WO2017040660A1

WO2017040660A1 - Combination therapy for treatment of disease

Info

Publication number: WO2017040660A1
Application number: PCT/US2016/049697
Authority: WO
Inventors: Timothy Charles Hoey; Jennifer Anne Cain
Original assignee: Oncomed Pharmaceuticals, Inc.
Priority date: 2015-08-31
Filing date: 2016-08-31
Publication date: 2017-03-09
Also published as: WO2017040660A4

Abstract

The present invention provides methods comprising combination therapy for modulating immune responses, for inhibiting tumor growth, and/or for treating cancer. In particular, the present invention provides RSPO or LGR antagonists in combination with immunotherapeutic agents for the treatment of cancer and other diseases.

Description

COMBINATION THERAPY FOR TREATMENT OF DISEASE

CROSS-REFERENCE TO RELATED APPLICATONS

[0001] This application claims the priority benefit of U.S. Provisional Application No. 62/212,471, filed August 31, 2015, which is hereby incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

[0002] The present invention provides methods comprising combination therapy for modulating immune responses and treating cancer and other diseases. In particular, the present invention provides RSPO or LGR antagonists in combination with at least one additional immunotherapeutic agent for the treatment of cancer.

BACKGROUND OF THE INVENTION

[0003] 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).

[0004] Studies have shown that RSPO proteins have a role during vertebrate development (Kamata et al., 2004, Biochim. Biophys Acta, 1676:51-62) and inXenopus myogenesis (Kazanskaya et al., 2004, Dev. Cell, 7:525-534). RSPO 1 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). 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.

[0005] The focus of cancer drug research is shifting toward targeted therapies aimed at genes, proteins, and pathways involved in human cancer. There is a need for new agents targeting signaling pathways and new combinations of agents that target multiple pathways that could provide therapeutic benefit for cancer patients. Thus, biomolecules (e.g., anti-RSP03 antibodies) that disrupt β-catenin signaling are a potential source of new therapeutic agents for cancer, as well as other β-catenin- associated diseases.

[0006] It is one of the objectives of the present invention to provide improved methods for cancer treatment, particularly methods using RSPO or LGR antagonists in combination with

immunotherapeutic agents.

BRIEF SUMMARY OF THE INVENTION

[0007] The present invention provides methods for treating diseases such as cancer, where the methods include administering to a subject an RPSO or LGR antagonist, such as an anti-RSPO antibody or anti-LGR antibody, or a soluble LGR receptor, in combination with an

immunotherapeutic agent. Combination therapy can employ at least two different agents that work by different mechanisms of action and/or target different pathways. The combination may result in additive or synergetic effects. Combination therapy may allow for a lower dose of each agent than used in monotherapy, thereby reducing toxic side effects and/or increasing the therapeutic index of the agent(s). Combination therapy may decrease the likelihood that resistance to an agent will develop. Combination therapy may allow one agent to sensitize tumor cells (including cancer stem cells) to enhanced activity by a second agent. Combination therapy comprising an immunotherapeutic agent may allow one agent to enhance the immune response to a tumor or tumor cells while the second agent may be effective at killing tumor cells more directly. In addition, the order and/or timing of the administration of each therapeutic agent may affect the overall efficacy of a drug combination.

[0008] The invention provides an RPSO or LGR antagonist, including but not limited to, antibodies and other polypeptides that bind to at least one RPSO 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 RPSO 1, RSP02, RSP03, and RSP04. The LGR protein may be LGR5.

[0009] The invention provides immunotherapeutic agents, including but not limited to, a modulator of PD-1 activity, a modulator of PD-L 1 activity, a modulator of PD-L2 activity, a modulator of CTLA-4 activity, a modulator of CD28 activity, a modulator of CD 80 activity, a modulator of CD 86 activity, a modulator of 4- IBB activity, an modulator of OX40 activity, a modulator of KIR activity, a modulator of Tim-3 activity, a modulator of LAG3 activity, a modulator of CD27 activity, a modulator of CD40 activity, a modulator of GITR activity, a modulator of TIGIT activity, a modulator of CD20 activity, a modulator of CD96 activity, a modulator of IDO 1 activity, a cytokine, a chemokine, an interferon, an interleukin, a lymphokine, a member of the tumor necrosis factor (TNF) family, and an immunostimulatory oligonucleotide.

[0010] Compositions comprising an RSPO or LGR antagonist (e.g., an anti-RSPO, anti-LGR antibody, or soluble LGR receptor) and/or at least one additional immunotherapeutic agent are provided. Pharmaceutical compositions comprising the RSPO or LGR antagonist and/or the immunotherapeutic agents are provided.

[0011] In one aspect, the invention provides methods of inhibiting tumor growth. In some embodiments, a method comprises contacting tumor cells with an effective amount of a RSPO or LGR antagonist (e.g., an anti-RSPO, anti-LGR antibody, or soluble LGR receptor) in combination with an effective amount of an immunotherapeutic agent. The method may be in vivo or in vitro. In certain embodiments, the tumor is in a subject, and contacting tumor cells with the RSPO or LGR antagonist and the immunotherapeutic agent comprises administering a therapeutically effective amount of each of the agents to the subject.

[0012] In another aspect, the invention provides a method of treating cancer. In some embodiments, a method of treating cancer comprises administering to a subject a therapeutically effective amount of a RSPO or LGR antagonist (e.g., an anti-RSPO, anti-LGR antibody, or soluble LGR receptor) in combination with a therapeutically effective amount of an immunotherapeutic agent.

[0013] In another aspect, the invention provides a method of inhibiting the activity of regulatory T- cells (Tregs). In some embodiments, a method of inhibiting the activity of Tregs comprises administering to a subject a therapeutically effective amount of a RSPO or LGR antagonist (e.g., an anti-RSPO, anti-LGR antibody, or soluble LGR receptor) in combination with a therapeutically effective amount of an immunotherapeutic agent. In some embodiments, the inhibition of Treg activity comprises inhibiting the suppression of immune responses. In some embodiments, the inhibition of Treg activity results in the inhibition of suppression of immune responses.

[0014] In another aspect, the invention provides a method of increasing T cell infiltration into a tumor. In some embodiments, a method of increasing T cell infiltration into a tumor comprises administering to a subject a therapeutically effective amount of a RSPO or LGR antagonist (e.g., an anti-RSPO, anti-LGR antibody, or soluble LGR receptor) in combination with a therapeutically effective amount of an immunotherapeutic agent.

[0015] In another aspect, the invention provides a method of increasing T cell cytotoxicity to a tumor. In some embodiments, a method of increasing T cell cytotoxicity to a tumor comprises administering to a subject a therapeutically effective amount of a RSPO or LGR antagonist (e.g., an anti-RSPO, anti-LGR antibody, or soluble LGR receptor) in combination with a therapeutically effective amount of an immunotherapeutic agent.

[0016] In another aspect, the invention provides a method of increasing tumor cell lysis. In some embodiments, a method of increasing tumor cell lysis comprises administering to a subject a therapeutically effective amount of a RSPO or LGR antagonist (e.g., an anti-RSPO, anti-LGR antibody, or soluble LGR receptor) in combination with a therapeutically effective amount of an immunotherapeutic agent. [0017] In another aspect, the invention provides a method to increase the efficacy of an immune checkpoint modulator. In some embodiments, a method to increase the efficacy of an immune checkpoint modulator comprises administering to a subject a therapeutically effective amount of a RSPO or LGR antagonist (e.g., an anti-RSPO, anti-LGR antibody, or soluble LGR receptor) in combination with a therapeutically effective amount of an immune checkpoint modulator. In some embodiments, the immune checkpoint modulator is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint modulator is an immune checkpoint enhancer or stimulator.

