding agent comprises a heavy chain variable region consisting of SEQ ID NO:56 and a light chain variable region consisting of SEQ ID NO:57.

[0152] In certain embodiments, the invention provides methods comprising a RSPOl-binding agent (e.g., an antibody) that specifically binds RSPOl, wherein the RSPOl-binding agent comprises: (a) a heavy chain having at least 90% sequence identity to SEQ ID NO: 13 or SEQ ID NO: 14; and/or (b) a light chain having at least 90% sequence identity to SEQ ID NO: 15 or SEQ ID NO: 16. In some embodiments, the RSPO l-binding agent comprises: (a) a heavy chain having at least 95% sequence identity to SEQ ID NO: 13 or SEQ ID NO: 14; and/or (b) a light chain having at least 95% sequence identity to SEQ ID NO: 15 or SEQ ID NO: 16. In some embodiments, the RSPO 1 -binding agent comprises a heavy chain comprising SEQ ID NO: 14 and/or a light chain comprising SEQ ID NO: 16. In some embodiments, the RSPO l-binding agent comprises a heavy chain comprising SEQ ID NO: 14 and a light chain comprising SEQ ID NO: 16. [0153] In certain embodiments, the invention provides methods comprising a RSPO 1 -binding agent (e.g., an antibody) that specifically binds RSPOl, wherein the RSPO 1 -binding agent comprises: (a) a heavy chain having at least 90% sequence identity to SEQ ID NO:58 or SEQ ID NO:59; and/or (b) a light chain having at least 90%> sequence identity to SEQ ID NO: 60 or SEQ ID NO:61. In some embodiments, the RSPO l-binding agent comprises: (a) a heavy chain having at least 95% sequence identity to SEQ ID NO:58 or SEQ ID NO:59; and/or (b) a light chain having at least 95% sequence identity to SEQ ID NO: 60 or SEQ ID NO:61. In some embodiments, the RSPOl -binding agent comprises a heavy chain comprising SEQ ID NO:59 and/or a light chain comprising SEQ ID NO:61. In some embodiments, the RSPO l-binding agent comprises a heavy chain comprising SEQ ID NO:59 and a light chain comprising SEQ ID NO:61.

[0154] In certain embodiments, a RSPO 1 -binding agent comprises the heavy chain variable region and light chain variable region of antibody h89M5-H8L5. In certain embodiments, a RSPO l-binding agent comprises the heavy chain and light chain of antibody h89M5-H8L5 (with or without the leader sequence). In certain embodiments, a RSPOl-binding agent is antibody h89M5-H8L5. In certain embodiments, a RSPOl-binding agent comprises the heavy chain variable region and/or light chain variable region of antibody h89M5-H8L5 in a chimeric form of the antibody. In some embodiments, the anti-RSPOl antibody is h89M5-H8L5.

[0155] In certain embodiments, a RSPOl-binding agent comprises the heavy chain variable region and light chain variable region of antibody h89M5-H2L2. In certain embodiments, a RSPOl -binding agent comprises the heavy chain and light chain of antibody h89M5-H2L2 (with or without the leader sequence). In certain embodiments, a RSPOl-binding agent is antibody h89M5-H2L2. In certain embodiments, a RSPOl-binding agent comprises the heavy chain variable region and/or light chain variable region of antibody h89M5-H2L2 in a chimeric form of the antibody. In some embodiments, the anti-RSPOl antibody is h89M5-H2L2.

[0156] In certain embodiments of the methods described herein, a RSPOl-binding agent comprises the heavy chain CDRs and/or light chain CDRs of antibody 89M5. The hybridoma cell line producing the 89M5 antibody was deposited with American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, VA, USA, under the conditions of the Budapest Treaty on June 30, 201 1 and assigned ATCC deposit designation number PTA-1 1970.

[0157] Plasmids encoding the heavy chain and light chain of antibody h89M5-H8L5 were deposited with ATCC, 10801 University Boulevard, Manassas, VA, USA, under the conditions of the Budapest Treaty on August 15, 2014 and assigned ATCC deposit designation number PTA-121494 and PTA-121495. In some embodiments, the RSPO 1 -binding agent comprises a heavy chain variable region encoded by the plasmid deposited with ATCC and designated PTA-121494. In some embodiments, the RSPO 1 -binding agent comprises a light chain variable region encoded by the plasmid deposited with ATCC and designated PTA-121495. In some embodiments, the RSPO 1 -binding agent comprises a heavy chain variable region encoded by the plasmid deposited with ATCC and designated PTA-121494 and a light chain variable region encoded by the plasmid deposited with ATCC and designated PTA-121495. In some embodiments, the RSPO 1 -binding agent comprises a heavy chain encoded by the plasmid deposited with ATCC and designated PTA-121494. In some embodiments, the RSPO 1 -binding agent comprises a light chain encoded by the plasmid deposited with ATCC and designated PTA-121495. In some embodiments, the RSPO 1 -binding agent comprises a heavy chain encoded by the plasmid deposited with ATCC and designated PTA-121494 and a light chain encoded by the plasmid deposited with ATCC and designated PTA-121495.

[0158] In certain embodiments, a RSPO 1 -binding agent comprises, consists essentially of, or consists of, antibody h89M5-H8L5. In certain embodiments, a RSPO 1 -binding agent comprises, consists essentially of, or consists of, a variant of antibody 89M5. In certain embodiments, a RSPO 1 -binding agent comprises, consists essentially of, or consists of, a variant of antibody h89M5-H8L5.

[0159] In certain embodiments, a RSPO 1 -binding agent comprises, consists essentially of, or consists of, antibody h89M5-H2L2. In certain embodiments, a RSPO 1 -binding agent comprises, consists essentially of, or consists of, a variant of antibody 89M5. In certain embodiments, a RSPO 1 -binding agent comprises, consists essentially of, or consists of, a variant of antibody h89M5-H2L2.

[0160] In certain embodiments of the methods described herein, the invention provides methods comprising a RSP02-binding agent (e.g., an antibody) that specifically binds human RSP02, wherein the RSP02-binding agent (e.g., an antibody) comprises one, two, three, four, five, and/or six of the CDRs of antibody 130M23 (see Table 1).

[0161] In certain embodiments, the invention provides methods comprising a RSP02 -binding agent (e.g., an antibody) that specifically binds human RSP02, wherein the RSP02-binding agent comprises a heavy chain CDR1 comprising SSYAMS (SEQ ID NO:21), a heavy chain CDR2 comprising

SISSGGSTYYPDSVKG (SEQ ID NO:22), and a heavy chain CDR3 comprising

RGGDPGVYNGDYEDAMDY (SEQ ID NO:23). In some embodiments, the RSP02-binding agent further comprises a light chain CDR1 comprising KASQDVSSAVA (SEQ ID NO:24), a light chain CDR2 comprising WASTRHT (SEQ ID NO:25), and a light chain CDR3 comprising QQHYSTP (SEQ ID NO:26). In some embodiments, the RSP02-binding agent comprises a light chain CDR1 comprising KASQDVSSAVA (SEQ ID NO:24), a light chain CDR2 comprising WASTRHT (SEQ ID NO:25), and a light chain CDR3 comprising QQHYSTP (SEQ ID NO:26). In certain embodiments, the RSP02-binding agent comprises: (a) a heavy chain CDR1 comprising SSYAMS (SEQ ID NO:21), a heavy chain CDR2 comprising SISSGGSTYYPDSVKG (SEQ ID NO:22), and a heavy chain CDR3 comprising

RGGDPGVYNGDYEDAMDY (SEQ ID NO:23); and (b) a light chain CDR1 comprising

KASQDVSSAVA (SEQ ID NO:24), a light chain CDR2 comprising WASTRHT (SEQ ID NO:25), and a light chain CDR3 comprising QQHYSTP (SEQ ID NO:26).

[0162] In certain embodiments, the invention provides methods comprising a RSP02 -binding agent (e.g., an antibody or bispecific antibody) that specifically binds human RSP02, wherein the RSP02- binding agent comprises: (a) a heavy chain CDR1 comprising SSYAMS (SEQ ID NO:21) or a variant thereof comprising 1, 2, 3, or 4 amino acid substitutions; (b) a heavy chain CDR2 comprising

SISSGGSTYYPDSVKG (SEQ ID NO:22) or a variant thereof comprising 1, 2, 3, or 4 amino acid substitutions; (c) a heavy chain CDR3 comprising RGGDPGVYNGDYEDAMDY (SEQ ID NO:23) or a variant thereof comprising 1, 2, 3, or 4 amino acid substitutions; (d) a light chain CDR1 comprising KASQDVSSAVA (SEQ ID NO: 24) or a variant thereof comprising 1, 2, 3, or 4 amino acid substitutions; (e) a light chain CDR2 comprising WASTRHT (SEQ ID NO:25) or a variant thereof comprising 1, 2, 3, or 4 amino acid substitutions; and (f) a light chain CDR3 comprising QQHYSTP (SEQ ID NO:26) 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.

[0163] In certain embodiments, the invention provides methods comprising a RSP02 -binding agent (e.g., an antibody) that specifically binds RSP02, wherein the RSP02-binding agent comprises a heavy chain variable region having at least about 80% sequence identity to SEQ ID NO:27 and/or a light chain variable region having at least 80%> sequence identity to SEQ ID NO:28 or SEQ ID NO:66. In certain embodiments, the RSP02-binding agent comprises a heavy chain variable region having at least about 85%o, at least about 90%>, at least about 95%>, at least about 97%>, or at least about 99%> sequence identity to SEQ ID NO:27. In certain embodiments, the RSP02-binding agent comprises a light chain variable region having at least about 85%>, at least about 90%>, at least about 95%>, at least about 97%>, or at least about 99%) sequence identity to SEQ ID NO:28. In certain embodiments, the RSP02-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:66. In certain embodiments, the RSP02-binding agent comprises a heavy chain variable region having at least about 95%> sequence identity to SEQ ID NO:27 and/or a light chain variable region having at least about 95%> sequence identity to SEQ ID NO:28 or SEQ ID NO:66. In certain embodiments, the RSP02-binding agent comprises a heavy chain variable region comprising SEQ ID NO:27 and/or a light chain variable region comprising SEQ ID NO:28 or SEQ ID NO:66. In certain embodiments, the RSP02-binding agent comprises a heavy chain variable region comprising SEQ ID NO:27 and a light chain variable region comprising SEQ ID NO:28. In certain embodiments, the RSP02-binding agent comprises a heavy chain variable region comprising SEQ ID NO:27 and a light chain variable region comprising SEQ ID NO:66. In certain embodiments, the RSP02-binding agent comprises a heavy chain variable region consisting of SEQ ID NO:27 and a light chain variable region consisting of SEQ ID NO:28. In certain embodiments, the RSP02-binding agent comprises a heavy chain variable region consisting of SEQ ID NO:27 and a light chain variable region consisting of SEQ ID NO:66.

[0164] In certain embodiments, the invention provides methods comprising a RSP02-binding agent (e.g., an antibody) that specifically binds RSP02, wherein the RSP02-binding agent comprises: (a) a heavy chain having at least 90% sequence identity to SEQ ID NO:29 or SEQ ID NO:30; and/or (b) a light chain having at least 90%> sequence identity to SEQ ID NO:31 or SEQ ID NO:32. In some embodiments, the RSP02-binding agent comprises: (a) a heavy chain having at least 95% sequence identity to SEQ ID NO:29 or SEQ ID NO:30; and/or (b) a light chain having at least 95% sequence identity to SEQ ID NO:31 or SEQ ID NO:32. In some embodiments, the RSP02-binding agent comprises a heavy chain comprising SEQ ID NO:30 and/or a light chain comprising SEQ ID NO:32. In some embodiments, the RSP02-binding agent comprises a heavy chain comprising SEQ ID NO:30 and a light chain comprising SEQ ID NO:32.

[0165] In certain embodiments, the invention provides methods comprising a RSP02-binding agent (e.g., an antibody) that specifically binds RSP02, wherein the RSP02-binding agent comprises: (a) a heavy chain having at least 90% sequence identity to SEQ ID NO:29 or SEQ ID NO:30; and/or (b) a light chain having at least 90% sequence identity to SEQ ID NO:67 or SEQ ID NO:68. In some embodiments, the RSP02-binding agent comprises: (a) a heavy chain having at least 95% sequence identity to SEQ ID NO:29 or SEQ ID NO:30; and/or (b) a light chain having at least 95% sequence identity to SEQ ID NO:67 or SEQ ID NO:68. In some embodiments, the RSP02-binding agent comprises a heavy chain comprising SEQ ID NO:30 and/or a light chain comprising SEQ ID NO:68. In some embodiments, the RSP02-binding agent comprises a heavy chain comprising SEQ ID NO:30 and a light chain comprising SEQ ID NO:68.

[0166] In certain embodiments, a RSP02-binding agent comprises the heavy chain variable region and light chain variable region of antibody hl30M23-HlL6. In certain embodiments, a RSP02-binding agent comprises the heavy chain and light chain of antibody hl30M23-HlL6 (with or without the leader sequence). In certain embodiments, a RSP02-binding agent is antibody hl30M23-HlL6. In certain embodiments, a RSP02-binding agent comprises the heavy chain variable region and/or light chain variable region of antibody hl30M23-HlL6 in a chimeric form of the antibody. In some embodiments, the anti-RSP02 antibody is hl30M23-HlL6.

[0167] In certain embodiments, a RSP02-binding agent comprises the heavy chain variable region and light chain variable region of antibody hl30M23-HlL2. In certain embodiments, a RSP02-binding agent comprises the heavy chain and light chain of antibody hl30M23-HlL2 (with or without the leader sequence). In certain embodiments, a RSP02-binding agent is antibody hl30M23-HlL2. In certain embodiments, a RSP02-binding agent comprises the heavy chain variable region and/or light chain variable region of antibody hl30M23-HlL2 in a chimeric form of the antibody. In some embodiments, the anti-RSP02 antibody is hl30M23-HlL2.

[0168] In certain embodiments of the methods described herein, a RSP02-binding agent comprises the heavy chain CDRs and/or light chain CDRs of antibody 130M23. The hybridoma cell line producing the 130M23 antibody was deposited with ATCC, 10801 University Boulevard, Manassas, VA, USA, under the conditions of the Budapest Treaty on August 10, 201 1 and assigned ATCC deposit designation number PTA- 12021.

[0169] In certain embodiments, a RSP02-binding agent comprises, consists essentially of, or consists of, antibody hl30M23-HlL6. In certain embodiments, a RSP02-binding agent comprises, consists essentially of, or consists of, a variant of antibody 130M23. In certain embodiments, a RSP02-binding agent comprises, consists essentially of, or consists of, a variant of antibody hl30M23-HlL6.

[0170] In certain embodiments, a RSP02-binding agent comprises, consists essentially of, or consists of, antibody hl30M23-HlL2. In certain embodiments, a RSP02-binding agent comprises, consists essentially of, or consists of, a variant of antibody 130M23. In certain embodiments, a RSP02-binding agent comprises, consists essentially of, or consists of, a variant of antibody hl30M23-HlL2.

[0171] In certain embodiments of the methods described herein, the invention provides methods comprising a 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 131R01 1 (see Table 1 herein).