[0018] In another aspect, the invention provides a method of reducing or preventing metastasis in a subject. In some embodiments, a method of reducing or preventing metastasis in a subject comprises administering to the subject a therapeutically effective amount of a RSPO or LGR antagonist (e.g., an anti-RSPO, anti-LGR antibody, or soluble LGR receptor) and a therapeutically effective amount of an immunotherapeutic agent.

[0019] In another aspect, the invention provides a method of enhancing treatment for a subject who is being treated with an immune checkpoint inhibitor, the method comprising administering to the subject a therapeutically effective amount of a RSPO or LGR antagonist (e.g., an anti-RSPO, anti- LGR antibody, or soluble LGR receptor).

[0020] In another aspect, the invention provides a method of enhancing or inducing an anti-tumor immune response in a subject, the method comprising administering to the subject a therapeutically effective amount of a RSPO or LGR antagonist (e.g., an anti-RSPO, anti-LGR antibody, or soluble LGR receptor).

[0021] In some embodiments, the RSPO or LGR antagonist is an RSPO-binding agent. In some embodiments, the RSPO or LGR antagonist is an 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 the antibody OMP-131R10 (also referred to as OMP-131R010, 131R10, or 131R010). In some embodiments, the RSPO or LGR antagonist is a soluble receptor. In some embodiments, the RSPO or LGR antagonist is an LGR-Fc soluble receptor. In some embodiments, the RSPO or LGR antagonist is an LGR5-Fc soluble receptor. In certain embodiments, the LGR5-Fc soluble receptor comprises amino acid sequence of SEQ ID NO:57.

[0022] 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. In some embodiments, the antibody specifically binds at least one human RSP03.

[0023] In some embodiments, the antibody specifically binds at least human RSPOl . In some embodiments, the antibody includes (a) a heavy chain CDR1 including TGYTMH (SEQ ID NO:5), a heavy chain CDR2 including GINPN GGTTYNQNFKG (SEQ ID NO:6), and a heavy chain CDR3 including KEFSDGYYFFAY (SEQ ID NO:7); and/or (b) a light chain CDR1 including KASQDVIFAVA (SEQ ID NO: 8), a light chain CDR2 including WASTRHT (SEQ ID NO:9), and a light chain CDR3 including QQHYSTPW (SEQ ID NO: 10). In some embodiments, the antibody includes a heavy chain variable region including an amino acid sequence at least 80%, 85%, 90%, 90%, 95%, or 100% identical to SEQ ID NO: 11 or 44 (e.g., including one, two or three of the heavy chain CDRs mentioned above), and a light chain variable region including an amino acid sequence at least 80%, 85%, 90%, 90%, 95%, or 100% identical to SEQ ID NO: 12 or 45 (e.g., including one, two, or three of the light chain CDRs mentioned above). In some embodiments, the antibody includes a heavy chain variable region including SEQ ID NO: 1 1 and a light chain variable region including SEQ ID NO: 12.

[0024] In some embodiments, the antibody specifically binds at least human RSP02. In some embodiments, the antibody includes (a) a heavy chain CDR1 including SSYAMS (SEQ ID NO: 17), a heavy chain CDR2 including SISSGGSTYYPDSVKG (SEQ ID NO: 18), and a heavy chain CDR3 including RGGDPGVYNGDYEDAMDY (SEQ ID NO: 19); and (b) a light chain CDR1 including KASQDVSSAVA (SEQ ID NO:20), a light chain CDR2 including WASTRHT (SEQ ID NO:21), and a light chain CDR3 including QQHYSTP (SEQ ID NO:22). In some embodiments, the antibody includes a heavy chain variable region including an amino acid sequence at least 80%, 85%, 90%, 90%, 95%, or 100% identical to SEQ ID NO:23 (e.g., including one, two, or three of the heavy chain CDRs mentioned above)and a light chain variable region including an amino acid sequence at least 80%, 85%, 90%, 90%, 95%, or 100% identical to SEQ ID NO:24 (e.g., including one, two, or three of the light CDRs mentioned above).

[0025] In some embodiments, the antibody specifically binds at least human RSP03. In some embodiments, the antibody includes (a) a heavy chain CDR1 including DYSIH (SEQ ID NO:29), a heavy chain CDR2 including YIYPSNGDSGYNQKFK (SEQ ID NO:30), and a heavy chain CDR3 including TYFANNFD (SEQ ID NO:31) or ATYF ANNTD Y( SEQ ID NO:32); and (b) a light chain CDR1 including KASQSVDYDGDSYMN (SEQ ID NO:33), a light chain CDR2 including

AASNLES (SEQ ID NO:34) or AAS (SEQ ID NO:35), and a light chain CDR3 including

QQSNEDPLT (SEQ ID NO:36) or QQSNEDPLTF (SEQ ID NO:37). In some embodiments, the antibody includes (a) a heavy chain CDR1 including DYSIH (SEQ ID NO:29), a heavy chain CDR2 including YIYPSNGDSGYNQKFK (SEQ ID NO:30), and a heavy chain CDR3 including

TYFANNFD (SEQ ID NO:31); and (b) a light chain CDR1 including KASQSVDYDGDSYMN (SEQ ID NO:33), a light chain CDR2 including AASNLES (SEQ ID NO:34), and a light chain CDR3 including QQSNEDPLT (SEQ ID NO:36). In some embodiments, the antibody includes a heavy chain variable region including an amino acid sequence at least 80%, 85%, 90%, 90%, 95%, or 100% identical to SEQ ID NO:38 (e.g., including one, two, or three of the heavy chain CDRs mentioned above), and a light chain variable region including an amino acid sequence at least 80%, 85%, 90%, 90%, 95%, or 100% identical to SEQ ID NO:39 (e.g., including one, two, or three of the light chain CDRs mentioned above). In some embodiments, the antibody is 131R10.

[0026] In some embodiments, the RSPO or LGR antagonist is an antibody that specifically binds at least one human LGR protein (e.g., LGR4, LGR5, and LGR6). In some embodiments, the antibody specifically binds at least human LGR5. In certain embodiments, the antibody includes (a) 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 (b) 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 some embodiments, the antibody includes 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.

[0027] In some embodiments, the antibody is a monoclonal antibody, a recombinant antibody, a chimeric antibody, a humanized antibody, a human antibody, or an antibody fragment including an antigen-binding site. In some embodiments, the antibody is a monospecific antibody or a bispecific antibody. In some embodiments, the antibody is an IgGl antibody or an IgG2 antibody.

[0028] In some embodiments, the RSPO or LGR antagonist is a soluble receptor including an extracellular domain of a human LGR protein (e.g., LGR5), where the extracellular domain is capable of binding a human RSPO protein. In some embodiments, the extracellular domain of a human LGR protein includes amino acids 22-564 of human LGR5 (SEQ ID NO:56). In some embodiments, the soluble receptor includes 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 includes a human Fc region (e.g., includes SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, or SEQ ID NO:62, or an amino acid sequence at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, or SEQ ID NO:62).

[0029] In certain embodiments of each of the aforementioned aspects, as well as other aspects and embodiments described elsewhere herein, the RSPO or LGR antagonist enhances the activity of the immunotherapeutic agent.

[0030] In certain embodiments of each of the aforementioned aspects, as well as other aspects and embodiments described elsewhere herein, the immunotherapeutic agent enhances the activity of the RSPO or LGR antagonist.

[0031] In certain embodiments of each of the aforementioned aspects, as well as other aspects and embodiments described elsewhere herein, the RSPO or LGR antagonist and the immunotherapeutic agent act synergistically. [0032] In certain embodiments of each of the aforementioned aspects, as well as other aspects and embodiments described elsewhere herein, the immunotherapeutic agent is an agent that modulates immune responses. In some embodiments, the immunotherapeutic agent is an agent that enhances anti-tumor immune responses. In some embodiments, the immunotherapeutic agent is an agent that increases cell-mediated immunity. In some embodiments, the immunotherapeutic agent is an agent that increases T-cell activity. In some embodiments, the immunotherapeutic agent is an agent that increases cytolytic T-cell (CTL) activity. In some embodiments, the immunotherapeutic agent is an agent that increases natural killer (NK) cell activity. In some embodiments, the immunotherapeutic agent is an agent that inhibits suppression of an immune response. In some embodiments, the immunotherapeutic agent is an agent that inhibits suppressor cells or suppressor cell activity. In some embodiments, the immunotherapeutic agent is an agent that inhibits Treg activity. In some embodiments, the immunotherapeutic agent is an agent that inhibits the activity of inhibitory immune checkpoint receptors.

[0033] In some embodiments, the immunotherapeutic agent is a modulator of PD-1 activity, a modulator of PD-Ll activity, a modulator of PD-L2 activity, a modulator of CTLA-4 activity, a modulator of CD28 activity, a modulator of CD80 activity, a modulator of CD86 activity, a modulator of 4- IBB activity, an modulator of OX40 activity, a modulator of KIR activity, a modulator of Tim-3 activity, a modulator of LAG3 activity, a modulator of CD27 activity, a modulator of CD40 activity, a modulator of GITR activity, a modulator of TIGIT activity, a modulator of CD20 activity, a modulator of CD96 activity, a modulator of IDOl activity, a cytokine, a chemokine, an interferon, an interleukin, a lymphokine, a member of the tumor necrosis factor (TNF) family, or an

immunostimulatory oligonucleotide. In some embodiments, the immunotherapeutic agent is an immune checkpoint modulator (e.g., an immune checkpoint inhibitor).

[0034] In some embodiments, the immunotherapeutic agent is an agent that inhibits the activity of PD-1. In some embodiments, the immunotherapeutic agent is an agent that inhibits the activity of PD-Ll and/or PD-L2. In some embodiments, the immunotherapeutic agent is an agent that inhibits the activity of CTLA-4. In some embodiments, the immunotherapeutic agent is an agent that inhibits the activity of CD80 and/or CD86. In some embodiments, the immunotherapeutic agent is an agent that inhibits the activity of TIGIT. In some embodiments, the immunotherapeutic agent is an agent that inhibits the activity of KIR. In some embodiments, the immunotherapeutic agent is an agent that enhances or stimulates the activity of activating immune checkpoint receptors.

[0035] In some of the embodiments of the methods described herein, the immunotherapeutic agent is a PD-1 antagonist, a PD-Ll antagonist, a PD-L2 antagonist, a CTLA-4 antagonist, a CD80 antagonist, a CD86 antagonist, a KIR antagonist, a Tim-3 antagonist, a LAG3 antagonist, a TIGIT antagonist, a CD20 antagonist, a CD96 antagonist, or an IDOl antagonist. [0036] In some embodiments, the PD-1 antagonist is an antibody that specifically binds PD-1. In some embodiments, the antibody that binds PD-1 is pembrolizumab (KEYTRUDA, MK-3475;

Merck), pidilizumab (CT-01 1 ; Curetech Ltd.), nivolumab (OPDIVO, BMS-936558, MDX-1 106; Bristol Myer Squibb), MEDI0680 (AMP-514; AstraZenenca/Medlmmune), REGN2810 (Regeneron Pharmaceuticals), BGB-A3 17 (BeiGene Ltd.), PDR-001 (Novartis), or STI-A 1 1 10 (Sorrento

Therapeutics). In some embodiments, the antibody that binds PD-1 is described in PCT Publication WO 2014/179664, for example, an antibody identified as APE2058, APE1922, APE1923, APE1924, APE 1950, or APE 1963 (Anaptysbio), or an antibody containing the CDR regions of any of these antibodies. In other embodiments, the PD-1 antagonist is a fusion protein that includes the extracellular domain of PD-Ll or PD-L2, for example, AMP-224 (AstraZeneca/Medlmmune). In other embodiments, the PD-1 antagonist is a peptide inhibitor, for example, AUNP-12 (Aurigene).

[0037] In some embodiments, the PD-Ll antagonist is an antibody that specifically binds PD-Ll . In some embodiments, the antibody that binds PD-Ll is atezolizumab (RG7446, MPDL3280A;

Genentech), MEDI4736 (AstraZeneca/Medlmmune), BMS-936559 (MDX-1 105; Bristol Myers Squibb), avelumab (MSB0010718C; Merck KGaA), KD033 (Kadmon), the antibody portion of

KD033, or STI-A 1014 (Sorrento Therapeutics). In some embodiments, the antibody that binds PD- L l is described in PCT Publication WO 2014/055897, for example, Ab-14, Ab-16, Ab-30, Ab-31, Ab-42, Ab-50, Ab-52, or Ab-55, or an antibody that contains the CDR regions of any of these antibodies.

[0038] In some embodiments, the CTLA-4 antagonist is an antibody that specifically binds CTLA-4. In some embodiments, the antibody that binds CTLA-4 is ipilimumab (YERVOY; Bristol Myer Squibb) or tremelimumab (CP -675,206; Pfizer). In some embodiments, the CTLA-4 antagonist a CTLA-4 fusion protein or soluble CTLA-4 receptor, for example, KAHR-102 (Kahr Medical Ltd.).

[0039] In some embodiments, the LAG3 antagonist is an antibody that specifically binds LAG3. In some embodimetns, the antibody that binds LAG3 is IMP701 (Prima BioMed), IMP731 (Prima BioMed/GlaxoSmithKline), BMS-986016 (Bristol Myer Squibb), LAG525 (Novartis), and

GSK2831781 (Glaxo SmithKline). In some embodiments, the LAG3 antagonist includes a soluble LAG3 receptor, for example, IMP321 (Prima BioMed).

[0040] In some embodiments, the KIR antagonist is an antibody that specifically binds KIR. In some embodiments, the antibody that binds KIR is lirilumab (Bristol Myer Squibb/Innate Pharma).

[0041] In some embodiments, the immune checkpoint enhancer or stimulator is a CD28 agonist, a 4- 1BB agonist, an OX40 agonist, a CD27 agonist, a CD80 agonist, a CD86 agonist, a CD40 agonist, or a GITR agonist.

[0042] In some embodiments, the OX40 agonist includes OX40 ligand, or an OX40-binding portion thereof. For example, the OX40 agonist may be MEDI6383 (AstraZeneca). In some embodiments, the OX40 agonist is an antibody that specifically binds OX40. In some embodiments, the antibody that binds OX40 is MEDI6469 (AstraZeneca/Medlmmune), MEDI0562 (AstraZeneca/Medlmmune), or MOXR0916 (RG7888; Genentech). In some embodiments, the OX40 agonist is a vector (e.g., an expression vector or virus, such as an adenovirus) capable of expressing OX40 ligand. In some embodiments the OX40-expressing vector is Delta-24-RGDOX (DNAtrix) or DNX2401 (DNAtrix).

[0043] In some embodiments, the 4-1BB (CD137) agonist is a binding molecule, such as an anticalin. In some embodiments, the anticalin is PRS-343 (Pieris AG). In some embodiments, the 4- 1BB agonist is an antibody that specifically binds 4-1BB. In some embodiments, antibody that binds 4-1BB is PF-2566 (PF-05082566; Pfizer) or urelumab (BMS-663513; Bristol Myer Squibb).