[0172] In certain embodiments, the invention provides methods comprising a RSP03 -binding agent (e.g., an antibody) that specifically binds human RSP03, wherein the RSP03 -binding agent comprises a heavy chain CDR1 comprising DYSIH (SEQ ID NO:37), a heavy chain CDR2 comprising

YIYPSNGDSGYNQKFK (SEQ ID NO: 38), and a heavy chain CDR3 comprising TYFANNFD (SEQ ID NO:39) or ATYFANNFDY (SEQ ID NO:40). In some embodiments, the RSP03-binding agent further comprises a light chain CDR1 comprising KASQSVDYDGDSYMN (SEQ ID NO:41), a light chain CDR2 comprising AASNLES (SEQ ID NO:42) or AAS (SEQ ID NO:43), and a light chain CDR3 comprising QQSNEDPLT (SEQ ID NO:44) or QQSNEDPLTF (SEQ ID NO:45). In some embodiments, the RSP03-binding agent comprises a light chain CDR1 comprising KASQSVDYDGDSYMN (SEQ ID NO:41), a light chain CDR2 comprising AASNLES (SEQ ID NO:42) or AAS (SEQ ID NO:43), and a light chain CDR3 comprising QQSNEDPLT (SEQ ID NO:44) or QQSNEDPLTF (SEQ ID NO:45). In certain embodiments, the RSP03-binding agent comprises: (a) a heavy chain CDR1 comprising DYSIH (SEQ ID NO:37), a heavy chain CDR2 comprising YIYPSNGDSGYNQKFK (SEQ ID NO:38), and a heavy chain CDR3 comprising TYFANNFD (SEQ ID NO:39); and (b) a light chain CDR1 comprising KASQSVDYDGDSYMN (SEQ ID NO:41), a light chain CDR2 comprising AASNLES (SEQ ID NO:42), and a light chain CDR3 comprising QQSNEDPLT (SEQ ID NO:44).

[0173] In certain embodiments, the invention provides methods comprising a 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 CDR1 comprising DYSIH (SEQ ID NO:37) or a variant thereof comprising 1, 2, 3, or 4 amino acid substitutions; (b) a heavy chain CDR2 comprising

YIYPSNGDSGYNQKFK (SEQ ID NO:38) or a variant thereof comprising 1, 2, 3, or 4 amino acid substitutions; (c) a heavy chain CDR3 comprising TYFANNFD (SEQ ID NO:39), ATYFANNFDY (SEQ ID NO:40), or a variant thereof comprising 1, 2, 3, or 4 amino acid substitutions; (d) a light chain CDR1 comprising KASQSVDYDGDSYMN (SEQ ID NO:41) or a variant thereof comprising 1, 2, 3, or 4 amino acid substitutions; (e) a light chain CDR2 comprising AASNLES (SEQ ID NO:42), AAS (SEQ ID NO:43), or a variant thereof comprising 1, 2, 3, or 4 amino acid substitutions; and (f) a light chain CDR3 comprising QQSNEDPLT (SEQ ID NO:44), QQSNEDPLTF (SEQ ID NO:45), 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.

[0174] In certain embodiments, the invention provides a 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:46 and/or a light chain variable region having at least 80%> sequence identity to SEQ ID NO:47. 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:46. In certain embodiments, the RSP03-binding agent comprises a light chain variable region having at least about 85%o, at least about 90%>, at least about 95%>, at least about 97%>, or at least about 99%> sequence identity to SEQ ID NO:47. In certain embodiments, the RSP03-binding agent comprises a heavy chain variable region having at least about 95%> sequence identity to SEQ ID NO:46 and/or a light chain variable region having at least about 95% sequence identity to SEQ ID NO:47. In certain embodiments, the RSP03- binding agent comprises a heavy chain variable region comprising SEQ ID NO:46, and/or a light chain variable region comprising SEQ ID NO:47. In certain embodiments, the RSP03-binding agent comprises a heavy chain variable region comprising SEQ ID NO:46 and a light chain variable region comprising SEQ ID NO:47. In certain embodiments, the RSP03-binding agent comprises a heavy chain variable region consisting of SEQ ID NO:46 and a light chain variable region consisting of SEQ ID NO:47.

[0175] In certain embodiments, the invention provides a 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:48 or SEQ ID NO:49; and/or (b) a light chain having at least 90% sequence identity to SEQ ID NO:50 or SEQ ID NO:51. In some embodiments, the RSP03-binding agent comprises: (a) a heavy chain having at least 95% sequence identity to SEQ ID NO:48 or SEQ ID NO:49; and/or (b) a light chain having at least 95% sequence identity to SEQ ID NO:50 or SEQ ID NO:51. In some embodiments, the RSP03-binding agent comprises a heavy chain comprising SEQ ID NO:49 and/or a light chain comprising SEQ ID NO:51. In some embodiments, the RSP03-binding agent comprises a heavy chain comprising SEQ ID NO:49 and a light chain comprising SEQ ID NO:51.

[0176] In certain embodiments, a RSP03-binding agent comprises the heavy chain variable region and light chain variable region of antibody 131R010. In certain embodiments, a RSP03 -binding agent comprises the heavy chain and light chain of antibody 131R010 (with or without the leader sequence). In certain embodiments, a RSP03-binding agent is antibody 131R010. In certain embodiments, a RSP03- binding agent comprises the heavy chain variable region and/or light chain variable region of antibody 131 R010 in a chimeric form of the antibody. In certain embodiments, a 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.

[0177] 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 RSPO 3 -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 RSPO 3 -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.

[0178] In certain embodiments, a RSP03-binding agent comprises, consists essentially of, or consists of, antibody 131R010. In certain embodiments, a RSP03-binding agent comprises, consists essentially of, or consists of, a variant of antibody 131R010.

[0179] The invention provides methods comprising polypeptides, including, but not limited to, antibodies that specifically bind at least one human RSPO protein or antibodies that specifically bind at least one human LGR protein. In some embodiments, a polypeptide binds human RSPOl . In some embodiments, a polypeptide binds human RSP02. In some embodiments, a polypeptide binds human RSP03. In some embodiments, a polypeptide binds human LGR4. In some embodiments, a polypeptide binds human LGR5. In some embodiments, a polypeptide binds human LGR6. The invention also provides methods comprising polypeptides, wherein the polypeptide comprises a soluble receptor that specifically binds at least one human RSPO protein.

[0180] In certain embodiments, the polypeptide comprises one, two, three, four, five, and/or six of the CDRs of antibody 89M5 (see Table 1 herein). In certain embodiments, the polypeptide comprises one, two, three, four, five, and/or six of the CDRs of antibody 130M23 (see Table 1 herein). 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.

[0181] In some embodiments, the invention provides methods comprising a polypeptide that specifically binds a human RSPOl, wherein the polypeptide comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO: 1 1 and/or SEQ ID NO: 12. In some embodiments, the polypeptide comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO: 13 and/or an amino acid sequence having at least about 80%> sequence identity to 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: 14 and/or an amino acid sequence having at least about 80% sequence identity to SEQ ID NO: 16. 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: l 1 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16. In certain embodiments, the polypeptide comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 1 1 and/or an amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 12. In certain embodiments, the polypeptide comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 13 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: 14 and/or an amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 16. In certain embodiments, the polypeptide comprises an amino acid sequence of SEQ ID NO: 1 1 and/or an amino acid sequence of SEQ ID NO: 12. In certain embodiments, the polypeptide comprises an amino acid sequence of SEQ ID NO: 13 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: 14 and/or an amino acid sequence of SEQ ID NO: 16.

[0182] In some embodiments, the invention provides methods comprising a polypeptide that specifically binds a human RSPO l, wherein the polypeptide comprises an amino acid sequence having at least about 80%) sequence identity to SEQ ID NO:56 and/or SEQ ID NO:57. In some embodiments, the polypeptide comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:58 and/or an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:60. In some embodiments, the polypeptide comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:59 and/or an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:61. 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:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, or SEQ ID NO:61. In certain embodiments, the polypeptide comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO:56 and/or an amino acid sequence having at least about 95%) sequence identity to SEQ ID NO:57. In certain embodiments, the polypeptide comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO:58 and/or an amino acid sequence having at least about 95% sequence identity to SEQ ID NO:60. In certain embodiments, the polypeptide comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO:59 and/or an amino acid sequence having at least about 95% sequence identity to SEQ ID NO:61. In certain embodiments, the polypeptide comprises an amino acid sequence of SEQ ID NO:56 and/or an amino acid sequence of SEQ ID NO:57. In certain embodiments, the polypeptide comprises an amino acid sequence of SEQ ID NO:58 and/or an amino acid sequence of SEQ ID NO:60. In certain embodiments, the polypeptide comprises an amino acid sequence of SEQ ID NO:59 and/or an amino acid sequence of SEQ ID NO:61.

[0183] In some embodiments, the invention provides methods comprising a polypeptide that specifically binds a human RSP02, wherein the polypeptide comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:27 and/or SEQ ID NO:28. In some embodiments, the invention provides methods comprising a polypeptide that specifically binds a human RSP02, wherein the polypeptide comprises an amino acid sequence having at least about 80%> sequence identity to SEQ ID NO:27 and/or SEQ ID NO:66. In some embodiments, the polypeptide comprises an amino acid sequence having at least about 80%> sequence identity to SEQ ID NO:29 and/or an amino acid sequence having at least about 80%> sequence identity to SEQ ID NO:31 or SEQ ID NO:67. In some embodiments, the polypeptide comprises an amino acid sequence having at least about 80%> sequence identity to SEQ ID NO:30 and/or an amino acid sequence having at least about 80%> sequence identity to SEQ ID NO:32 or SEQ ID NO:68. In certain embodiments, the polypeptide comprises an amino acid sequence having at least about 85%>, at least about 90%>, at least about 95%>, at least about 97%>, or at least about 99%> sequence identity to SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:3 1 , SEQ ID NO:32, SEQ ID NO:66, SEQ ID NO:67, or SEQ ID NO:68. In certain embodiments, the polypeptide comprises an amino acid sequence having at least about 95%> sequence identity to SEQ ID NO:27 and/or an amino acid sequence having at least about 95%> sequence identity to SEQ ID NO:28 or SEQ ID NO:66. In certain embodiments, the polypeptide comprises an amino acid sequence having at least about 95%> sequence identity to SEQ ID NO:29 and/or an amino acid sequence having at least about 95%) sequence identity to SEQ ID NO:31 or SEQ ID NO:67. In certain embodiments, the polypeptide comprises an amino acid sequence having at least about 95%> sequence identity to SEQ ID NO:30 and/or an amino acid sequence having at least about 95%> sequence identity to SEQ ID NO:32 or SEQ ID NO:68. In certain embodiments, the polypeptide comprises an amino acid sequence of SEQ ID NO:27 and/or an amino acid sequence of SEQ ID NO:28. In certain embodiments, the polypeptide comprises an amino acid sequence of SEQ ID NO:27 and/or an amino acid sequence of SEQ ID NO:66. In certain embodiments, the polypeptide comprises an amino acid sequence of SEQ ID NO:29 and/or an amino acid sequence of SEQ ID NO:31. In certain embodiments, the polypeptide comprises an amino acid sequence of SEQ ID NO:29 and/or an amino acid sequence of SEQ ID NO:67. In certain embodiments, the polypeptide comprises an amino acid sequence of SEQ ID NO:30 and/or an amino acid sequence of SEQ ID NO:32. In certain embodiments, the polypeptide comprises an amino acid sequence of SEQ ID NO:30 and/or an amino acid sequence of SEQ ID NO:68. [0184] In some embodiments, the invention provides methods comprising 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:46 and/or SEQ DI NO:47. In some embodiments, the polypeptide comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:48 and/or an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:50. In some embodiments, the polypeptide comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:49 and/or an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:51. 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:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, or SEQ ID NO:51. In certain embodiments, the polypeptide comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO:46 and/or an amino acid sequence having at least about 95% sequence identity to SEQ ID NO:47. In certain embodiments, the polypeptide comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO:48 and/or an amino acid sequence having at least about 95% sequence identity to SEQ ID NO:50. In certain embodiments, the polypeptide comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO:49 and/or an amino acid sequence having at least about 95% sequence identity to SEQ ID NO:51. In certain embodiments, the polypeptide comprises an amino acid sequence of SEQ ID NO:46 and/or an amino acid sequence of SEQ ID NO:47. In certain embodiments, the polypeptide comprises an amino acid sequence of SEQ ID NO:48 and/or an amino acid sequence of SEQ ID NO:50. In certain embodiments, the polypeptide comprises an amino acid sequence of SEQ ID NO:49 and/or an amino acid sequence of SEQ ID NO: 51.

[0185] 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. 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 may be recognized and/or may 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 may comprise a mixture of polypeptides with different N-termini. In some embodiments, the N-termini differ in length by 1, 2, 3, 4, or 5 amino acids. In some 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.

[0186] In certain embodiments, the invention provides methods comprising a RSPO 1 -binding agent (e.g., antibody) that competes for specific binding to RSPOl with an antibody that comprises the CDRs of antibody 89M5. In certain embodiments, the invention provides methods comprising a RSP02-binding agent (e.g., antibody) that competes for specific binding to RSP02 with an antibody that comprises the CDRs of antibody 130M23. In certain embodiments, the invention provides methods comprising a RSP03-binding agent (e.g., antibody) that competes for specific binding to RSP03 with an antibody that comprises the CDRs of antibody 131R010.

[0187] In certain embodiments, the invention provides methods comprising a RSPO 1 -binding agent (e.g., an antibody) that binds the same epitope, or essentially the same epitope on RSPOl, as an antibody that comprises the CDRs of antibody 89M5. In certain embodiments, the invention provides methods comprising a RSP02-binding agent (e.g., an antibody) that binds the same epitope, or essentially the same epitope on RSP02, as an antibody that comprises the CDRs of antibody 89M5. In certain embodiments, the invention provides methods comprising a 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.

[0188] In certain embodiments, the invention provides methods comprising a RSPO 1 -binding agent (e.g., an antibody) that binds an epitope on RSPO l that overlaps with the epitope on RSPOl bound by an antibody comprising the CDRs of antibody 89M5. In certain embodiments, the invention provides methods comprising a RSP02-binding agent (e.g., an antibody) that binds an epitope on RSP02 that overlaps with the epitope on RSP02 bound by an antibody comprising the CDRs of antibody 130M23. In certain embodiments, the invention provides methods comprising a 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.

[0189] In certain embodiments of the methods described herein, a RSPO-binding agent (e.g., an antibody or soluble receptor) described herein binds at least one human RSPO protein and modulates RSPO activity. In some embodiments, the RSPO-binding agent is a RSPO antagonist and decreases RSPO activity. In some embodiments, the RSPO-binding agent is a RSPO antagonist and decreases β-catenin activity.

[0190] In certain embodiments, a RSPO 1 -binding agent (e.g., an antibody or soluble receptor) described herein binds human RSPO l and modulates RSPO l activity. In some embodiments, a RSPO 1 -binding agent is a RSPO 1 antagonist and decreases RSPO 1 activity. In some embodiments, a RSPO 1 -binding agent is a RSPO 1 antagonist and decreases β-catenin activity. In certain embodiments, a RSP02-binding agent (e.g., an antibody or soluble receptor) described herein binds human RSP02 and modulates RSP02 activity. In some embodiments, a RSP02-binding agent is a RSP02 antagonist and decreases RSP02 activity. In some embodiments, a RSP02-binding agent is a RSP02 antagonist and decreases β-catenin activity. In certain embodiments, a RSP03-binding agent (e.g., an antibody or soluble receptor) described herein binds human RSP03 and modulates RSP03 activity. In some embodiments, a RSP03- binding agent is a RSP03 antagonist and decreases RSP03 activity. In some embodiments, a RSP03- binding agent is a RSP03 antagonist and decreases β-catenin activity.

[0191] In certain embodiments, the RSPO-binding agent (e.g., an antibody or soluble receptor) 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 RSPO l , RSP02, RSP03, and/or RSP04.