[0044] In some embodiments, the CD27 agonist is an antibody that specifically binds CD27. In some embodiments, the antibody that binds CD27 is varlilumab (CDX-1127; Celldex).

[0045] In some embodiments, the GITR agonist comprises GITR ligand or a GITR-binding portion thereof. In some embodiments, the GITR agonist is an antibody that specifically binds GITR. In some embodiments, the antibody that binds GITR is TRX518 (GITR, Inc.), MK-4166 (Merck), or INBRX-110 (Five Prime Therapeutics/Inhibrx).

[0046] In some embodiments, where the RSPO or LGR antagonist and immunotherapeutic agent together are a bispecific antibody. For example, the bispecific antibody may specifically bind a RSPO or LGR protein and immune checkpoint (e.g., any described herein, such as PD-1, PD-L1, PD-L2, or CTLA-4, LAG-3, OX40, or CD27). In particular embodiments, the bispecific antibody specifically binds a human RSPO protein and one of PD-1, PD-L1, and CTLA-4. In other embodiments, the bispecific antibody does not have both RSP03 as a first antigen target and an effector molecule on a leukocyte of (e.g., CD2, CD3, CD28, CTLA-4, CD80, or CD86) or a Fc receptor (e.g., CD64, CD32, or CD 16) as a second antigen target.

[0047] In some embodiments, the immunotherapeutic agent is a cytokine, for example, a chemokine, an interferon, an interleukin, lymphokine, or a member of the tumor necrosis factor family. In some embodiments, the cytokine is IL-2, IL15, or interferon-gamma.

[0048] In some embodiments of any of the above aspects or those described elsewhere herein, the cancer is selected from the group consisting of lung cancer, pancreatic cancer, breast cancer, colon cancer, colorectal cancer, melanoma, gastrointestinal cancer, gastric cancer, renal cancer, ovarian cancer, liver cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer,

neuroblastoma, glioma, glioblastoma, glioblastoma multiforme, cervical cancer, stomach cancer, bladder cancer, head and neck cancer, and hepatoma.

[0049] In some embodiments of any of the above aspects or those described elsewhere herein, the tumor is selected from the group consisting of lung tumor, pancreatic tumor, breast tumor, colon tumor, colorectal tumor, melanoma, gastrointestinal tumor, gastric tumor, renal tumor, ovarian tumor, liver tumor, endometrial tumor, kidney tumor, prostate tumor, thyroid tumor, neuroblastoma, glioma, glioblastoma, glioblastoma multiforme, cervical tumor, stomach tumor, bladder tumor, head and neck tumor, and hepatoma. In some embodiments, the cancer expresses an RSPO protein (e.g., RSP03). In other embodiments, the cancer does not express an RSPO protein, for example, does not express RSPO l, RSP02, RSP03, or RSP04. In some embodiments, the cancer does not express RSP03.

[0050] In some embodiments of any of the above aspects or those described elsewhere herein, the subject's cancer or tumor does not respond to immune checkpoint inhibition (e.g., to any immune checkpoint inhibitor described herein, such as a PD-1 antagonist or PD-Ll antagonist) or the subject's cancer or tumor has progressed following an initial response to immune checkpoint inhibition (e.g., to any immune checkpoint inhibitor described herein, such as a PD-1 antagonist or PD-Ll antagonist).

[0051] In some embodiments of any of the above aspects or those described elsewhere herein, the subj ect is a human.

BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figures 1A-1D show the effect of anti-CTLA-4 and anti -PD-Ll antibody treatment on tumor growth in the presence or absence of the anti-RSP03 antibody 131R10. Figure 1A shows average results. Figures IB-ID show tumor measure growth measurements from individual control animals, animals receiving a control antibody with anti-CTLA-4 and anti -PD-Ll antibodies, and animals receiving 131R10 with anti-CTLA-4 and anti-PD-Ll antibodies, respectively.

[0053] Figures 2A and 2B show changes in interferon-gamma (Figure 2A) and IL-2 (Figure 2B) in splenocytes following treatment with control antibodies, anti-CTLA-4 and anti-PD-Ll antibodies, or 131R10 with anti-CTLA-4 and anti-PD-Ll antibodies.

[0054] Figure 3 shows changes in T cell cytotoxicity following treatment with control antibodies, anti-CTLA-4 and anti-PD-Ll antibodies, or 131R10 with anti-CTLA-4 and anti-PD-Ll antibodies.

[0055] Figures 4A and 4B show increases in tumor-infiltrating CD4⁺ (Figure 4A) and CD8⁺ (Figure 4B) T-cells in cells receiving 131R10 with anti-CTLA-4 and anti-PD-Ll antibodies, as compared to those receiving anti-CTLA-4 and anti-PD-Ll antibodies without 131R10 or control antibodies.

[0056] Figures 5A-5C show reduction in breast tumor cell growth in mice receiving anti-RSP03 antibody 131R10. Figure 5A shows the average in the control group (left) and 131R10 group (right). Figures 5B and 5C show the individual results from the control and 131R10 treatment groups, respectively.

[0057] Figures 6A-6D show changes in splenocyte cytokine expression of interferon gamma (Figure 6A), IL-2 (Figure 6B), IL-10 (Figure 6C), and IL-17A (Figure 6D) in mice implanted with breast tumor cells and receiving either control or 131R10.

[0058] Figures 7A-7C show changes in splenic CD8+ T cells (Figure 7A), splenic CD4+ T cells (Figure 7B), and splenic regulatory T cells (Tregs; Figure 7C) in mice implanted with breast tumor cells and receiving either saline control or 131R10.

[0059] Figures 8A-8D show reduction in breast tumor cell growth in mice receiving (left to right) control, anti-PD 1 , or anti-PD 1+131R10. Figure 8 A shows the average of each treatment group . Figures 8B-8D show the individual results from the control, anti-PDl, and 131R10+anti-PD l treatment groups, respectively.

[0060] Figures 9A-9D show changes in splenocyte cytokine expression of interferon gamma (Figure 9A), IL-10 (Figure 9B), IL-2 (Figure 9C), and IL-17A (Figure 9D) in mice implanted with breast tumor cells and receiving control, anti-PDl, or 13 lRlO+anti-PDl treatment.

[0061] Figure 10A shows changes in T cell cytotoxicity in mice implanted with breast tumor cells and receiving control, anti-PDl, or 131R10+anti-PDl treatment.

[0062] Figures 10B and IOC show changes in tumor T cell numbers in mice implanted with breast tumor cells and receiving control, anti-PDl, or 131R10+anti-PDl treatment.

[0063] Figures 10D-10F show changes in splenic CD8+ T cells (Figure 7A), splenic CD4+ T cells (Figure 7B), and splenic regulatory T cells (Tregs; Figure 7C) in mice implanted with breast tumor cells and receiving control, anti-PDl, or 131R10+anti-PD l treatment.

[0064] Figures 11A-11D show reduction in breast tumor cell growth in mice receiving (left to right) control, anti-CTLA-4, or anti-CTLA-4+131R10. Figure 11A shows the average of each treatment group. Figures 1 lB-1 ID show the individual results from the control, anti-CTLA-4, and

13 lRlO+anti-CTLA-4 treatment groups, respectively.

[0065] Figures 12A-12D show changes in splenocyte cytokine expression of interferon gamma (Figure 12A), IL-10 (Figure 12B), IL-2 (Figure 12C), and IL-17A (Figure 12D) in mice implanted with breast tumor cells and receiving control, anti-CLTA-4, or 13 lRlO+anti-CTLA-4 treatment.