[0192] In certain embodiments, the RSPO-binding agent (e.g., antibody or soluble receptor) 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 RSPO l , RSP02, RSP03, and/or RSP04.

[0193] In certain embodiments, the RSPO-binding agent (e.g., antibody or soluble receptor) 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%.

[0194] In certain embodiments, the RSPO-binding agent (e.g., antibody or soluble receptor) inhibits binding of at least one RSPO protein to a receptor. In certain embodiments, the RSPO-binding agent inhibits binding of a human RSPO protein to one or more of its receptors. In some embodiments, the RSPO-binding agent inhibits binding of a RSPO protein to at least one LGR protein. In some embodiments, the RSPO-binding agent inhibits binding of a RSPO protein to LGR4, LGR5, and/or LGR6. In certain embodiments, the inhibition of binding of a RSPO-binding agent to at least one LGR protein is at least about 10%, at least about 25%, at least about 50%, at least about 75%, at least about 90%, or at least about 95%. In certain embodiments, a RSPO-binding agent that inhibits binding of at least one RSPO to at least one LGR protein further inhibits β-catenin signaling.

[0195] In certain embodiments, the RSPO-binding agent (e.g., antibody or soluble receptor) blocks binding of at least one RSPO 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 a RSPO to at least one LGR protein. In some embodiments, the RSPO- binding agent blocks binding of at least one RSPO protein to LGR4, LGR5, and/or LGR6. In certain embodiments, the blocking of binding of a RSPO-binding agent to at least one LGR protein is at least about 10%), at least about 25%, at least about 50%, at least about 75%, at least about 90%, or at least about 95%). In certain embodiments, a RSPO-binding agent that blocks binding of at least one RSPO protein to at least one LGR protein further inhibits β-catenin signaling.

[0196] In certain embodiments, the RSPO-binding agent (e.g., an antibody or soluble receptor) inhibits β-catenin signaling. It is understood that a RSPO-binding agent that inhibits β-catenin signaling may, 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 may be inhibited. In certain embodiments, β-catenin signaling by one or more receptors selected from the group consisting of LGR4, LGR5, and LGR6 is inhibited. In certain embodiments, the inhibition of β-catenin signaling by a 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%.

[0197] In certain embodiments, the RSPO-binding agent (e.g., an antibody or soluble receptor) inhibits activation of β-catenin. It is understood that a RSPO-binding agent that inhibits activation of β-catenin may, 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 may be inhibited. In certain embodiments, activation of β-catenin by one or more receptors selected from the group consisting of LGR4, LGR5, and LGR6 is inhibited. In certain

embodiments, the inhibition of activation of β-catenin by a 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%.

[0198] In vivo and in vitro assays for determining whether a RSPO-binding agent or a LGR-binding agent (or candidate binding 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 may 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 a RSPO protein or RSPO- conditioned media in the presence of a RSPO-binding agent is compared to the level of signaling without the RSPO-binding agent present. In addition to the TCF/Luc reporter assay, the effect of a RSPO-binding agent or LGR-binding agent (or a candidate agent) on β-catenin signaling may 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 a RSPO-binding agent or LGR-binding agent on β-catenin signaling may 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.

[0199] In certain embodiments, the RSPO-binding agents or LGR-binding agents described herein have a circulating half-life in mice, cynomolgus monkeys, or humans of at least about 2 hours, at least about 5 hours, at least about 10 hours, at least about 24 hours, at least about 3 days, at least about 1 week, or at least about 2 weeks. In certain embodiments, the RSPO-binding agent or LGR-binding 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 2 hours, at least about 5 hours, at least about 10 hours, at least about 24 hours, at least about 3 days, at least about 1 week, or at least about 2 weeks. Methods of increasing (or decreasing) the 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) at pH 6.0. Known methods of increasing the circulating half-life of antibody fragments lacking the Fc region include such techniques as PEGylation. [0200] In some embodiments, the RSPO-binding agents or LGR-binding agents are polyclonal antibodies. Polyclonal antibodies can be prepared by any known method. In some embodiments, polyclonal antibodies are produced by immunizing an animal (e.g., a rabbit, rat, mouse, goat, donkey) with an antigen of interest (e.g., a purified peptide fragment, full-length recombinant protein, or fusion protein) using multiple subcutaneous or intraperitoneal injections. The antigen can be optionally conjugated to a carrier such as keyhole limpet hemocyanin (KLH) or serum albumin. The antigen (with or without a carrier protein) is diluted in sterile saline and usually combined with an adjuvant (e.g., Complete or Incomplete Freund's Adjuvant) to form a stable emulsion. After a sufficient period of time, polyclonal antibodies are recovered from the immunized animal, usually from blood or ascites. The polyclonal antibodies can be purified from serum or ascites according to standard methods in the art including, but not limited to, affinity chromatography, ion-exchange chromatography, gel electrophoresis, and dialysis.

[0201] In some embodiments, the RSPO-binding agents or LGR-binding agents are monoclonal antibodies. Monoclonal antibodies can be prepared using hybridoma methods known to one of skill in the art. In some embodiments, using the hybridoma method, a mouse, hamster, or other appropriate host animal, is immunized as described above to elicit from lymphocytes the production of antibodies that specifically bind the immunizing antigen. In some embodiments, lymphocytes can be immunized in vitro. In some embodiments, the immunizing antigen can be a human protein or a portion thereof. In some embodiments, the immunizing antigen can be a mouse protein or a portion thereof.

[0202] Following immunization, lymphocytes are isolated and fused with a suitable myeloma cell line using, for example, polyethylene glycol. The hybridoma cells are selected using specialized media as known in the art and unfused lymphocytes and myeloma cells do not survive the selection process.

Hybridomas that produce monoclonal antibodies directed specifically against a chosen antigen may be identified by a variety of methods including, but not limited to, immunoprecipitation, immunoblotting, and in vitro binding assays (e.g., flow cytometry, FACS, ELISA, and radioimmunoassay). The hybridomas can be propagated either in in vitro culture using standard methods or in vivo as ascites tumors in an animal. The monoclonal antibodies can be purified from the culture medium or ascites fluid according to standard methods in the art including, but not limited to, affinity chromatography, ion- exchange chromatography, gel electrophoresis, and dialysis.

[0203] In certain embodiments, monoclonal antibodies can be made using recombinant DNA techniques as known to one skilled in the art. The polynucleotides encoding a monoclonal antibody are isolated from mature B-cells or hybridoma cells, such as by RT-PCR using oligonucleotide primers that specifically amplify the genes encoding the heavy and light chains of the antibody, and their sequence is determined using standard techniques. The isolated polynucleotides encoding the heavy and light chains are then cloned into suitable expression vectors which produce the monoclonal antibodies when transfected into host cells such as E. coli, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin proteins.

[0204] In certain other embodiments, recombinant monoclonal antibodies, or fragments thereof, can be isolated from phage display libraries expressing variable domains or CDRs of a desired species.

[0205] The polynucleotide(s) encoding a monoclonal antibody can be modified, for example, by 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. Site- directed or high-density mutagenesis of the variable region can be used to optimize specificity, affinity, etc. of a monoclonal antibody.

[0206] In some embodiments of the methods described herein, a monoclonal antibody against a human RSPO protein or a LGR protein is a humanized antibody. Typically, humanized antibodies are human immunoglobulins in which residues from the CDRs are replaced by residues from a CDR 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 Fv framework region residues of a human immunoglobulin are replaced with the corresponding residues in an antibody from a non-human species. In some embodiments, a humanized antibody can be further modified by the substitution of additional residues either in the Fv framework region and/or within the replaced non- human residues to refine and optimize antibody specificity, affinity, and/or capability. In general, a 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, a 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 may reduce antigenicity and HAMA (human anti-mouse antibody) responses when administered to a human subject.

[0207] In certain embodiments of the methods described herein, the RSPO-binding agent or LGR- binding agent is a human antibody. Human antibodies can be directly prepared using various techniques known in the art. In some embodiments, human antibodies may be generated from immortalized human B lymphocytes immunized in vitro or from lymphocytes isolated from an immunized individual. In either case, cells that produce an antibody directed against a target antigen can be generated and isolated. In some embodiments, the human antibody can be selected from a phage library, where that phage library expresses human antibodies. Alternatively, phage display technology can be used to produce human antibodies and antibody fragments in vitro, from immunoglobulin variable domain gene repertoires from unimmunized donors. Techniques for the generation and use of antibody phage libraries are well known and phage libraries are commercially available. Once antibodies are identified, affinity maturation strategies known in the art, including but not limited to, chain shuffling, domain shuffling, and site- directed mutagenesis, may be employed to generate high affinity human antibodies.

[0208] In some embodiments, human antibodies can be made in transgenic mice that contain human immunoglobulin loci. Upon immunization these mice are capable of producing the full repertoire of human antibodies in the absence of endogenous immunoglobulin production.

[0209] This invention also encompasses bispecific antibodies that specifically recognize at least one human RSPO protein and/or at least one human LGR protein. Bispecific antibodies are capable of specifically recognizing and binding at least two different antigens or epitopes. The different epitopes can either be within the same molecule (e.g., two epitopes on human RSPOl) or on different molecules (e.g., one epitope on RSPOl and one epitope on LGR5). In some embodiments, a bispecific antibody has enhanced potency as compared to an individual antibody or to a combination of more than one antibody. In some embodiments, a bispecific antibody has reduced toxicity as compared to an individual antibody or to a combination of more than one antibody. It is known to those of skill in the art that any binding agent (e.g., antibody) may have unique pharmacokinetics (PK) (e.g., circulating half-life). In some embodiments, a bispecific antibody has the ability to synchronize the PK of two active binding agents wherein the two individual binding agents have different PK profiles. In some embodiments, a bispecific antibody has the ability to concentrate the actions of two binding agents (e.g., antibodies) in a common area (e.g., a specific tissue). In some embodiments, a bispecific antibody has the ability to concentrate the actions of two binding agents (e.g., antibodies) to a common target (e.g., a specific tissue). In some embodiments, a bispecific antibody has the ability to target the actions of two binding agents (e.g., antibodies) to more than one biological pathway or function.

[0210] In some embodiments, the bispecific antibody is a monoclonal antibody. In some embodiments, the bispecific antibody is a humanized antibody. In some embodiments, the bispecific antibody is a human antibody. In some embodiments, the bispecific antibody is an IgGl antibody. In some embodiments, the bispecific antibody is an IgG2 antibody. In some embodiments, the bispecific antibody has decreased toxicity and/or side effects. In some embodiments, the bispecific antibody has decreased toxicity and/or side effects as compared to a mixture of the two individual antibodies or the antibodies as single agents. In some embodiments, the bispecific antibody has an increased therapeutic index. In some embodiments, the bispecific antibody has an increased therapeutic index as compared to a mixture of the two individual antibodies or the antibodies as single agents.

[0211] In some embodiments, the antibodies can specifically recognize and bind a first antigen target, (e.g., RSP02) as well as a second antigen target, such as an effector molecule on a leukocyte (e.g., CD2, CD3, CD28, CTLA-4, 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 and/or producing 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, DPT A, DOT A, or TETA.

[0212] Techniques for making bispecific antibodies are known by those skilled in the art. In some embodiments, the bispecific antibodies comprise heavy chain constant regions with modifications in the amino acids which are part of the interface between the two heavy chains. In some embodiments, the bispecific antibodies can be generated using a "knobs-into-holes" strategy. In some cases, the "knobs" and "holes" terminology is replaced with the terms "protuberances" and "cavities". In some

embodiments, the bispecific antibodies may comprise variant hinge regions incapable of forming disulfide linkages between the heavy chains. In some embodiments, the modifications may comprise changes in amino acids that result in altered electrostatic interactions. In some embodiments, the modifications may comprise changes in amino acids that result in altered hydrophobic/hydrophilic interactions.

[0213] Bispecific antibodies can be intact antibodies or antibody fragments comprising antigen-binding sites. Antibodies with more than two valencies are also contemplated. Thus, in certain embodiments the antibodies to RSPO proteins are multispecific.

[0214] In certain embodiments, the antibodies (or other polypeptides) described herein may be monospecific. 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 RSPO proteins or a homologous epitope on LGR proteins. In certain embodiments, an antigen-binding site of a monospecific antibody described herein is capable of binding (or binds), for example, RSP02 and RSP03 (i.e., the same epitope is found on both RSP02 and RSP03 proteins).

[0215] In certain embodiments of the methods described herein, a RSPO-binding agent or LGR-binding agent is an antibody fragment. Antibody fragments may have different functions or capabilities than intact antibodies; for example, antibody fragments can have increased tissue 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. Fab, Fv, and scFv antibody fragments can be expressed in and secreted from E. coli or other host cells, allowing for the production of large amounts of these fragments. In some embodiments, antibody fragments are isolated from antibody phage libraries as discussed herein. For example, methods can be used for the construction of Fab expression libraries to allow rapid and effective identification of monoclonal Fab fragments with the desired specificity for a RSPO protein or derivatives, fragments, analogs or homologs thereof. In some embodiments, antibody fragments are linear antibody fragments. In certain embodiments, antibody fragments are monospecific or bispecific. In certain embodiments, the RSPO-binding agent is a scFv. Various techniques can be used for the production of single-chain antibodies specific to one or more human RSPO proteins or one or more human LGR proteins.

[0216] It can further be desirable, especially in the case of antibody fragments, to modify an antibody in order to alter (e.g., increase or decrease) 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).

[0217] Heteroconjugate antibodies are also within the scope of the present invention. Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune cells to unwanted cells. It is also contemplated that 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.

[0218] For the purposes of the present invention, it should be appreciated that modified antibodies can comprise any type of variable region that provides for the association of the antibody with the target (i.e., human RSPO). In this regard, the variable region may comprise or be derived from any type of mammal that can be induced to mount a humoral response and generate immunoglobulins against the desired 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 useful in the present invention can be humanized or otherwise altered through the inclusion of imported amino acid sequences.

[0219] 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 may be derived from an antibody of the same class or even subclass as the antibody from which the framework regions are derived, it is envisaged that the CDRs may be derived from an antibody of different class and often 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 required to maintain the activity of the antigen- binding site.

[0220] Alterations to the variable region notwithstanding, those skilled in the art will appreciate that the modified antibodies of this invention will comprise antibodies (e.g., full-length antibodies or

immunoreactive fragments 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 tissue localization or increased serum half-life when compared with an antibody of

approximately the same immunogenicity comprising a native or unaltered constant region. In some embodiments, the constant region of the modified antibodies will comprise a human constant region. Modifications to the constant region compatible with this invention comprise additions, deletions or substitutions of one or more amino acids in one or more domains. The modified antibodies disclosed herein may 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 (ACH2 constructs). In some embodiments, the omitted constant region domain is replaced by a short amino acid spacer (e.g., 10 amino acid residues) that provides some of the molecular flexibility typically imparted by the absent constant region.

[0221] 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 may be expressed wherein the CH2 domain has been deleted and the remaining CH3 domain (modified or unmodified) is joined to the hinge region with a 5-20 amino acid spacer. Such a spacer may be added to ensure that the regulatory elements of the constant domain remain free and accessible or that the hinge region remains flexible. However, it should be noted that amino acid spacers may, in some cases, prove to be immunogenic and elicit an unwanted immune response against the construct. Accordingly, in certain embodiments, any spacer added to the construct will be relatively non-immunogenic so as to maintain the desired biological qualities of the modified antibodies.

[0222] In some embodiments, the modified antibodies may have only a partial deletion of a constant domain or substitution of a few or even a single amino acid. For example, the mutation of a single amino acid in selected areas of the CH2 domain may be enough to substantially reduce Fc binding and thereby increase cell localization and/or tissue penetration. Similarly, it may be desirable to simply delete the part of one or more constant region domains that control a specific effector function (e.g. complement Clq binding) to be modulated. Such partial deletions of the constant regions may improve selected characteristics of the antibody (serum half-life) while leaving other desirable functions associated with the subject constant region domain intact. Moreover, as alluded to above, the constant regions of the disclosed antibodies may 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 may be possible to disrupt the activity provided by a conserved binding site (e.g., Fc binding) while substantially maintaining the configuration and immunogenic profile of the 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.

[0223] It is known in the art that 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 a 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 (called antibody- dependent cell cytotoxicity or ADCC), release of inflammatory mediators, placental transfer, and control of immunoglobulin production.

[0224] In certain embodiments, the modified antibodies provide for altered effector functions that, in turn, affect the biological profile of the administered antibody. For example, in some embodiments, the deletion or inactivation (through point mutations or other means) of a constant region domain may reduce Fc receptor binding of the circulating modified antibody thereby increasing cell localization and/or tissue penetration. In other embodiments, the constant region modifications increase the serum half-life of the antibody. In other embodiments, the constant region modifications reduce the serum half-life of the antibody. In some embodiments, the constant region is modified to eliminate disulfide linkages or oligosaccharide moieties. Modifications to the constant region in accordance with this invention may easily be made using well known biochemical or molecular engineering techniques.

[0225] In certain embodiments, a RSPO-binding agent or LGR-binding agent that is an antibody does not have one or more effector functions. For instance, in some embodiments, the antibody has no ADCC activity, and/or no complement-dependent cytotoxicity (CDC) activity. In certain embodiments, the antibody does not bind an Fc receptor, and/or complement factors. In certain embodiments, the antibody has no effector function.

[0226] The present invention further embraces variants and equivalents which are substantially homologous to the chimeric, humanized, and human antibodies, or antibody fragments thereof, set forth herein. These can contain, for example, conservative substitution mutations, i.e. the substitution of one or more amino acids by similar amino acids.

[0227] In certain embodiments, the antibodies described herein are isolated. In certain embodiments, the antibodies described herein are substantially pure.

[0228] In some embodiments of the present invention, the RSPO-binding agents or LGR-binding agents are polypeptides. The polypeptides can be recombinant polypeptides, natural polypeptides, or synthetic polypeptides comprising an antibody, or fragment thereof, that bind at least one RSPO protein and/or at least one LGR protein. It will be recognized in the art that some amino acid sequences of the invention can be varied without significant effect of the structure or function of the protein. Thus, the invention further includes variations of the polypeptides which show substantial activity or which include regions of an antibody, or fragment thereof, against a human RSPO protein or a human LGR protein. In some embodiments, amino acid sequence variations of RSPO-binding polypeptides or LGR-binding polypeptides include deletions, insertions, inversions, repeats, and/or other types of substitutions.

[0229] In certain embodiments, the polypeptides described herein are isolated. In certain embodiments, the polypeptides described herein are substantially pure. [0230] 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 or otherwise modulate the solubility, the biological half-life, and/or absorption of the polypeptide. The moieties can also reduce or eliminate undesirable side effects of the polypeptides and variants. An overview for chemical moieties can be found in Remington: The Science and Practice of Pharmacy, 22 ^nd Edition, 2012, Pharmaceutical Press, London.

[0231] The 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.

[0232] In some embodiments, a DNA sequence encoding a polypeptide of interest may be constructed by chemical synthesis using an oligonucleotide synthesizer. Oligonucleotides can be designed based on the amino acid sequence of the desired polypeptide and selecting those codons that are favored in the host cell in which the recombinant polypeptide of interest will be produced. Standard methods can be applied to synthesize a polynucleotide sequence encoding an isolated polypeptide of interest. For example, a complete amino acid sequence can be used to construct a back-translated gene. Further, a DNA oligomer containing a nucleotide sequence coding for the particular isolated polypeptide can be synthesized. For example, several small oligonucleotides coding for portions of the desired polypeptide can be synthesized and then ligated. The individual oligonucleotides typically contain 5' or 3' overhangs for complementary assembly.

[0233] Once assembled (by synthesis, site-directed mutagenesis, or another method), the polynucleotide sequences encoding a particular polypeptide of interest can be inserted into an expression vector and operatively linked to an expression control sequence appropriate for expression of the protein in a desired host. Proper assembly can be confirmed by nucleotide sequencing, restriction enzyme mapping, and/or expression of a biologically active polypeptide in a suitable host. As is well-known in the art, in order to obtain high expression levels of a transfected gene in a host, the gene must be operatively linked to transcriptional and translational expression control sequences that are functional in the chosen expression host.

[0234] In certain embodiments, recombinant expression vectors are used to amplify and express DNA encoding antibodies, or fragments thereof, against a human RSPO protein or a human LGR protein. For example, recombinant expression vectors can be replicable DNA constructs which have synthetic or cDNA-derived DNA fragments encoding a polypeptide chain of a RSPO-binding agent, such as an anti- RSPO antibody, or fragment thereof, operatively linked to suitable transcriptional and/or translational regulatory elements derived from mammalian, microbial, viral or insect genes. A transcriptional unit generally comprises an assembly of (1) a genetic element or elements having a regulatory role in gene expression, for example, transcriptional promoters or enhancers, (2) a structural or coding sequence which is transcribed into mRNA and translated into protein, and (3) appropriate transcription and translation initiation and termination sequences. Regulatory elements can include an operator sequence to control transcription. The ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants can additionally be incorporated. DNA regions are "operatively linked" when they are functionally related to each other. For example, DNA for a signal peptide (secretory leader) is operatively linked to DNA for a polypeptide if it is expressed as a precursor which participates in the secretion of the polypeptide; a promoter is operatively linked to a coding sequence if it controls the transcription of the sequence; or a ribosome binding site is operatively linked to a coding sequence if it is positioned so as to permit translation. In some embodiments, structural elements intended for use in yeast expression systems include a leader sequence enabling extracellular secretion of translated protein by a host cell. In other embodiments, in situations where recombinant protein is expressed without a leader or transport sequence, it can include an N-terminal methionine residue. This residue can optionally be subsequently cleaved from the expressed recombinant protein to provide a final product.

[0235] The choice of an expression control sequence and an expression vector depends upon the choice of host. A wide variety of expression host/vector combinations can be employed. Useful expression vectors for eukaryotic hosts include, for example, vectors comprising expression control sequences from SV40, bovine papilloma virus, adenovirus, and cytomegalovirus. Useful expression vectors for bacterial hosts include known bacterial plasmids, such as plasmids from E. coli, including pCRl, pBR322, pMB9 and their derivatives, and wider host range plasmids, such as Ml 3 and other filamentous single-stranded DNA phages.

[0236] The RSPO-binding agents or the LGR-binding agents (e.g., polypeptides or antibodies) of the present invention can be expressed from one or more vectors. For example, in some embodiments, one heavy chain polypeptide is expressed by one vector, a second heavy chain polypeptide is expressed by a second vector and a light chain polypeptide is expressed by a third vector. In some embodiments, a first heavy chain polypeptide and a light chain polypeptide is expressed by one vector and a second heavy chain polypeptide is expressed by a second vector. In some embodiments, two heavy chain polypeptides are expressed by one vector and a light chain polypeptide is expressed by a second vector. In some embodiments, three polypeptides are expressed from one vector. Thus, in some embodiments, a first heavy chain polypeptide, a second heavy chain polypeptide, and a light chain polypeptide are expressed by a single vector.

[0237] Suitable host cells for expression of a RSPO-binding agent or LGR-binding agent (or a RSPO or LGR protein to use as an antigen) include prokaryotes, yeast cells, insect cells, or higher eukaryotic cells under the control of appropriate promoters. Prokaryotes include gram-negative or gram-positive organisms, for example E. coli or Bacillus. Higher eukaryotic cells include established cell lines of mammalian origin as described below. Cell-free translation systems may also be employed. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts are known to one of skill in the art.

[0238] Various mammalian culture systems may be used to express recombinant polypeptides.

Expression of recombinant proteins in mammalian cells may be desirable because these proteins are generally correctly folded, appropriately modified, and biologically functional. Examples of suitable mammalian host cell lines include, but are not limited to, COS-7 (monkey kidney-derived), L-929 (murine fibrob last-derived), CI 27 (murine mammary tumor-derived), 3T3 (murine fibroblast-derived), CHO (Chinese hamster ovary-derived), HeLa (human cervical cancer-derived), BHK (hamster kidney fibroblast-derived), HEK-293 (human embryonic kidney-derived) cell lines and variants thereof.

Mammalian expression vectors can comprise non-transcribed elements such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, and other 5' or 3' flanking non- transcribed sequences, and 5' or 3' non-translated sequences, such as necessary ribosome binding sites, a polyadenylation site, splice donor and acceptor sites, and transcriptional termination sequences.

[0239] Expression of recombinant proteins in insect cell culture systems (e.g., baculovirus) also offers a robust method for producing correctly folded and biologically functional proteins. Baculovirus systems for production of heterologous proteins in insect cells are well-known to those of skill in the art.

[0240] The proteins produced by a transformed host can be purified according to any suitable method. Standard methods include chromatography (e.g., ion exchange, affinity, and sizing column

chromatography), centrifugation, differential solubility, or by any other standard technique for protein purification. Affinity tags such as hexa-histidine, maltose binding domain, influenza coat sequence, and glutathione-S-transferase can be attached to the protein to allow easy purification by passage over an appropriate affinity column. Affinity chromatography used for purifying immunoglobulins can include Protein A, Protein G, and Protein L chromatography. Isolated proteins can be physically characterized using such techniques as proteolysis, size exclusion chromatography (SEC), mass spectrometry (MS), nuclear magnetic resonance (NMR), isoelectric focusing (IEF), high performance liquid chromatography (HPLC), and x-ray crystallography. The purity of isolated proteins can be determined using techniques known to those of skill in the art, including but not limited to, SDS-PAGE, SEC, capillary gel electrophoresis, IEF, and capillary isoelectric focusing (cIEF).

[0241] In some embodiments, supernatants from expression systems which secrete recombinant protein into culture media can be first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. Following the concentration step, the concentrate can be applied to a suitable purification matrix. In some embodiments, an anion exchange resin can be employed, for example, a matrix or substrate having pendant diethylaminoethyl (DEAE) groups. The matrices can be acrylamide, agarose, dextran, cellulose, or other types commonly employed in protein purification. In some embodiments, a cation exchange step can be employed. Suitable cation exchangers include various insoluble matrices comprising sulfopropyl or carboxymethyl groups. In some embodiments, a hydroxyapatite media can be employed, including but not limited to, ceramic hydroxyapatite (CHT). In certain embodiments, one or more reverse-phase HPLC steps employing hydrophobic RP-HPLC media, e.g., silica gel having pendant methyl or other aliphatic groups, can be employed to further purify a recombinant protein (e.g., a RSPO-binding agent). Some or all of the foregoing purification steps, in various combinations, can be employed to provide a homogeneous recombinant protein.

[0242] In some embodiments, heterodimeric proteins such as bispecific antibodies are purified according the any of the methods described herein. In some embodiments, anti-RSPO or anti-LGR bispecific antibodies are isolated and/or purified using at least one chromatography step. In some embodiments, the at least one chromatography step comprises affinity chromatography. In some embodiments, the at least one chromatography step further comprises anion exchange chromatography. In some embodiments, the isolated and/or purified antibody product comprises at least 90% heterodimeric antibody. In some embodiments, the isolated and/or purified antibody product comprises at least 95%, 96%>, 97%, 98% or 99% heterodimeric antibody. In some embodiments, the isolated and/or purified antibody product comprises about 100% heterodimeric antibody.

[0243] In some embodiments, recombinant protein produced in bacterial culture can be isolated, for example, by initial extraction from cell pellets, followed by one or more concentration, salting-out, aqueous ion exchange, or size exclusion chromatography steps. HPLC can be employed for final purification steps. Microbial cells employed in expression of a recombinant protein can be disrupted by any convenient method, including freeze -thaw cycling, sonication, mechanical disruption, or use of cell lysing agents. [0244] In certain embodiments, the RSPO-binding agents or LGR-binding agents can be used in any one of a number of conjugated (i.e. an immunoconjugate or radioconjugate) or non-conjugated forms. In certain embodiments, the antibodies can be used in a non-conjugated form to harness the subject's natural defense mechanisms including complement-dependent cytotoxicity and antibody dependent cellular toxicity to eliminate hyperproliferative cells.

[0245] In some embodiments, the RSPO-binding agent or LGR-binding agent 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, non-binding 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, ⁹⁰ Y, ¹²⁵ I, ¹³¹ I, ¹²³ I, ^m In, ¹³¹ In, ¹⁰⁵ Rh, ¹⁵³ Sm, ⁶⁷ Cu, ⁶⁷ Ga, ¹⁶⁶ Ho, ¹⁷⁷ Lu, ¹⁸⁶ Re,

188 212

Re and Bi. Conjugates of an antibody and one or more small molecule toxins, such as

calicheamicins, maytansinoids, trichothenes, and CC1065, and the derivatives of these toxins that have toxin activity, can also be used. Conjugates of an antibody and cytotoxic agent may be 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 HQ), 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).

IV. Polynucleotides

[0246] In certain embodiments, the invention encompasses polynucleotides comprising polynucleotides that encode a polypeptide (or a fragment of a polypeptide) that specifically binds at least one human RSPO protein or at least one human LGR protein. The term "polynucleotides that encode a polypeptide" encompasses a polynucleotide which includes only coding sequences for the polypeptide as well as a polynucleotide which includes additional coding and/or non-coding sequences. For example, in some embodiments, the invention provides a polynucleotide comprising a polynucleotide sequence that encodes an antibody to a human RSPO protein or encodes a fragment of such an antibody (e.g., a fragment comprising the antigen-binding site). In some embodiments, the invention provides a polynucleotide comprising a polynucleotide sequence that encodes an antibody to a human LGR protein or encodes a fragment of such an antibody (e.g., a fragment comprising the antigen-binding site). The polynucleotides of the invention can be in the form of RNA or in the form of DNA. DNA includes cDNA, genomic DNA, and synthetic DNA; and can be double-stranded or single-stranded, and if single stranded can be the coding strand or non-coding (anti-sense) strand.

[0247] In certain embodiments, the polynucleotide comprises a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 1 1, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:66, SEQ ID NO:67, and SEQ ID NO:68. In some embodiments, the polynucleotide comprises a polynucleotide sequence selected from the group consisting of: SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:69, and SEQ ID NO:70.

[0248] In certain embodiments, the polynucleotide comprises a polynucleotide having a nucleotide sequence at least about 80% identical, at least about 85%> identical, at least about 90%> identical, at least about 95%) identical, and in some embodiments, at least about 96%>, 97%>, 98%> or 99%> identical to a polynucleotide comprising a sequence selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, and SEQ ID NO:55. Also provided is a polynucleotide that comprises a polynucleotide that hybridizes to SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:69, and SEQ ID NO:70. Also provided is a polynucleotide that comprises a polynucleotide that hybridizes to the complement of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:69, and SEQ ID NO:70. In certain embodiments, the hybridization is under conditions of high stringency. [0249] In certain embodiments, the polynucleotides comprise the coding sequence for the mature polypeptide fused in the same reading frame to a polynucleotide which aids, for example, in expression and secretion of a polypeptide from a host cell (e.g., a leader sequence which functions as a secretory sequence for controlling transport of a polypeptide from the cell). The polypeptide having a leader sequence is a preprotein and can have the leader sequence cleaved by the host cell to form the mature form of the polypeptide. The polynucleotides can also encode for a proprotein which is the mature protein plus additional 5' amino acid residues. A mature protein having a prosequence is a proprotein and is an inactive form of the protein. Once the prosequence is cleaved an active mature protein remains.