[0066] Figure 13 shows changes in T cell cytotoxicity in mice implanted with breast tumor cells and receiving control, anti-CTLA-4, or 131R10+anti-CTLA-4 treatment.

[0067] Figures 14A-14C show changes in splenic CD8+ T cells (Figure 14A), splenic CD4+ T cells (Figure 14B), and splenic regulatory T cells (Tregs; Figure 14C) in mice implanted with breast tumor cells and receiving the control, anti-CLTA-4, or 131R10+anti-CTLA-4.

[0068] Figure 15 shows changes in colon tumor size in mice receiving (left to right) saline, 5- fluorouracil (5-FU), 131R10+5-FU, anti-PD l+5-FU, or 131R10+anti-PD l+5-FU.

[0069] Figure 16 shows changes in 4T1 tumor size in mice receiving saline, 131R10, anti-PD-1 antibody, docetaxel, anti-PDl+docetaxel, or 131R10+anti-PDl+docetaxel. The lower six graphs show the results from each individual animal treated.

[0070] Figures 17A-17D show changes in tumor dendritic cell frequency (CD103⁺ and CD8a⁺CD3⁺; Figures 17A and 17B), CD8a⁺ cell tumor infiltration (Figure 17C), and expression of the activation marker CD69 on CD8a⁺ cells (Figure 17D) in 4T1 tumor-implanted mice receiving saline, 131R10, anti-PDl antibody, docetaxel, anti-PDl+docetaxel, or 131R10+anti-PD l+docetaxel.

[0071] Figures 18A-18B show changes in splenic dendritic cells (Figure 18A) and T regulatory cells (Figure 18B) in 4T1 tumor-implanted mice receiving saline, 131R10, anti-PDl antibody, docetaxel, anti-PDl+docetaxel, or 131R10+anti-PDl+docetaxel. [0072] Figures 19A-19D show changes in IL-17a (Figures 19A and 19B) and IL-2 (Figures 19C and Figure 19D) secreted by splenocytes from 4T1 tumor-implanted mice.

[0073] Figure 20 shows changes in MC38 tumor size in mice receiving saline, 131R10, anti-PDl antibody, or 13 lRlO+anti-PD 1. The lower four graphs show the results from each individual animal treated.

[0074] Figure 21 shows the percentage of tumors in each experimental group that were below 500 mm³ in volume following treatment in MC38-implated mice.

[0075] Figures 22A-22B show changes in IL-2 (Figure 22A) and IL-17a (Figure 22B) secreted by splenocytes from MC38-implanted mice.

DETAILED DESCRIPTION OF THE INVENTION

[0076] The present invention provides methods of modulating immune responses, particularly antitumor immune responses, methods of inhibiting tumor growth, and methods of treating cancer. The methods provided herein comprise administering to a subject a therapeutically effective amount of a RSPO or LGR antagonist in combination with a therapeutically effective amount of an

immunotherapeutic agent. In some embodiments, the RSPO or LGR antagonist is an antibody that specifically binds a human RSPO protein (e.g., RSP03), an antibody that specifically binds a human LGR protein (e.g., LGR5), or an LGR soluble receptor (e.g., an LGR5 soluble receptor). In some embodiments, the anti-RSP03 antibody is 131R10 or an antibody containing the CDR regions of 131R10. In some embodiments, the immunotherapeutic agent includes but is not limited to, a modulator of PD-1 activity, a modulator of PD-L1 activity, a modulator of PD-L2 activity, a modulator of CTLA-4 activity, a modulator of CD28 activity, a modulator of CD80 activity, a modulator of CD86 activity, a modulator of 4-1BB activity, an modulator of OX40 activity, a modulator of KIR activity, a modulator of Tim -3 activity, a modulator of LAG3 activity, a modulator of CD27 activity, a modulator of CD40 activity, a modulator of GITR activity, a modulator of TIGIT activity, a modulator of CD20 activity, a modulator of CD96 activity, a modulator of IDO 1 activity, a cytokine, a chemokine, an interferon, an interleukin, a lymphokine, a member of the tumor necrosis factor (TNF) family, and an immunostimulatory oligonucleotide.

I. Definitions

[0077] To facilitate an understanding of the present invention, a number of terms and phrases are defined below.

[0078] 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. Suitable antagonist molecules include, but are not limited to, antagonist antibodies, antibody fragments, soluble receptors, and small molecules. [0079] The terms "agonist" and "agonistic" as used herein refer to or describe an agent that is capable of, directly or indirectly, substantially inducing, activating, promoting, increasing, or enhancing the biological activity of a target and/or a signaling pathway. The term "agonist" is used herein to include any agent that partially or fully induces, activates, promotes, increases, or enhances the activity of a protein. Suitable agonists specifically include, but are not limited to, agonist antibodies or fragments thereof, soluble receptors, other fusion proteins, and small molecules.

[0080] The term "biomarker" as used herein may include but is not limited to, nucleic acids and proteins, and variants and fragments thereof. A biomarker may include DNA comprising the entire or partial nucleic acid sequence encoding the biomarker, or the complement of such a sequence.

Biomarker nucleic acids useful in the invention are considered to include both DNA and RNA comprising the entire or partial sequence of any of the nucleic acid sequences of interest. Biomarker proteins are considered to comprise the entire or partial amino acid sequence of any of the biomarker proteins or polypeptides.

[0081] The term "antibody" as used herein refers to an immunoglobulin molecule that recognizes and specifically binds a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combinations of the foregoing, through at least one antigen-binding site within the variable region of the immunoglobulin molecule. As used herein, the term encompasses intact polyclonal antibodies, intact monoclonal antibodies, antibody fragments comprising an antigen-binding site (such as Fab, Fab', F(ab')2, and Fv fragments), single chain Fv (scFv) antibodies, multispecific antibodies such as bispecific antibodies, monospecific antibodies, monovalent antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising an antigen-binding site of an antibody, and any other modified immunoglobulin molecule comprising an antigen-binding site as long as the antibodies exhibit the desired biological activity. An antibody can be any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g., IgGl, IgG2, IgG3, IgG4, IgA l 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 -characterized subunit structures and three-dimensional configurations. Antibodies can be naked or conjugated to other molecules, including but not limited to, toxins and radioisotopes.

[0082] The term "antibody fragment" as used herein refers to a portion of an intact antibody and generally includes the antigenic determining variable region or antigen-binding site of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, single chain antibodies, and multispecific antibodies formed from antibody fragments. "Antibody fragment" as used herein comprises at least one antigen-binding site or epitope -binding site. [0083] The term "variable region" of an antibody as used herein refers to the variable region of the antibody light chain, or the variable region of the antibody heavy chain, either alone or in

combination. The variable region of the heavy or light chain generally consists of four framework regions connected by three complementarity determining regions (CDRs), also known as

"hypervariable regions". The CDRs in each chain are held together in close proximity by the framework regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of the antibody. There are at least two techniques for determining CDRs: (1) an approach based on cross-species sequence variability (i.e., Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Edition, National Institutes of Health, Bethesda MD), and (2) an approach based on crystallographic studies of antigen-antibody complexes (Al-Lazikani et al., 1997, J. Mol. Biol, 273:927-948). Combinations of these two approaches are sometimes used in the art to determine CDRs.