[0250] In certain embodiments, the polynucleotides comprise the coding sequence for the mature polypeptide fused in the same reading frame to a marker sequence that allows, for example, for purification of the encoded polypeptide. For example, the marker sequence can be a hexa-histidine tag supplied by a pQE-9 vector to provide for purification of the mature polypeptide fused to the marker in the case of a bacterial host, or the marker sequence can be a hemagglutinin (HA) tag derived from the influenza hemagglutinin protein when a mammalian host (e.g., COS-7 cells) is used. In some embodiments, the marker sequence is a FLAG-tag which can be used in conjunction with other affinity tags.

[0251] The present invention further relates to variants of the hereinabove described polynucleotides encoding, for example, fragments, analogs, and/or derivatives.

[0252] In certain embodiments, the present invention provides polynucleotides comprising

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

[0253] As used herein, the phrase a polynucleotide having a nucleotide sequence at least, for example, 95%) "identical" to a reference nucleotide sequence is intended to mean that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence can include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence can be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence can be inserted into the reference sequence. These mutations of the reference sequence can occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.

[0254] The polynucleotide variants can contain alterations in the coding regions, non-coding regions, or both. In some embodiments, a polynucleotide variant contains alterations which produce silent substitutions, additions, or deletions, but does not alter the properties or activities of the encoded polypeptide. In some embodiments, a polynucleotide variant comprises silent substitutions that result in no change to the amino acid sequence of the polypeptide (due to the degeneracy of the genetic code). In some embodiments, nucleotide variants comprise nucleotide sequences which result in expression differences (e.g., increased or decreased expression) at the transcript level. Polynucleotide variants can be produced for a variety of reasons, for example, to optimize codon expression for a particular host (i.e., change codons in the human mRNA to those preferred by a bacterial host such as E. coli). In some embodiments, a polynucleotide variant comprises at least one silent mutation in a non-coding or a coding region of the sequence.

[0255] In some embodiments, a polynucleotide variant is produced to modulate or alter expression (or expression levels) of the encoded polypeptide. In some embodiments, a polynucleotide variant is produced to increase expression of the encoded polypeptide. In some embodiments, a polynucleotide variant is produced to decrease expression of the encoded polypeptide. In some embodiments, a polynucleotide variant has increased expression of the encoded polypeptide as compared to a parental polynucleotide sequence. In some embodiments, a polynucleotide variant has decreased expression of the encoded polypeptide as compared to a parental polynucleotide sequence.

[0256] In some embodiments, at least one polynucleotide variant is produced (without changing the amino acid sequence of the encoded polypeptide) to increase production of a heteromultimeric molecule. In some embodiments, at least one polynucleotide variant is produced (without changing the amino acid sequence of the encoded polypeptide) to increase production of a bispecific antibody.

[0257] In certain embodiments, the polynucleotides are isolated. In certain embodiments, the polynucleotides are substantially pure.

[0258] Vectors comprising the polynucleotides described herein are also provided. Cells comprising the polynucleotides described herein are also provided. In some embodiments, an expression vector comprises a polynucleotide molecule. In some embodiments, a host cell comprises an expression vector comprising the polynucleotide molecule. In some embodiments, a host cell comprises a polynucleotide molecule. V. Kits comprising RSPO-binding agents

[0259] The present invention provides kits that comprise the RSPO-binding agents or LGR-binding agents (e.g., antibodies or soluble receptors) described herein and that can be used to perform the methods described herein. In certain embodiments, a kit comprises at least one purified antibody against at least one human RSPO protein in one or more containers. In certain embodiments, a kit comprises at least one purified antibody against at least one human LGR protein in one or more containers. In certain embodiments, a kit comprises at least one purified soluble receptor that binds at least one human RSPO protein in one or more containers. In some embodiments, the kits contain all of the components necessary and/or sufficient to perform a detection assay, including all controls, directions for performing assays, and any necessary software for analysis and presentation of results. One skilled in the art will readily recognize that the disclosed RSPO-binding agents and/or LGR-binding proteins of the present invention can be readily incorporated into one of the established kit formats which are well known in the art.

[0260] Further provided are kits comprising a RSPO-binding agent or a LGR-binding agent (e.g., an antibody or a soluble receptor), as well as at least one additional therapeutic agent. In certain

embodiments, the second (or more) therapeutic agent is an anti-inflammatory agent. In certain embodiments, the second (or more) therapeutic agent is a Wnt pathway inhibitor.

[0261] Embodiments of the present disclosure can be further defined by reference to the following non- limiting examples, which describe in detail preparation of certain antibodies of the present disclosure and methods for using antibodies of the present disclosure. It will be apparent to those skilled in the art that many modifications, both to materials and methods, may be practiced without departing from the scope of the present disclosure.

EXAMPLES

Example 1

Lung fibrosis model

[0262] Induction of lung fibrosis in a mouse model is described in Baran et al., 201 1, Am. J. Respir. Cell Mol. Biol, 45:999-1006 and Dakhlallah et al., 2013, Am J. Respir. Crit. Care Med., 187:397-405 and this model was generally followed. Briefly, 6 to 8-week-old C57BL/6N mice were injected intraperitoneally with 0.035U/g bleomycin or vehicle (saline). ΙΟΟμΙ of bleomycin dissolved in 0.9% NaCl at a concentration of 7U/ml was administered to mice on Day 1, 4, 9, 12, 15, 18, 22, 25, 29, and 32. On Day 9, 16, 22, 29, and 33 five representative mice were euthanized. Tissue fragments from the lungs were harvested for gene expression analysis. The remaining lung tissue was perfused with formalin and stored for histological analysis.

[0263] Histological analysis of the lung tissue from mice injected with bleomycin showed evidence of fibrosis at Day 16, with maximum fibrosis observed at Day 29. Gene expression of Rspol, Rspo2, and Rspo3 in lung tissue from mice injected with bleomycin was assessed by quantitative RT-PCR. Gene expression in lung tissue from mice injected with bleomycin at a given timepoint is relative to gene expression in lung tissue from mice injected with saline. For analysis, glucoronidase beta (Gusb) was used for normalization.

[0264] As shown in Figure 1, Rspo2 expression in lung tissue from mice injected with bleomycin was up-regulated at Day 9 and Day 16, followed by down-regulation on Day 22, 26, and 33. In contrast, gene expression of Rspol and Rspo3 was down-regulated at Day 9 and continued to be down-regulated through Day 33.

[0265] The elevation of Rspo2 gene expression detected at Day 9 and Day 16 coincided with the presence of infiltrating inflammatory cells into the lung tissue and the onset of lung fibrosis, suggesting that RSPO proteins, particularly RSP02, may be involved in the fibrotic process.

Example 2

Skin fibrosis model

[0266] Induction of dermal fibrosis in a mouse model using bleomycin is described in Distler et al., 2007, Arthritis & Rheumatism, 56(1 ):31 1-322 and this model was generally followed. Briefly, 6 to 8- week-old mice were injected with bleomycin by subcutaneous injection into defined regions of the upper back. Injection site regions were approximately 1cm ² in size and located on the dorsolateral region of the mouse's back (approximately 2cm laterally from the spinal cord). There were two injection sites per mouse. ΙΟΟμΙ of bleomycin dissolved in 0.9% NaCl at a concentration of 0.5mg/ml was administered on Day 1, 3, 5, 8, and 10. On Day 2, 5, 8, and 12, four representative mice were euthanized. Tissue fragments from the injection sites were harvested for gene expression analysis and formalin- fixed for histological analysis.

[0267] Gene expression in skin tissue from mice injected with bleomycin at a given timepoint is relative to gene expression in skin tissue from na ^' ive mice. For analysis, glucuronidase beta was used for normalization.

[0268] As shown in Figure 2, Rspo3 expression in skin tissue at the bleomycin injection site was up- regulated 24 hours after the first injection (Day 2) and peaked at Day 5. Rspo3 was still up-regulated on Day 8 and Day 12, but expression levels had decreased from Day 5. Expression levels of Rspol did not appear to be affected at the bleomycin injection site in this model, while Rspo2 appeared to be slightly up-regulated.

[0269] The observed elevation of Rspo3 gene expression as early as 24 hours after the first injection of bleomycin suggests that RSPO proteins may be involved in the early events of fibrosis. The strong up- regulation of Rspo3 gene expression in the dermal fibrosis model contrasts with the induction of Rspo2 gene expression in the lung fibrosis model. These results suggest that there may be tissue-specific roles for RSPO proteins in the onset and progression of different types of fibrosis.

Example 3

Effect of anti-RSPO antibodies on induction of skin lesions by bleomycin

[0270] Dermal fibrosis was induced in mice as described above in Example 2. Six to 8-week-old mice were injected with bleomycin by subcutaneous injection into two defined regions of the upper back. Ι ΟΟμΙ of bleomycin dissolved in 0.9% NaCl at a concentration of 0.5mg/ml was administered on Day 1, 3, 5, 8, and 10. Control antibody 1B7.1 1 , anti-RSP02 antibody 130M23, anti-RSP03 antibody 131R010, or a combination of anti-RSP02 antibody 130M23 and anti-RSP03 antibody 131R010 were administered at a dose of 25mg/kg on Day 1 and 8. Four mice were treated in the control antibody group and eight mice were treated in each anti-RSPO antibody group.

[0271] On Day 12, palpable skin lesions were present at injection sites on each of the control antibody mice, 8 of 8 injection sites, for a frequency of 100%. In mice treated with anti-RSP02 antibody 130M23 there were palpable skin lesions at 13 of 16 injection sites, for a frequency of 81%. In mice treated with anti-RSP03 antibody 131R010, there were palpable lesions at 6 of 16 injection sites for a frequency of 38%). In mice treated with a combination of anti-RSP02 antibody 130M23 and anti-RSP03 antibody 131R010, there were palpable lesions at only 2 of 16 injection sites for a frequency of 13%>. These results are represented in Figure 3.

[0272] Tissue from the injection site regions was harvested on Day 12 and the tissue was used for gene expression analysis and for histological analysis. Gene expression of several genes was assessed by quantitative RT-PCR. Genes included chemokine (C-X-C motif) ligand 1 (Cxcll), endoglin (Eng), interferon gamma (Ifng), interleukin 16 (1116), tumor necrosis factor (Tnf), axin2 (Axin2), leucine-rich repeat containing G protein-coupled receptor 5 (Lgr5), R-spondin 1 (Rspol), R-spondin 3 (Rspo3), and smooth muscle actin (Acta2).

[0273] As shown in Figure 4, treatment with a combination of anti-RSP02 antibody 130M23 and anti- RSP03 131R010 reduced expression of genes involved in inflammation including interferon gamma, tumor necrosis factor, and endoglin (as assessed on Day 12). This treatment also reduced gene expression of Wnt-pathway related gene Lgr5 and to a lesser extent Axin2. At Day 12, treatment with anti-RSP02 antibody 130M23 and anti-RSP03 antibody 131R010 as single agents did not reduce expression of these genes. Interestingly, 24 hours after administration of a single dose of bleomycin and a single dose of anti- RSPO antibodies, gene expression of interferon gamma was significantly reduced by the anti-RSPO antibodies as single agents as well as in combination (Figure 5).

[0274] These results show that treatment with an anti-RSP03 antibody either alone, or in combination with an anti-RSP02 antibody has the ability to strongly inhibit formation of dermal lesions induced by bleomycin. The reduction in the incidence of palpable dermal lesions and the down-regulation of genes associated with Wnt signaling and inflammation by administration with anti-RSP02 antibody 130M23 in combination with anti-RSP03 antibody 131R010 suggests that multiple RSPO proteins may be involved in the onset and development of fibrosis. These results support the hypothesis that anti-RSPO

antagonists, such as anti-RSPO antibodies, may be efficacious in treating fibrotic diseases.

[0275] 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 person skilled in the art and are to be included within the spirit and purview of this application.

[0276] All publications, patents, patent applications, internet sites, and accession numbers/database sequences including both polynucleotide and polypeptide sequences cited herein are hereby incorporated by reference herein in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, internet site, or accession number/database sequence were specifically and individually indicated to be so incorporated by reference.

[0277] The sequences disclosed in the application are:

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

MRLGLCWALVLSWTHLTI SSRGIKGKRQRRISAEGSQACAKGCELCSEVNGCLKCSPKL FILLERNDIRQVGVCLPSCPPGYFDARNPDMNKCIKCKIEHCEACFSHNFCTKCKEGLYL HKGRCYPACPEGSSAANGTMECSSPAQCEMSEWSPWGPCSKKQQLCGFRRGSEERTRRVL HAPVGDHAACSDTKETRRCTVRRVPCPEGQKRRKGGQGRRENANRNLARKESKEAGAGSR RRKGQQQQQQQGTVGPLTSAGPA

Human RSP02 amino acid sequence with signal sequence (SEQ ID NO:2)

MQFRLFSFALI ILNCMDYSHCQGNRWRRSKRASYVSNPICKGCLSCSKDNGCSRCQQKLF FFLRREGMRQYGECLHSCPSGYYGHRAPDMNRCARCRIENCDSCFSKDFCTKCKVGFYLH RGRCFDECPDGFAPLEETMECVEGCEVGHWSEWGTCSRNNRTCGFKWGLETRTRQIVKKP VKDTIPCPTIAESRRCKMTMRHCPGGKRTPKAKEKRNKKKKRKLIERAQEQHSVFLATDR ANQ

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

MHLRLI SWLFI ILNFMEYIGSQNASRGRRQRRMHPNVSQGCQGGCATCSDYNGCLSCKPR LFFALERIGMKQIGVCLSSCPSGYYGTRYPDINKCTKCKADCDTCFNKNFCTKCKSGFYL HLGKCLDNCPEGLEANNHTMECVS IVHCEVSEWNPWSPCTKKGKTCGFKRGTETRVREI I QHPSAKGNLCPPTNETRKCTVQRKKCQKGERGKKGRERKRKKPNKGESKEAIPDSKSLES SKEIPEQRENKQQQKKRKVQDKQKSVSVSTVH

Human RSP04 amino acid sequence with signal sequence (SEQ ID N0:4)

MRAPLCLLLLVAHAVDMLALNRRKKQVGTGLGGNCTGCI ICSEENGCSTCQQRLFLFIRR EGIRQYGKCLHDCPPGYFGIRGQEVNRCKKCGATCESCFSQDFCIRCKRQFYLYKGKCLP TCPPGTLAHQNTRECQGECELGPWGGWSPCTHNGKTCGSAWGLESRVREAGRAGHEEAAT CQVLSESRKCPIQRPCPGERSPGQKKGRKDRRPRKDRKLDRRLDVRPRQPGLQP

89M5 Heavy chain CDR1 (SEQ ID N0:5)

TGYTMH

89M5 Heavy chain CDR2 (SEQ ID N0:6)

GINPNNGGTTYNQNFKG

89M5 Heavy chain CDR3 (SEQ ID N0:7)

KEFSDGYYFFAY

89M5 Light chain CDR1 (SEQ ID N0:8)

KASQDVIFAVA

89M5 Light chain CDR2 (SEQ ID N0:9)