[0084] The term "monoclonal antibody" as used herein refers to a homogenous antibody population involved in the highly specific recognition and binding of a single antigenic determinant or epitope. This is in contrast to polyclonal antibodies that typically include a mixture of different antibodies directed against different antigenic determinants. The term "monoclonal antibody" encompasses both intact and full-length antibodies as well as antibody fragments (e.g., Fab, Fab', F(ab')2, Fv), single chain (scFv) antibodies, fusion proteins comprising an antibody portion, and any other modified immunoglobulin molecule comprising at least one antigen-binding site. Furthermore, "monoclonal antibody" refers to such antibodies made by any number of techniques, including but not limited to, hybridoma production, phage selection, recombinant expression, and transgenic animals.

[0085] The term "humanized antibody" as used herein refers to antibodies that are specific immunoglobulin chains, chimeric immunoglobulins, or fragments thereof that contain minimal non- human sequences. Typically, humanized antibodies are human immunoglobulins in which amino acid residues of the CDRs are replaced by amino acid residues from the CDRs of a non-human species (e.g., mouse, rat, rabbit, or hamster) that have the desired specificity, affinity, and/or binding capability.

[0086] The term "human antibody" as used herein refers to an antibody produced by a human or an antibody having an amino acid sequence corresponding to an antibody produced by a human made using any of the techniques known in the art.

[0087] The term "chimeric antibody" as used herein refers to an antibody where the amino acid sequence of the immunoglobulin molecule is derived from two or more species. Typically, the variable region of both light and heavy chains corresponds to the variable region of antibodies derived from one species of mammals (e.g., mouse, rat, rabbit, etc.) with the desired specificity, affinity, and/or binding capability, while the constant regions are homologous to the sequences in antibodies derived from another species (usually human). [0088] The term "affinity-matured antibody" as used herein refers to an antibody with one or more alterations in one or more CDRs that result in an improvement in the affinity of the antibody for antigen, compared to a parent antibody that does not possess those alterations(s). In some instances, alterations are made in the framework regions. Preferred affinity-matured antibodies will have nanomolar or even picomolar affinities for the target antigen. Affinity-matured antibodies are produced by procedures known in the art including heavy chain and light chain variable region shuffling, random mutagenesis of CDR and/or framework residues, or site-directed mutagenesis of CDR and/or framework residues.

[0089] The terms "epitope" and "antigenic determinant" are used interchangeably herein and refer to that portion of an antigen capable of being recognized and specifically bound by a particular antibody. When the antigen is a polypeptide, epitopes can be formed both from contiguous amino acids and non-contiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids (also referred to as linear epitopes) are typically retained upon protein denaturing, whereas epitopes formed by tertiary folding (also referred to as conformational epitopes) are typically lost upon protein denaturing. An epitope typically includes at least 3, and more usually, at least 5, or 8-10 amino acids in a unique spatial conformation.

[0090] The terms "selectively binds" or "specifically binds" as used herein mean that a binding agent or an antibody reacts or associates more frequently, more rapidly, with greater duration, with greater affinity, or with some combination of the above to the epitope, protein, or target molecule than with alternative substances, including unrelated or related proteins. In certain embodiments "specifically binds" means, for instance, that an antibody binds a target with a K_D of about 0. ImM or less, but more usually less than about Ι μΜ. In certain embodiments, "specifically binds" means that an antibody binds a target with a K_D of at least about 0. Ι μΜ or less, at least about 0.0 Ι μΜ or less, or at least about InM or less. Because of the sequence identity between homologous proteins in different species, specific binding can include an antibody that recognizes a protein in more than one species.

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. It is understood that, in certain embodiments, an antibody or binding agent 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. In certain alternative embodiments, an antibody may be bispecific and comprise at least two antigen-binding sites with differing specificities. Generally, but not necessarily, reference to binding means specific binding. [0091] The term "soluble receptor" as used herein refers to an extracellular fragment (or a portion thereof) of a receptor protein preceding the first transmembrane domain of the receptor that can be secreted from a cell in soluble form.

[0092] 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.

[0093] The term "amino acid" as used herein refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine. The phrase "amino acid analog" refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, e.g., an alpha carbon that is bound to an hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs can have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. The phrase "amino acid mimetic" refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that function similarly to a naturally occurring amino acid.

[0094] 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.

[0095] 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 and BLAST variations, ALIGN and ALIGN variations, Megalign, BestFit, GCG Wisconsin Package, etc. 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, at least about 60-80 nucleotides or amino acid residues in length or any integral value therebetween. In some embodiments, identity exists over a longer region than 60-80 nucleotides or amino acid residues, such as at least about 80-100 nucleotides or amino acid 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.

[0096] The term "conservative amino acid substitution" as used herein refers to a substitution in which one amino acid residue is replaced with another amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For example, substitution of a phenylalanine for a tyrosine is a conservative substitution. Preferably, conservative substitutions in the sequences of the polypeptides and antibodies of the invention do not abrogate the binding of the polypeptide or antibody containing the amino acid sequence to the antigen(s). Methods of identifying nucleotide and amino acid conservative substitutions which do not eliminate antigen binding are well- known in the art.

[0097] 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.

[0098] As used herein, a polypeptide, antibody, polynucleotide, vector, cell, or composition which is "isolated" is a polypeptide, antibody, polynucleotide, vector, cell, or composition which is in a form not found in nature. Isolated polypeptides, antibodies, polynucleotides, vectors, cells, or compositions include those which have been purified to a degree that they are no longer in a form in which they are found in nature. In some embodiments, a polypeptide, antibody, polynucleotide, vector, cell, or composition which is isolated is substantially pure. [0099] 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.

[00100] The terms "cancer" and "cancerous" as used herein refer to or describe the physiological condition in mammals in which a population of cells is characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, blastema, sarcoma, and hematologic cancers such as lymphoma and leukemia.

[00101] The terms "proliferative disorder" and "proliferative disease" as used herein refer to disorders associated with abnormal cell proliferation such as cancer.

[00102] The terms "tumor" and "neoplasm" as used herein refer to any mass of tissue that results from excessive cell growth or proliferation, either benign (non -cancerous) or malignant (cancerous), including pre-cancerous lesions.

[00103] The term "metastasis" as used herein refers to the process by which a cancer spreads or transfers from the site of origin to other regions of the body with the development of a similar cancerous lesion at the new location. A "metastatic" or "metastasizing" cell is generally one that loses adhesive contacts with neighboring cells and migrates from the primary site of disease to invade neighboring tissue sites.

[00104] The terms "cancer stem cell" and "CSC" and "tumor stem cell" and "tumor initiating cell" are used interchangeably herein and refer to cells from a cancer or tumor that: ( 1) have extensive proliferative capacity; 2) are capable of asymmetric cell division to generate one or more types of differentiated cell progeny where the differentiated cells have reduced proliferative or developmental potential; and (3) are capable of symmetric cell divisions for self-renewal or self-maintenance. These properties confer on the cancer stem cells the ability to form or establish a tumor or cancer upon serial transplantation into an immunocompromised host (e.g., a mouse) compared to the majority of tumor cells that fail to form tumors. Cancer stem cells undergo self-renewal versus differentiation in a chaotic manner to form tumors with abnormal cell types that can change over time as mutations occur.

[00105] The terms "cancer cell" and "tumor cell" as used herein refer to the total population of cells derived from a cancer or tumor or pre-cancerous lesion, including both non-tumorigenic cells, which comprise the bulk of the cancer cell population, and tumorigenic cells (cancer stem cells). As used herein, the terms "cancer cell" or "tumor cell" will be modified by the term "non-tumorigenic" when referring solely to those cells lacking the capacity to renew and differentiate to distinguish those tumor cells from cancer stem cells.

[00106] The term "subject" as used herein refers to any animal (e.g., a mammal), including, but not limited to, humans, non-human primates, canines, felines, rodents, and the like, which is to be the recipient of a particular treatment. Typically, the terms "subject" and "patient" are used

interchangeably herein in reference to a human subject. [00107] The term "pharmaceutically acceptable" refers to an agent, compound, molecule, etc.

approved or approvable by a regulatory agency of the Federal government, a state government, and/or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, including humans.

[00108] The phrases "pharmaceutically acceptable excipient, carrier or adjuvant" and "acceptable pharmaceutical carrier" refer to an excipient, carrier, or adjuvant that can be administered to a subject, together with a therapeutic agent, and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic effect. In general, those of skill in the art and the FDA consider a pharmaceutically acceptable excipient, carrier, or adjuvant to be an inactive ingredient of any formulation or pharmaceutical composition.

[00109] The terms "effective amount" and "therapeutically effective amount" and "therapeutic effect" as used herein refer to an amount of a binding agent, an antibody, a polypeptide, a polynucleotide, a small molecule, or other therapeutic agent effective to "treat" a disease or disorder in a subject or mammal. In the case of cancer, the therapeutically effective amount of an agent (e.g., an antibody) has a therapeutic effect and as such can reduce the number of cancer cells; decrease tumorigenicity, tumorigenic frequency, or tumorigenic capacity; reduce the number or frequency of cancer stem cells; reduce tumor size; reduce the cancer cell population; inhibit and/or stop cancer cell infiltration into peripheral organs including, for example, the spread of cancer into soft tissue and bone; inhibit and stop tumor or cancer cell metastasis; inhibit and/or stop tumor or cancer cell growth; relieve to some extent one or more of the symptoms associated with the cancer; reduce morbidity and mortality; improve quality of life; or a combination of such effects. To the extent the agent prevents growth and/or kills existing cancer cells, it can be referred to as cytostatic and/or cytotoxic.

[00110] The terms "treating" and "treatment" and "to treat" and "alleviating" and "to alleviate" refer to both 1) therapeutic measures that cure, slow down, lessen symptoms of, and/or halt progression of a diagnosed pathologic condition or disorder and 2) prophylactic or preventative measures that prevent or slow the development of a targeted pathologic condition or disorder. Thus, those in need of treatment include those who already have a disorder; those prone to have a disorder; and those in whom a disorder is to be prevented. In some embodiments, a subject is successfully "treated" according to the methods of the present invention if the patient shows one or more of the following: a reduction in the number of or complete absence of cancer cells; a reduction in tumor size; inhibition of or an absence of cancer cell infiltration into peripheral organs including the spread of cancer cells into soft tissue and bone; inhibition of or an absence of tumor or cancer cell metastasis; inhibition or an absence of cancer growth; inhibition or an absence of tumor growth; relief of one or more symptoms associated with the specific cancer; reduced morbidity and mortality; improvement in quality of life; reduction in tumorigenicity; reduction in the number or frequency of cancer stem cells; or some combination of effects. [00111] The term "biomarker" as used herein may include but is not limited to, nucleic acids and proteins, and variants and fragments thereof. A biomarker may include DNA comprising the entire or partial nucleic acid sequence encoding the biomarker, or the complement of such a sequence.

[00112] As used in the present disclosure and claims, the singular forms "a", "an" and "the" include plural forms unless the context clearly dictates otherwise.

[00113] 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.

[00114] 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

[00115] A RSPO or LGR antagonist described herein in combination with an immunotherapeutic agent is useful in a variety of applications including, but not limited to, therapeutic treatment methods, such as immunotherapy for cancer. In certain embodiments, the combination of a RSPO or LGR antagonist (e.g., an anti-RSPO or anti-LGR antibody or a soluble LGR receptor) with an

immunotherapeutic agent, is useful for activating, promoting, increasing, and/or enhancing an immune response, inhibiting tumor growth, reducing tumor volume, increasing tumor cell apoptosis, and/or reducing the tumorigenicity of a tumor. The methods of use may be in vitro, ex vivo, or in vivo methods. In some embodiments, the combination of a RSPO or LGR antagonist (e.g., an anti-RSPO or anti-LGR antibody or a soluble LGR receptor) with an immunotherapeutic agent acts as an agonist of an immune response. In some embodiments, the combination of a RSPO or LGR antagonist (e.g., an anti-RSPO or anti-LGR antibody or a soluble LGR receptor) with an immunotherapeutic agent acts as an enhancer, activator, or stimulator of an immune response. In some embodiments, the combination of a RSPO or LGR antagonist (e.g., an anti-RSPO or anti-LGR antibody or a soluble LGR receptor) with an immunotherapeutic agent acts as an agonist of an anti-tumor immune response. In some embodiments, the combination of a RSPO or LGR antagonist (e.g., an anti-RSPO or anti- LGR antibody or a soluble LGR receptor) with an immunotherapeutic agent works as an antagonist of the PD-1 pathway. In some embodiments, the combination of a RSPO or LGR antagonist (e.g., an anti-RSPO or anti-LGR antibody or a soluble LGR receptor) with an immunotherapeutic agent works as an antagonist of PD-1 or PD-1 activity. In some embodiments, the combination of a RSPO or LGR antagonist (e.g., an anti-RSPO or anti-LGR antibody or a soluble LGR receptor) with an

immunotherapeutic agent works as an antagonist of PD-L1 or PD-L1 activity. In some embodiments, the combination of a RSPO or LGR antagonist (e.g., an anti-RSPO or anti-LGR antibody or a soluble LGR receptor) with an immunotherapeutic agent works as an antagonist of the CTLA-4 pathway. In some embodiments, the combination of a RSPO or LGR antagonist (e.g., an anti-RSPO or anti-LGR antibody or a soluble LGR receptor) with an immunotherapeutic agent works as an antagonist of CTLA-4 or CTLA-4 activity. In some embodiments, the combination of a RSPO or LGR antagonist (e.g., an anti-RSPO or anti-LGR antibody or a soluble LGR receptor) with an immunotherapeutic agent works as an antagonist of Tim -3 or Tim-3 activity. In some embodiments, the combination of a RSPO or LGR antagonist (e.g., an anti-RSPO or anti-LGR antibody or a soluble LGR receptor) with an immunotherapeutic agent works as an antagonist of LAG3 or LAG3 activity. In some

embodiments, the combination of a RSPO or LGR antagonist (e.g., an anti-RSPO or anti-LGR antibody or a soluble LGR receptor) with an immunotherapeutic agent works as an antagonist of TIGIT or TIGIT activity. In some embodiments, the combination of a RSPO or LGR antagonist (e.g., an anti-RSPO or anti-LGR antibody or a soluble LGR receptor) with an immunotherapeutic agent works as an antagonist of KIR or KIR activity. In any of these embodiments, the RSPO antagonist may be an anti-RSP03 antibody, such as 131R10 or an antibody containing the 131R10 CDR regions.