WASTRHT

89M5 Light chain CDR3 (SEQ ID NO: 10)

QQHYSTPW h89M5-H8L5 Heavy chain variable region amino acid sequence (SEQ ID NO: l 1)

EVQLVQSGAEVKKPGESLRI SCKGSGYSFTGYTMHWVRQMPGKGLEWMGGINPNNGGTTY NQNFKGHVTISADKSI STAYLQWSSLKASDTAMYYCARKEFSDGYYFFAYWGQGTLVTVS

S

h89M5-H8L5 Light chain variable region amino acid sequence (SEQ ID NO: 12)

DIVMTQSPDSLAVSLGERATINCKASQDVIFAVAWYQQKPGQPPKLLIYWASTRHTGVPD RFSGSGSGTDFTLTISSLQAEDVAVYYCQQHYSTPWTFGGGTKVEIK

h89M5-H8L5 Heavy chain amino acid sequence with predicted signal sequence underlined (SEQ ID NO: 13)

MDWTWRILFLVAAATGAHSEVQLVQSGAEVKKPGESLRISCKGSGYSFTGYTMHWVRQMP GKGLEWMGGINPNNGGTTYNQNFKGHVTISADKSISTAYLQWSSLKASDTAMYYCARKEF SDGYYFFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEP KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCVWDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK h89M5-H8L5 Heavy chain amino acid sequence without predicted signal sequence (SEQ ID NO: 14) EVQLVQSGAEVKKPGESLRISCKGSGYSFTGYTMHWVRQMPGKGLEWMGGINPNNGGTTY

NQNFKGHVTI SADKS ISTAYLQWSSLKASDTAMYYCARKEFSDGYYFFAYWGQGTLVTVS SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLG GPSVFLFPPKPKDTLMI SRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

h89M5-H8L5 Light chain amino acid sequence with predicted signal sequence underlined (SEQ ID NO: 15)

MVLQTQVFISLLLWISGAYGDIVMTQSPDSLAVSLGERATINCKASQDVIFAVAWYQQKP GQPPKLLIYWASTRHTGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHYSTPWTFGG GTKVEIKRTVAAPSVFI FPPSDEQLKSGTASWCLLN FYPREAKVQWKVDNALQSGNSQ ESVTEQDSKDSTYSLSNTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

h89M5-H8L5 Light chain amino acid sequence without predicted signal sequence (SEQ ID NO: 16)

DIVMTQSPDSLAVSLGERATI CKASQDVI FAVAWYQQKPGQPPKLLIYWASTRHTGVPD RFSGSGSGTDFTLTI SSLQAEDVAVYYCQQHYSTPWTFGGGTKVEIKRTVAAPSVFI FPP SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSNTLT LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

h89M5-H8L5 Heavy chain variable region nucleotide sequence (SEQ ID NO: 17)

GAAGTGCAGCTGGTGCAGTCTGGAGCAGAGGTCAAAAAGCCCGGGGAGTCTCTGAGGATC TCCTGCAAGGGTTCTGGATACAGCTTTACTGGATACACCATGCACTGGGTGCGCCAGATG CCCGGGAAAGGACTGGAGTGGATGGGGGGTATTAATCCTAACAATGGTGGTACTACTTAC AACCAGAACTTCAAGGGCCACGTCACCATCTCAGCTGACAAGTCCATCAGCACTGCCTAC CTGCAATGGAGCAGCCTGAAGGCTTCTGACACCGCCATGTATTACTGTGCAAGAAAGGAG TTCTCTGATGGATACTACTTTTTTGCTTACTGGGGCCAAGGGACCCTGGTGACCGTCAGC TCA

h89M5-H8L5 Light chain variable region nucleotide sequence (SEQ ID NO: 18)

GACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACC ATCAACTGCAAGGCTTCCCAGGACGTGATTTTTGCTGTTGCCTGGTATCAGCAGAAACCA GGACAGCCTCCTAAGCTGCTCATTTACTGGGCATCTACCCGCCACACTGGGGTCCCTGAC CGCTTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTCCAGGCT GAAGATGTGGCAGTTTATTACTGTCAGCAACATTATAGCACTCCTTGGACTTTCGGCGGA GGGACCAAGGTGGAGATCAAA h89M5-H8L5 Heavy chain nucleotide sequence (SEQ ID NO: 19)

ATGGACTGGACCTGGAGGATACTCTTTCTCGTGGCGGCAGCCACAGGAGCCCACTCCGAA GTGCAGCTGGTGCAGTCTGGAGCAGAGGTCAAAAAGCCCGGGGAGTCTCTGAGGATCTCC TGCAAGGGTTCTGGATACAGCTTTACTGGATACACCATGCACTGGGTGCGCCAGATGCCC GGGAAAGGACTGGAGTGGATGGGGGGTATTAATCCTAACAATGGTGGTACTACTTACAAC CAGAACTTCAAGGGCCACGTCACCATCTCAGCTGACAAGTCCATCAGCACTGCCTACCTG CAATGGAGCAGCCTGAAGGCTTCTGACACCGCCATGTATTACTGTGCAAGAAAGGAGTTC TCTGATGGATACTACTTTTTTGCTTACTGGGGCCAAGGGACCCTGGTGACCGTCAGCTCA GCCAGCACAAAGGGCCCCTCCGTGTTCCCTCTGGCCCCTTCCTCCAAGTCCACCTCCGGC GGCACCGCCGCTCTGGGCTGCCTGGTGAAGGACTACTTCCCTGAGCCTGTGACCGTGTCC TGGAACTCTGGCGCACTGACCTCTGGCGTGCACACCTTCCCAGCCGTGCTCCAGTCCTCC GGCCTGTACTCCCTGTCCTCCGTCGTCACCGTGCCTTCCTCCTCCCTGGGCACCCAGACC TACATCTGCAACGTGAACCACAAGCCTTCCAACACAAAGGTGGACAAGCGGGTGGAGCCT AAGTCCTGCGACAAGACCCACACCTGCCCTCCCTGCCCTGCCCCTGAGCTGCTGGGCGGA CCTTCCGTGTTCCTGTTCCCTCCTAAGCCTAAGGACACCCTGATGATCTCCCGGACCCCT GAAGTGACATGCGTGGTGGTGGACGTGTCCCACGAGGACCCTGAGGTGAAGTTCAACTGG TATGTGGACGGCGTGGAGGTGCACAACGCTAAGACCAAGCCGAGGGAGGAGCAGTACAAC TCCACCTACCGGGTGGTGTCTGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAA GAATACAAGTGCAAGGTCTCCAACAAGGCCCTGCCCGCTCCCATCGAGAAAACCATCAGC AAGGCAAAGGGCCAGCCTCGCGAGCCTCAGGTGTACACCCTGCCACCCAGCCGGGAGGAG ATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAGGGCTTTTACCCTTCCGATATT GCCGTGGAGTGGGAGTCTAACGGCCAGCCCGAGAACAACTACAAGACCACCCCTCCTGTG CTGGACTCCGACGGCTCCTTCTTCCTGTACTCCAAGCTGACCGTGGACAAGTCCCGGTGG CAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACC CAGAAGAGCCTGTCTCTGTCTCCTGGCAAGTGA h89M5-H8L5 Light chain nucleotide sequence (SEQ ID NO:20)

ATGGTGCTCCAGACCCAGGTCTTCATTTCTCTGCTCCTCTGGATCTCTGGTGCCTACGGG GACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACC ATCAACTGCAAGGCTTCCCAGGACGTGATTTTTGCTGTTGCCTGGTATCAGCAGAAACCA GGACAGCCTCCTAAGCTGCTCATTTACTGGGCATCTACCCGCCACACTGGGGTCCCTGAC CGCTTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTCCAGGCT GAAGATGTGGCAGTTTATTACTGTCAGCAACATTATAGCACTCCTTGGACTTTCGGCGGA GGGACCAAGGTGGAGATCAAACGGACTGTGGCTGCACCATCTGTCTTCATCTTCCCTCCA TCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTAT CCCAGAGAGGCCAAAGTCCAGTGGAAGGTGGATAACGCCCTCCAATCCGGTAACTCCCAG GAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAACACCCTGACA CTGAGCAAAGCAGACTACGAGAAACACAAAGTCTATGCCTGCGAAGTCACCCATCAGGGC CTGTCTTCCCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGCTAA

130M23 Heavy chain CDR1 (SEQ ID NO:21)

SSYAMS

130M23 Heavy chain CDR2 (SEQ ID NO:22)

SISSGGSTYYPDSVKG

130M23 Heavy chain CDR3 (SEQ ID NO:23)

RGGDPGVYNGDYEDAMDY

130M23 Light chain CDR1 (SEQ ID NO:24)

KASQDVSSAVA

130M23 Light chain CDR2 (SEQ ID NO:25)

WASTRHT

130M23 Light chain CDR3 (SEQ ID NO:26)

QQHYSTP hl30M23-HlL6 Heavy chain variable region amino acid sequence (SEQ ID NO:27)

EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSI SSGGSTYYP DSVKGRFTI SRDNAKNSLYLQMNSLRAEDTAVYYCARGGDPGVYNGDYEDAMDYWGQGTT VTVSS hl30M23-HlL6 Light chain variable region amino acid sequence (SEQ ID NO:28)

DIQMTQSPSSLSASVGDRVTITCKASQDVSSAVAWYQQKPGKAPKLLIYWASTRHTGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQHYSTPWTFGQGTKVEIK

hl30M23-HlL6 Heavy chain amino acid sequence with predicted signal sequence underlined (SEQ ID NO:29)

MELGLRWVFLVAILEGVQCEVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMSWVRQAP GKGLEWVSS I SSGGSTYYPDSVKGRFTI SRDNAKNSLYLQMNSLRAEDTAVYYCARGGDP GVYNGDYEDAMDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSNFGTQTYTCNVDHKPSNTKVDK TVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVQFNW YVDGVEVHNAKTKPREEQFNSTFRWSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTIS KTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPM LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK hl30M23-HlL6 Heavy chain amino acid sequence without predicted signal sequence (SEQ ID NO:30)

EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSI SSGGSTYYP DSVKGRFTI SRDNAKNSLYLQMNSLRAEDTAVYYCARGGDPGVYNGDYEDAMDYWGQGTT VTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSWTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVA GPSVFLFPPKPKDTLMI SRTPEVTCVWDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQF NSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTI SKTKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK hl30M23-HlL6 Light chain amino acid sequence with predicted signal sequence underlined (SEQ ID NO:31)

MGIKMESQIQAFVFVFLWLSGVDGDIQMTQSPSSLSASVGDRVTITCKASQDVSSAVAWY QQKPGKAPKLLIYWASTRHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHYSTPW TFGQGTKVEIKRTVAAPSVFI FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSNTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

hl30M23-HlL6 Light chain amino acid sequence without predicted signal sequence (SEQ ID NO:32)

DIQMTQSPSSLSASVGDRVTITCKASQDVSSAVAWYQQKPGKAPKLLIYWASTRHTG VPS RFSGSGSGTDFTLTI SSLQPEDFATYYCQQHYSTPWTFGQGTKVEIKRTVAAPSVFI FPP SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSNTLT LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

hl30M23-HlL6 Heavy chain variable region nucleotide sequence (SEQ ID NO:33)

GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCCTGGTCAAGCCTGGAGGATCTCTGCGGCTC TCCTGTGCAGCCTCTGGATTCACCTTCTCCTCTTATGCCATGTCTTGGGTCCGGCAGGCT CCAGGGAAGGGGCTGGAATGGGTCTCATCCATTTCTAGTGGAGGTAGCACATATTATCCT GACAGCGTGAAGGGCCGGTTCACCATCTCCAGAGACAACGCCAAGAACAGCCTGTATCTG CAAATGAACAGCCTGAGAGCCGAGGACACAGCTGTGTATTACTGTGCTAGAGGTGGAGAT CCTGGGGTCTACAATGGAGATTACGAAGATGCTATGGACTACTGGGGGCAAGGAACAACA GTCACAGTCAGCTCA hl30M23-HlL6 Light chain variable region nucleotide sequence (SEQ ID NO:34)

GACATCCAGATGACCCAGTCCCCTTCCTCCCTGTCTGCTTCCGTCGGAGACAGAGTCACC ATCACTTGCAAGGCCTCCCAGGATGTGTCCTCTGCTGTCGCTTGGTATCAGCAGAAACCA GGAAAAGCTCCTAAGCTCCTGATCTATTGGG^ATrrArrA rACACAGGAGTCCCTTCC AGGTTCTCCGGCTCTGGATCTGGGACAGATTTCACTCTCACCATCAGCTCCCTGCAACCT GAAGATTTTGCAACTTACTACTGTCAGCAACATTATAGCACTCCTTGGACATTCGGACAA GGGACCAAGGTGGAAATCAAA hl30M23-HlL6 Heavy chain nucleotide sequence (SEQ ID NO: 35)

ATGGAACTGGGACTCAGATGGGTTTTCCTCGTTGCTATTCTGGAAGGAGTCCAGTGTGAG GTGCAGCTGGTGGAGTCTGGGGGAGGCCTGGTCAAGCCTGGAGGATCTCTGCGGCTCTCC TGTGCAGCCTCTGGATTCACCTTCTCCTCTTATGCCATGTCTTGGGTCCGGCAGGCTCCA GGGAAGGGGCTGGAATGGGTCTCATCCATTTCTAGTGGAGGTAGCACATATTATCCTGAC AGCGTGAAGGGCCGGTTCACCATCTCCAGAGACAACGCCAAGAACAGCCTGTATCTGCAA ATGAACAGCCTGAGAGCCGAGGACACAGCTGTGTATTACTGTGCTAGAGGTGGAGATCCT GGGGTCTACAATGGAGATTACGAAGATGCTATGGACTACTGGGGGCAAGGAACAACAGTC ACAGTCAGCTCAGCCAGCACAAAGGGCCCTAGCGTCTTCCCTCTGGCTCCCTGCAGCAGG AGCACCAGCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCG GTGACGGTGTCGTGGAACTCAGGCGCTCTGACCAGCGGCGTGCACACCTTCCCAGCTGTC CTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAACTTC GGCACCCAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAG ACAGTTGAGCGCAAATGTTGTGTCGAGTGCCCACCGTGCCCAGCACCACCTGTGGCAGGA CCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCT GAGGTCACGTGCGTGGTGGTGGACGTGAGCCACGAAGACCCCGAGGTCCAGTTCAACTGG TACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCACGGGAGGAGCAGTTCAAC AGCACGTTCCGTGTGGTCAGCGTCCTCACCGTTGTGCACCAGGACTGGCTGAACGGCAAG GAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCAGCCCCCATCGAGAAAACCATCTCC AAAACCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAG ATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATC GCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACACCTCCCATG CTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGG CAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACG CAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA hl30M23-HlL6 Light chain nucleotide sequence (SEQ ID NO: 36)

ATGGGCATCAAGATGGAGTCACAGATTCAGGCATTTGTATTCGTGTTTCTCTGGTTGTCT GGTGTTGACGGAGACATCCAGATGACCCAGTCCCCTTCCTCCCTGTCTGCTTCCGTCGGA GACAGAGTCACCATCACTTGCAAGGCCTCCCAGGATGTGTCCTCTGCTGTCGCTTGGTAT CAGCAGAAACCAGGAAAAGCTCCTAAGCTCCTGATCTATTGGGCATCCACCAGGCACACA GGAGTCCCTTCCAGGTTCTCCGGCTCTGGATCTGGGACAGATTTCACTCTCACCATCAGC TCCCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAGCAACATTATAGCACTCCTTGG ACATTCGGACAAGGGACCAAGGTGGAAATCAAAAGAACTGTGGCTGCACCTTCTGTCTTC ATCTTCCCTCCATCTGATGAGCAGCTCAAATCTGGAACTGCCTCCGTTGTGTGCCTGCTG AATAACTTCTATCCTAGAGAGGCCAAAGTCCAGTGGAAGGTGGATAACGCCCTCCAATCC GGTAACTCCCAGGAGTCTGTCACAGAGCAGGACTCCAAGGACAGCACCTACTCCCTCAGC AACACCCTGACACTGTCTAAAGCTGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTC ACCCATCAGGGACTGAGCTCCCCCGTCACAAAATCCTTCAACAGGGGAGAGTGCTAA

131R010 Heavy chain CDR1 (SEQ ID NO:37)

DYSIH

131R010 Heavy chain CDR2 (SEQ ID NO:38)

YIYPSNGDSGYNQKFK

131R010 Heavy chain CDR3 (SEQ ID ΝΟ:39 TYFANNFD

131R010 Alternative Heavy chain CDR3 (SEQ ID NO:40)

ATYFANNFDY

131R010 Light chain CDR1 (SEQ ID N0:41)

KASQSVDYDGDSYMN

131R010 Light chain CDR2 (SEQ ID NO:42)

AASNLES

131R010 Alternative Light chain CDR2 (SEQ ID NO:43)

AAS

131R010 Light chain CDR3 (SEQ ID NO:44)

QQSNEDPLT

131R010 Alternative Light chain CDR3 (SEQ ID NO:45)

QQSNEDPLTF

131R010 Heavy chain variable region amino acid sequence (SEQ ID NO:46)

QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYSIHWVRQAPGQGLEWIGYIYPSNGDSGY NQKFKNRVTMTRDTSTSTAYMELSRLRSEDTAVYYCATYFANNFDYWGQGTTLTVSS

131R010 Light chain variable region amino acid sequence (SEQ ID NO:47)

DIQMTQSPSSLSASVGDRVTITCKASQSVDYDGDSYMNWYQQKPGKAPKLLIYAASNLES GVPSRFSGSGSGTDFTLTI SPVQAEDFATYYCQQSNEDPLTFGAGTKLELKR

131R010 Heavy chain amino acid sequence with predicted signal sequence underlined (SEQ ID NO:48)

MKHLWFFLLLVAAPRWVLSQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYS IHWVRQAP GQGLEWIGYIYPSNGDSGYNQKFKNRVTMTRDTSTSTAYMELSRLRSEDTAVYYCATYFA NNFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

131 RO 10 Heavy chain amino acid sequence without predicted signal sequence (SEQ ID NO :49)

QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYSIHWVRQAPGQGLEWIGYIYPSNGD SGY NQKFKNRVTMTRDTSTSTAYMELSRLRSEDTAVYYCATYFANNFDYWGQGTTLTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY SLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK

131R010 Light chain amino acid sequence with predicted signal sequence underlined (SEQ ID NO:50)

MKHLWFFLLLVAAPRWVLSDIQMTQSPSSLSASVGDRVTITCKASQSVDYDGDSYMN WYQ QKPGKAPKLLIYAASNLESGVPSRFSGSGSGTn^TT.TT.qPA/nA .OFATYYCQQSNEDPLT FGAGTKLELKRTVAAPSVFI FPPS DEQLKS GTASWCLLNNFYPREAKVQWKVDNALQSG NSQE SVTEQDSKDSTYS LS STLTLSKADYEKHKVYACEVTHQGLS S PVTKS FNRGEC

131 RO 10 Light chain amino acid sequence without predicted signal sequence (SEQ ID NO : 51 )

DIQMTQS PS S LSASVGDRVT I TCKASQSVDYDGDSYMNWYQQKPGKAPKLL IYAASNLES GVPSRFSGS GSGTDFTLT I S PVQAEDFATYYCQQSNEDPLTFGAGTKLELKRTVAAPSVF I FPPS DEQLKSGTASWCLLNNFYPREAKVQWKVDNALQS GNSQE SVTEQDSKDSTYSLS S TLTLSKADYEKHKVYACEVTHQGLS S PVTKS FNRGEC

131R010 Heavy chain variable region nucleotide sequence (SEQ ID NO:52)

CAAGTGCAATTGGTGCAGTCCGGAGCGGAAGTGAAGAAGCCTGGTGCCTCGGTCAAAGTC TCATGCAAGGCCAGCGGATACACTTTCACCGACTACTCCATCCATTGGGTGAGGCAGGCT CCGGGCCAGGGCCTGGAGTGGATTGGGTACATCTACCCGTCGAACGGAGATTCGGGGTAC AATCAGAAGTTCAAGAACCGCGTGACCATGACTCGGGACACCTCAACTTCCACGGCTTAT ATGGAACTGAGCCGCCTGAGATCCGAGGACACTGCGGTGTACTACTGTGCCACCTACTTT GCGAACAATTTCGATTACTGGGGACAAGGAACCACGCTCACTGTCAGCTC

131R010 Light chain variable region nucleotide sequence (SEQ ID NO:53)

GATATCCAGATGACTCAGTCGCCCTCATCGTTGAGCGCCTCGGTCGGGGATCGCGTGACT ATTACTTGTAAAGCGTCCCAGAGCGTGGACTACGACGGAGATTCCTACATGAACTGGTAT CAGCAAAAACCGGGAAAGGCTCCTAAACTTCTCATCTACGCAGCCTCGAATCTGGAATCA GGAGTCCCGAGCCGGTTCAGCGGATCAGGCTCCGGTACTGATTTTACCCTCACGATCTCG CCAGTGCAAGCCGAGGACTTCGCGACCTACTACTGCCAACAGTCCAACGAGGACCCGCTG ACCTTCGGCGCAGGGACCAAGCTGGAACTGAAGCGT

131 RO 10 Heavy chain nucleotide sequence (SEQ ID NO :54)

ATGAAACACTTGTGGTTCTTTCTGCTCCTTGTCGCAGCACCACGGTGGGTGCTGTCGCAA GTGCAATTGGTGCAGTCCGGAGCGGAAGTGAAGAAGCCTGGTGCCTCGGTCAAAGTCTCA TGCAAGGCCAGCGGATACACTTTCACCGACTACTCCATCCATTGGGTGAGGCAGGCTCCG GGCCAGGGCCTGGAGTGGATTGGGTACATCTACCCGTCGAACGGAGATTCGGGGTACAAT CAGAAGTTCAAGAACCGCGTGACCATGACTCGGGACACCTCAACTTCCACGGCTTATATG GAACTGAGCCGCCTGAGATCCGAGGACACTGCGGTGTACTACTGTGCCACCTACTTTGCG AACAATTTCGATTACTGGGGACAAGGAACCACGCTCACTGTCAGCTCAGCCAGCACCAAG GGCCCCTCCGTGTTCCCTCTGGCCCCTTCCTCCAAGTCCACCTCCGGCGGCACCGCCGCT CTGGGCTGCCTGGTGAAGGACTACTTCCCTGAGCCTGTGACCGTGTCCTGGAACTCTGGC GCCCTGACCTCTGGCGTGCACACCTTCCCAGCCGTGCTGCAGTCCTCCGGCCTGTACTCC CTGTCCTCCGTGGTGACCGTGCCTTCCTCCTCCCTGGGCACCCAGACCTACATCTGCAAC GTGAACCACAAGCCTTCCAACACCAAGGTGGACAAGCGGGTGGAGCCTAAGTCCTGCGAC AAGACCCACACCTGCCCTCCCTGCCCTGCCCCTGAGCTGCTGGGCGGACCTTCCGTGTTC CTGTTCCCTCCTAAGCCTAAGGACACCCTGATGATCTCCCGGACCCCTGAGGTGACCTGC GTGGTGGTGGACGTGTCCCACGAGGATCCTGAGGTGAAGTTCAATTGGTACGTGGACGGC GTGGAGGTGCACAACGCTAAGACCAAGCCAAGGGAGGAGCAGTACAACTCCACCTACCGG GTGGTGTCTGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAATACAAGTGC AAGGTCTCCAACAAGGCCCTGCCCGCTCCCATCGAGAAAACCATCTCCAAGGCCAAGGGC CAGCCTCGCGAGCCTCAGGTGTACACCCTGCCACCCAGCCGGGAGGAGATGACCAAGAAC CAGGTGTCCCTGACCTGTCTGGTGAAGGGCTTCTACCCTTCCGATATCGCCGTGGAGTGG GAGTCTAACGGCCAGCCCGAGAACAACTACAAGACCACCCCTCCTGTGCTGGACTCCGAC GGCTCCTTCTTCCTGTACTCCAAGCTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAAC GTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTG TCTCTGTCTCCTGGCAAGTGATAA 131R010 Light chain nucleotide sequence (SEQ ID NO:55)

ATGAAACACCTGTGGTTCTTCCTCCTGCTGGTGGCAGCTCCCAGATGGGTCCTGTCCGAT ATCCAGATGACTCAGTCGCCCTCATCGTTGAGCGCCTCGGTCGGGGATCGCGTGACTATT ACTTGTAAAGCGTCCCAGAGCGTGGACTACGACGGAGATTCCTACATGAACTGGTATCAG CAAAAACCGGGAAAGGCTCCTAAACTTCTCATCTACGCAGCCTCGAATCTGGAATCAGGA GTCCCGAGCCGGTTCAGCGGATCAGGCTCCGGTACTGATTTTACCCTCACGATCTCGCCA GTGCAAGCCGAGGACTTCGCGACCTACTACTGCCAACAGTCCAACGAGGACCCGCTGACC TTCGGCGCAGGGACCAAGCTGGAACTGAAGCGTACGGTGGCCGCTCCATCCGTGTTTATC TTTCCGCCGTCCGATGAGCAGCTCAAGTCGGGCACTGCCAGCGTGGTCTGCCTGCTTAAC AATTTCTACCCTAGGGAAGCCAAGGTGCAGTGGAAGGTGGATAACGCGCTCCAATCCGGT AACTCGCAAGAGAGCGTGACCGAACAGGACTCAAAGGACTCGACGTACAGCCTGTCATCG ACCTTGACTCTCTCAAAGGCCGACTACGAAAAGCACAAGGTCTACGCGTGCGAAGTCACC CATCAGGGACTGTCCTCGCCTGTGACCAAGAGCTTCAATCGCGGAGAGTGCTGA h89M5-H2L2 Heavy chain variable region amino acid sequence (SEQ ID NO:56)

QVQLVQSGAEVKKPGASVKVSCKTSGYTFTGYTMHWVRQAPGQRLEWMGGINPNNGGTTY NQNFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCARKEFSDGYYFFAYWGQGTLVTVS

S

h89M5-H2L2 Light chain variable region amino acid sequence (SEQ ID NO:57)

DIQMTQSPSSLSASVGDRVTITCKASQDVIFAVAWYQQKPGKAPKLLIYWASTRHTGVPS RFSGSGSGTDYTLTI SSLQPEDFATYYCQQHYSTPWTFGGGTKVEIK

h89M5-H2L2 Heavy chain amino acid sequence with predicted signal sequence underlined (SEQ ID NO:58)

MDWTWRILFLVAAATGAHSQVQLVQSGAEVKKPGASVKVSCKTSGYTFTGYTMHWVRQAP GQRLEWMGGINPNNGGTTYNQNFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCARKEF SDGYYFFAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVER KCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVQFNWYVDG VEVHNAKTKPREEQFNSTFRVVSVLTWHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK h89M5-H2L2 Heavy chain amino acid sequence without predicted signal sequence (SEQ ID NO:59)

QVQLVQSGAEVKKPGASVKVSCKTSGYTFTGYTMHWVRQAPGQRLEWMGGINPNNGG TTY NQNFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCARKEFSDGYYFFAYWGQGTLVTVS SASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSV FLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTF RWSVLTWHQDWLNGKEYKCKVSNKGLPAPIEKTI SKTKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK h89M5-H2L2 Light chain amino acid sequence with predicted signal sequence underlined (SEQ ID NO: 60)

MDMRVPAQLLGLLLLWLRGARCDIQMTQSPSSLSASVGDRVTITCKASQDVIFAVAWYQQ KPGKAPKLLIYWASTRHTGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQHYSTPWTF GGGTKVEIKRTVAAPSVFI FPPSDEQLKSGTASWCLLN FYPREAKVQWKVDNALQSGN SQESVTEQDSKDSTYSLSNTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC h89M5-H2L2 Light chain amino acid sequence without predicted signal sequence (SEQ ID NO:61)

DIQMTQSPSSLSASVGDRVTITCKASQDVIFAVAWYQQKPGKAPKLLIYWASTRHTG VPS RFSGSGSGTDYTLTI SSLQPEDFATYYCQQHYSTPWTFGGGTKVEIKRTVAAPSVFI FPP SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSNTLT LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

h89M5-H2L2 Heavy chain variable region nucleotide sequence (SEQ ID NO:62)

CAGGTCCAGCTCGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCTGTGAAGGTT TCCTGCAAGACTTCTGGATACACCTTCACTGGATACACCATGCACTGGGTTAGACAGGCC CCCGGACAAAGGCTGGAGTGGATGGGAGGTATTAATCCTAACAATGGTGGTACTACTTAC AACCAGAACTTCAAGGGCAGAGTCACCATTACCAGGGACACATCCGCAAGCACAGCCTAC ATGGAGCTGTCCAGCCTGAGATCTGAAGACACAGCTGTGTATTACTGTGCAAGAAAGGAG TTCTCTGATGGATACTACTTTTTTGCTTACTGGGGCCAAGGGACCCTGGTCACCGTCAGC TCA h89M5-H2L2 Light chain variable region nucleotide sequence (SEQ ID NO:63)

GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTCGGAGACAGAGTCACC ATCACTTGCAAGGCCTCCCAGGATGTGATTTTTGCTGTTGCCTGGTATCAGCAGAAACCA GGGAAAGCCCCTAAGCTCCTGATCTATTGGGCATCCACCCGGCACACTGGGGTCCCATCA AGGTTCAGTGGCAGTGGATCTGGGACAGATTACACTCTCACCATCAGCAGTCTGCAACCT GAAGATTTTGCAACTTACTACTGTCAGCAACATTATAGCACTCCTTGGACTTTCGGCGGA GGGACCAAGGTGGAGATCAAA

h89M5-H2L2 Heavy chain nucleotide sequence (SEQ ID NO:64)

ATGGACTGGACCTGGAGGATACTCTTTCTCGTGGCAGCAGCCACAGGAGCCCACTCCCAG GTCCAGCTCGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCTGTGAAGGTTTCC TGCAAGACTTCTGGATACACCTTCACTGGATACACCATGCACTGGGTTAGACAGGCCCCC GGACAAAGGCTGGAGTGGATGGGAGGTATTAATCCTAACAATGGTGGTACTACTTACAAC CAGAACTTCAAGGGCAGAGTCACCATTACCAGGGACACATCCGCAAGCACAGCCTACATG GAGCTGTCCAGCCTGAGATCTGAAGACACAGCTGTGTATTACTGTGCAAGAAAGGAGTTC TCTGATGGATACTACTTTTTTGCTTACTGGGGCCAAGGGACCCTGGTCACCGTCAGCTCA GCCAGCACAAAGGGCCCTAGCGTCTTCCCTCTGGCTCCCTGCAGCAGGAGCACCAGCGAG AGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCG TGGAACTCAGGCGCTCTGACCAGCGGCGTGCACACCTTCCCAGCTGTCCTACAGTCCTCA GGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAACTTCGGCACCCAGACC TACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGACAGTTGAGCGC AAATGTTGTGTCGAGTGCCCACCGTGCCCAGCACCACCTGTGGCAGGACCGTCAGTCTTC CTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACGTGC GTGGTGGTGGACGTGAGCCACGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGC GTGGAGGTGCATAATGCCAAGACAAAGCCACGGGAGGAGCAGTTCAACAGCACGTTCCGT GTGGTCAGCGTCCTCACCGTTGTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGC AAGGTCTCCAACAAAGGCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGG CAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAAC CAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGG GAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACACCTCCCATGCTGGACTCCGAC GGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAAC GTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTC TCCCTGTCTCCGGGTAAATGA

h89M5-H2L2 Light chain nucleotide sequence (SEQ ID NO:65)

ATGGACATGAGGGTCCCCGCACAGCTCCTGGGGCTCCTGCTCCTCTGGCTCCGGGGTGCC AGATGTGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTCGGAGACAGA GTCACCATCACTTGCAAGGCCTCCCAGGATGTGATTTTTGCTGTTGCCTGGTATCAGCAG AAACCAGGGAAAGCCCCTAAGCTCCTGATCTATTGGGCATCCACCCGGCACACTGGGGTC CCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTACACTCTCACCATCAGCAGTCTG CAACCTGAAGATTTTGCAACTTACTACTGTCAGCAACATTATAGCACTCCTTGGACTTTC GGCGGAGGGACCAAGGTGGAGATCAAACGGACTGTGGCTGCACCATCTGTCTTCATCTTC CCTCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAAC TTCTATCCCAGAGAGGCCAAAGTCCAGTGGAAGGTGGATAACGCCCTCCAATCCGGTAAC TCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAACACC CTGACACTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCAT CAGGGCCTGAGCTCCCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGCTAA

hl30M23-HlL2 Light chain variable region amino acid sequence (SEQ ID NO:66)

DIQMTQSPSSLSASVGDRVTITCKASQDVSSAVAWYQQKPGKAPKLLIYWASTRHTGVPS RFSGSGSGTDFTLTISSVQAEDFATYYCQQHYSTPWTFGQGTKVEIK

hl30M23-HlL2 Light chain amino acid sequence with predicted signal sequence underlined (SEQ ID NO: 67)

MKYLLPTAAAGLLLLAAQPAMADIQMTQSPSSLSASVGDRVTITCKASQDVSSAVAWYQQ KPGKAPKLLIYWASTRHTGVPSRFSGSGSGTDFTLTISSVQAEDFATYYCQQHYSTPWTF GQGTKVEIKRTVAAPSVFI FPPSDEQLKSGTASWCLLN FYPREAKVQWKVDNALQSGN SQESVTEQDSKDSTYSLSNTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

hl30M23-HlL2 Light chain amino acid sequence without predicted signal sequence underlined (SEQ ID NO:68)

DIQMTQSPSSLSASVGDRVTITCKASQDVSSAVAWYQQKPGKAPKLLIYWASTRHTGVPS RFSGSGSGTDFTLTI SSVQAEDFATYYCQQHYSTPWTFGQGTKVEIKRTVAAPSVFI FPP SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSNTLT LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

hl30M23-HlL2 Light chain variable region nucleotide sequence (SEQ ID NO:69)

GACATCCAGATGACCCAGTCCCCTTCCTCCCTGTCTGCTTCCGTCGGAGACAGAGTCACC ATCACTTGCAAGGCCTCCCAGGATGTGTCCTCTGCTGTCGCTTGGTATCAGCAGAAACCA GGAAAAGCTCCTAAGCTCCTGATCTATTGGGCATCCACCAGGCACACAGGAGTCCCTTCC AGGTTCTCCGGCTCTGGATCTGGGACAGATTTCACTCTCACCATCAGCTCCGTGCAAGCT GAAGATTTTGCAACTTACTACTGTCAGCAACATTATAGCACTCCTTGGACATTCGGACAA GGGACCAAGGTGGAAATCAAA

hl30M23-HlL2 Light chain nucleotide sequence (SEQ ID NO:70)

ATGAAATACCTCCTCCCTACAGCTGCCGCTGGACTCCTCCTCCTCGCTGCCCAGCCTGCC ATGGCCGACATCCAGATGACCCAGTCCCCTTCCTCCCTGTCTGCTTCCGTCGGAGACAGA GTCACCATCACTTGCAAGGCCTCCCAGGATGTGTCCTCTGCTGTCGCTTGGTATCAGCAG AAACCAGGAAAAGCTCCTAAGCTCCTGATCTATTGGGCATCCACCAGGCACACAGGAGTC CCTTCCAGGTTCTCCGGCTCTGGATCTGGGACAGATTTCACTCTCACCATCAGCTCCGTG CAAGCTGAAGATTTTGCAACTTACTACTGTCAGCAACATTATAGCACTCCTTGGACATTC GGACAAGGGACCAAGGTGGAAATCAAAAGAACTGTGGCTGCACCTTCTGTCTTCATCTTC CCTCCATCTGATGAGCAGCTCAAATCTGGAACTGCCTCCGTTGTGTGCCTGCTGAATAAC TTCTATCCTAGAGAGGCCAAAGTCCAGTGGAAGGTGGATAACGCCCTCCAATCCGGTAAC TCCCAGGAGTCTGTCACAGAGCAGGACTCCAAGGACAGCACCTACTCCCTCAGCAACACC CTGACACTGTCTAAAGCTGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCAT CAGGGACTGAGCTCCCCCGTCACAAAATCCTTCAACAGGGGAGAGTGCTAA Human LGR4 protein sequence (SEQ ID NO:71)

MPGPLGLLCFLALGLLGSAGPSGAAPPLCAAPCSCDGDRRVDCSGKGLTAVPEGLSAFTQ ALDISMNNITQLPEDAFKNFPFLEELQLAGNDLSFIHPKALSGLKELKVLTLQNNQLKTV PSEAIRGLSALQSLRLDANHITSVPEDSFEGLVQLRHLWLDDNSLTEVPVHPLSNLPTLQ ALTLALNKI SSI PDFAFTNLSSLVVLHLHNNKIRSLSQHCFDGLDNLETLDLNYNNLGEF PQAIKALPSLKELGFHSNSISVIPDGAFDGNPLLRTIHLYDNPLSFVGNSAFHNLSDLHS LVIRGASMVQQFPNLTGTVHLESLTLTGTKI SS IPNNLCQEQKMLRTLDLSYNNIRDLPS FNGCHALEEI SLQRNQIYQIKEGTFQGLISLRILDLSRNLIHEIHSRAFATLGPITNLDV SFNELTSFPTEGLNGLNQLKLVGNFKLKEALAAKDFVNLRSLSVPYAYQCCAFWGCDSYA NLNTEDNSLQDHSVAQEKGTADAANVTSTLENEEHSQI I IHCTPSTGAFKPCEYLLGSWM IRLTVWFIFLVALFFNLLVILTTFASCTSLPSSKLFIGLI SVSNLFMGIYTGILTFLDAV SWGRFAEFGIWWETGSGCKVAGFLAVFSSESAI FLLMLATVERSLSAKDIMKNGKSNHLK QFRVAALLAFLGATVAGCFPLFHRGEYSASPLCLPFPTGETPSLGFTVTLVLLNSLAFLL MAVIYTKLYCNLEKEDLSENSQSSMIKHVAWLIFTNCIFFCPVAFFSFAPLITAIS I SPE IMKSVTLIFFPLPACLNPVLYVFFNPKFKEDWKLLKRRVTKKSGSVSVSISSQGGCLEQD FYYDCGMYSHLQGNLTVCDCCESFLLTKPVSCKHLIKSHSCPALAVASCQRPEGYWSDCG TQSAHSDYADEEDSFVSDSSDQVQACGRACFYQSRGFPLVRYAYNLPRVKD

Human LGR5 protein sequence (SEQ ID NO: 72)

MDTSRLGVLLSLPVLLQLATGGSSPRSGVLLRGCPTHCHCEPDGRMLLRVDCSDLGLSEL PSNLSVFTSYLDLSMN ISQLLPNPLPSLRFLEELRLAGNALTYI PKGAFTGLYSLKVLM LQNNQLRHVPTEALQNLRSLQSLRLDANHI SYVPPSCFSGLHSLRHLWLDDNALTEI PVQ AFRSLSALQAMTLALNKIHHI PDYAFGNLSSLVVLHLHNNRIHSLGKKCFDGLHSLETLD LNYNNLDEFPTAIRTLSNLKELGFHSNNIRS IPEKAFVGNPSLITIHFYDNPIQFVGRSA FQHLPELRTLTLNGASQITEFPDLTGTANLESLTLTGAQI SSLPQTVCNQLPNLQVLDLS YNLLEDLPSFSVCQKLQKI DLRHNEIYEIKVDTFQQLLSLRSLNLAWNKIAI IHPNAFST LPSLIKLDLSSNLLSSFPITGLHGLTHLKLTGNHALQSLI SSENFPELKVIEMPYAYQCC AFGVCENAYKISNQWNKGDNSSMDDLHKKDAGMFQAQDERDLEDFLLDFEEDLKALHSVQ CSPSPGPFKPCEHLLDGWLIRIGVWTIAVLALTCNALVTSTVFRSPLYISPIKLLIGVIA AVNMLTGVSSAVLAGVDAFTFGSFARHGAWWENGVGCHVIGFLSI FASESSVFLLTLAAL ERGFSVKYSAKFETKAPFSSLKVI ILLCALLALTMAAVPLLGGSKYGASPLCLPLPFGEP STMGYMVALILLNSLCFLMMTIAYTKLYCNLDKGDLENIWDCSMVKHIALLLFTNCILNC PVAFLSFSSLINLTFISPEVIKFILLWVPLPACLNPLLYILFNPHFKEDLVSLRKQTYV WTRSKHPSLMSINSDDVEKQSCDSTQALVTFTSSSITYDLPPSSVPSPAYPVTESCHLSS VAFVPCL

Human LGR6 protein sequence (SEQ ID NO: 73)

MGRPRLTLVCQVSI I ISARDLSMNNLTELQPGLFHHLRFLEELRLSGNHLSHIPGQAFSG LYSLKILMLQNNQLGGI PAEALWELPSLQSLRLDANLI SLVPERSFEGLSSLRHLWLDDN ALTEIPVRALNNLPALQAMTLALNRI SHIPDYAFQNLTSLWLHLHNNRIQHLGTHSFEG LHNLETLDLNYNKLQEFPVAIRTLGRLQELGFHNN IKAI PEKAFMGNPLLQTIHFYDNP IQFVGRSAFQYLPKLHTLSLNGAMDIQEFPDLKGTTSLEILTLTRAGIRLLPSGMCQQLP RLRVLELSHNQIEELPSLHRCQKLEEIGLQHNRIWEIGADTFSQLSSLQALDLSWNAIRS IHPEAFSTLHSLVKLDLTDNQLTTLPLAGLGGLMHLKLKGNLALSQAFSKDSFPKLRILE VPYAYQCCPYGMCASFFKASGQWEAEDLHLDDEESSKRPLGLLARQAENHYDQDLDELQL EMEDSKPHPSVQCSPTPGPFKPCEYLFESWGIRLAVWAIVLLSVLCNGLVLLTVFAGGPV PLPPVKFWGAIAGANTLTGI SCGLLASVDALTFGQFSEYGARWETGLGCRATGFLAVLG SEASVLLLTLAAVQCSVSVSCVRAYGKSPSLGSVRAGVLGCLALAGLAAALPLASVGEYG ASPLCLPYAPPEGQPAALGFTVALVMMNSFCFLWAGAYIKLYCDLPRGDFEAVWDCAMV RHVAWLIFADGLLYCPVAFLSFASMLGLFPVTPEAVKSVLLVVLPLPACLNPLLYLLFNP HFRDDLRRLRPRAGDSGPLAYAAAGELEKSSCDSTQALVAFSDVDLILEASEAGRPPGLE TYGFPSVTLI SCQQPGAPRLEGSHCVEPEGNHFGNPQPSMDGELLLRAEGSTPAGGGLSG GGGFQPSGLAFASHV

LGR5 ECD amino acids 22-564 (SEQ ID NO:74)

GSSPRSGVLLRGCPTHCHCEPDGRMLLRVDCSDLGLSELPSNLSVFTSYLDLSMN I SQL LPNPLPSLRFLEELRLAGNALTYI PKGAFTGLYSLKVLMLQNNQLRHVPTEALQNLRSLQ SLRLDANHI SYVPPSCFSGLHSLRHLWLDDNALTEI PVQAFRSLSALQAMTLALNKIHHI PDYAFGNLSSLVVLHLHNNRIHSLGKKCFDGLHSLETLDLNYNNLDEFPTAIRTLSNLKE LGFHSNNIRS IPEKAFVGNPSLITIHFYDNPIQFVGRSAFQHLPELRTLTLNGASQITEF PDLTGTANLESLTLTGAQI SSLPQTVCNQLPNLQVLDLSYNLLEDLPSFSVCQKLQKIDL RHNEIYEIKVDTFQQLLSLRSLNLAWNKIAI IHPNAFSTLPSLIKLDLSSNLLSSFPITG LHGLTHLKLTGNHALQSLI SSENFPELKVIEMPYAYQCCAFGVCENAYKISNQWNKGDNS SMDDLHKKDAGMFQAQDERDLEDFLLDFEEDLKALHSVQCSPSPGPFKPCEHLLDGWLIR IGV

LGR5-Fc protein sequence (SEQ ID NO:75)

MDTSRLGVLLSLPVLLQLATGGSSPRSGVLLRGCPTHCHCEPDGRMLLRVDCSDLGLSEL PSNLSVFTSYLDLSMN ISQLLPNPLPSLRFLEELRLAGNALTYI PKGAFTGLYSLKVLM LQNNQLRHVPTEALQNLRSLQSLRLDANHI SYVPPSCFSGLHSLRHLWLDDNALTEI PVQ AFRSLSALQAMTLALNKIHHI PDYAFGNLSSLVVLHLHNNRIHSLGKKCFDGLHSLETLD LNYNNLDEFPTAIRTLSNLKELGFHSNNIRSI PEKAFVGNPSLITIHFYDNPIQFVGRSA FQHLPELRTLTLNGASQITEFPDLTGTANLESLTLTGAQI SSLPQTVCNQLPNLQVLDLS YNLLEDLPSFSVCQKLQKI DLRHNEIYEIKVDTFQQLLSLRSLNLAWNKIAI IHPNAFST LPSLIKLDLSSNLLSSFPITGLHGLTHLKLTGNHALQSLI SSENFPELKVIEMPYAYQCC AFGVCENAYKISNQWNKGDNSSMDDLHKKDAGMFQAQDERDLEDFLLDFEEDLKALHSVQ CSPSPGPFKPCEHLLDGWLIRIGVGRADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDW LNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK

Human Igd Fc region (SEQ ID NO:76)

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCVWDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAK GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Human IgGi Fc region (SEQ ID NO:77)

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCVWDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAK GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Human Igd Fc region (SEQ ID NO:78)

KSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCVWDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Human Igd Fc region (SEQ ID NO:79) EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT I SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Human IgG ₂ Fc region (SEQ ID NO:80)

CVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTFRVVSVLTWHQDWLNGKEYKCKVSNKGLPAPIEKTI SKTKGQP REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

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Indications are Made All designations Original (for SUBMISSION )

Indications are Made All designations

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