[00116] In certain embodiments of the methods described herein, a method of inhibiting tumor growth comprises contacting tumor cells with an effective amount of a RSPO or LGR antagonist in combination with an effective amount of an immunotherapeutic agent. The method may be in vivo or in vitro. In certain embodiments, the tumor is in a subject, and contacting tumor cells with the RSPO or LGR antagonist and the immunotherapeutic agent comprises administering a therapeutically effective amount of each of the agents to the subject. In some embodiments, a method of inhibiting tumor growth comprises administering to a subject a therapeutically effective amount of a RSPO or LGR antagonist and a therapeutically effective amount of an immunotherapeutic agent, where the RSPO or LGR antagonist is an anti-RSPO or anti-LGR antibody or a soluble LGR receptor. In some embodiments, the RSPO or LGR antagonist and the immunotherapeutic agent inhibit tumor growth by inhibiting or suppressing regulatory T-cell (Treg) activity. In some embodiments, the RSPO or LGR antagonist and the immunotherapeutic agent inhibit tumor growth by increasing cytolytic cell activity. In some embodiments, the RSPO or LGR antagonist and the immunotherapeutic agent inhibit tumor growth by increasing NK cell activity. In some embodiments, the RSPO or LGR antagonist and the immunotherapeutic agent inhibit tumor growth by increasing the number of CD4+ or CD8+ T-cells. In some embodiments, the RSPO or LGR antagonist and the immunotherapeutic agent inhibit tumor growth by increasing cytolytic T-cell activity. In some embodiments, the RSPO or LGR antagonist and the immunotherapeutic agent inhibit tumor growth by increasing CD8+ cytolytic T-cell activity. In some embodiments, the RSPO or LGR antagonist and the immunotherapeutic agent inhibit tumor growth by decreasing PD-1 expression on T-cells. In some embodiments, the RSPO or LGR antagonist and the immunotherapeutic agent inhibit tumor growth by decreasing the number or percentage of PD-1 expressing T-cells. In some embodiments, the RSPO or LGR antagonist and the immunotherapeutic agent inhibit tumor growth by decreasing the number or percentage of myeloid- derived suppressor cells (M-MDSCs). In some embodiments, the RSPO or LGR antagonist and the immunotherapeutic agent inhibit tumor growth by increasing the number or percentage of activated myeloid cells. In some embodiments, the RSPO or LGR antagonist and the immunotherapeutic agent inhibit tumor growth by increasing the number or percentage of memory T-cells. In some embodiments, the RSPO or LGR antagonist and the immunotherapeutic agent inhibit tumor growth by increasing IFN-gamma production. In some embodiments, the RSPO or LGR antagonist and the immunotherapeutic agent inhibit tumor growth by increasing IL-2 production. In some embodiments, the RSPO or LGR antagonist and the immunotherapeutic agent inhibit tumor growth by increasing IL- 10 production. In some embodiments, the RSPO or LGR antagonist and the immunotherapeutic agent inhibit tumor growth by increasing IL-17 production. In some embodiments, the RSPO or LGR antagonist and the immunotherapeutic agent inhibit tumor growth by decreasing IL-6 production.

[00117] In certain embodiments of the methods described herein, a method of treating cancer comprises administering to a subject a therapeutically effective amount of a RSPO or LGR antagonist in combination with a therapeutically effective amount of an immunotherapeutic agent. In some embodiments, the RSPO or LGR antagonist and the immunotherapeutic agent treat cancer by inhibiting or suppressing Treg activity. In some embodiments, the RSPO or LGR antagonist and the immunotherapeutic agent treat cancer by increasing cytolytic cell activity. In some embodiments, the RSPO or LGR antagonist and the immunotherapeutic agent treat cancer by increasing NK cell activity. In some embodiments, the RSPO or LGR antagonist and the immunotherapeutic agent treat cancer by increasing the number of CD4+ or CD8+ T-cells. In some embodiments, the RSPO or LGR antagonist and the immunotherapeutic agent treat cancer by increasing cytolytic T-cell activity. In some embodiments, the RSPO or LGR antagonist and the immunotherapeutic agent treat cancer by increasing CD8+ cytolytic T-cell activity. In some embodiments, the RSPO or LGR antagonist and the immunotherapeutic agent treat cancer by decreasing PD-1 expression on T-cells. In some embodiments, the RSPO or LGR antagonist and the immunotherapeutic agent treat cancer by decreasing the number or percentage of PD-1 expressing T-cells. In some embodiments, the RSPO or LGR antagonist and the immunotherapeutic agent treat cancer by decreasing the number or percentage of M-MDSCs. In some embodiments, the RSPO or LGR antagonist and the

immunotherapeutic agent treat cancer by increasing the number or percentage of activated myeloid cells. In some embodiments, the RSPO or LGR antagonist and the immunotherapeutic agent treat cancer by increasing the number or percentage of memory T-cells. In some embodiments, the RSPO or LGR antagonist and the immunotherapeutic agent treat cancer by increasing IFN-gamma production. In some embodiments, the RSPO or LGR antagonist and the immunotherapeutic agent treat cancer by increasing IL-2 production. In some embodiments, the RSPO or LGR antagonist and the immunotherapeutic agent treat cancer by increasing IL-10 production. In some embodiments, the RSPO or LGR antagonist and the immunotherapeutic agent treat cancer by increasing IL-17 production. In some embodiments, the RSPO or LGR antagonist and the immunotherapeutic agent treat cancer by decreasing IL-6 production.

[00118] In certain embodiments of the methods described herein, a method of cancer immunotherapy comprises administering to a subject a therapeutically effective amount of a RSPO or LGR antagonist in combination with a therapeutically effective amount of an immunotherapeutic agent, and where the combination results in enhanced therapeutic efficacy as compared to administration of either agent alone. In some embodiments, the RSPO or LGR antagonist is an anti-RSPO or anti-LGR antibody or a soluble LGR receptor.

[00119] In certain embodiments of the methods described herein, a method of inhibiting the activity of Tregs comprises administering to a subject a therapeutically effective amount of a RSPO or LGR antagonist in combination with a therapeutically effective amount of an immunotherapeutic agent. In some embodiments, a method of inhibiting the activity of Tregs comprises administering to a subject a therapeutically effective amount of a RSPO or LGR antagonist and a therapeutically effective amount of an immunotherapeutic agent, where the RSPO or LGR antagonist is an anti-RSPO or anti- LGR antibody or a soluble LGR receptor.

[00120] In certain embodiments of the methods described herein, a method of inhibiting the suppression of immune responses by Tregs comprises administering to a subject a therapeutically effective amount of a RSPO or LGR antagonist (e.g., an anti-RSPO antibody, anti-LGR antibody, or soluble LGR receptor) in combination with a therapeutically effective amount of an

immunotherapeutic agent.

[00121] In certain embodiments of the methods described herein, a method of increasing T cell infiltration into a tumor comprises administering to a subject a therapeutically effective amount of a RSPO or LGR antagonist (e.g., an anti-RSPO antibody, anti-LGR antibody, or soluble LGR receptor) in combination with a therapeutically effective amount of an immunotherapeutic agent.

[00122] In certain embodiments of the methods described herein, a method of increasing T cell cytotoxicity to a tumor comprises administering to a subject a therapeutically effective amount of a RSPO or LGR antagonist (e.g., an anti-RSPO antibody, anti-LGR antibody, or soluble LGR receptor) in combination with a therapeutically effective amount of an immunotherapeutic agent. [00123] In certain embodiments of the methods described herein, a method of increasing tumor cell lysis comprises administering to a subject a therapeutically effective amount of a RSPO or LGR antagonist (e.g., an anti-RSPO antibody, anti-LGR antibody, or soluble LGR receptor) and an immunotherapeutic agent.

[00124] In certain embodiments of the methods described herein, a method of inhibiting tumor relapse or tumor regrowth comprises administering to a subject a therapeutically effective amount of a RSPO or LGR antagonist (e.g., an anti-RSPO antibody, anti-LGR antibody, or soluble LGR receptor) in combination with a therapeutically effective amount of an immunotherapeutic agent.

[00125] In certain embodiments of the methods described herein, a method to increase the efficacy of an immune checkpoint modulator comprises administering to a subject a therapeutically effective amount of a RSPO or LGR antagonist (e.g., an anti-RSPO antibody, anti-LGR antibody, or soluble LGR receptor) in combination with a therapeutically effective amount of an immune checkpoint modulator. In some embodiments, the immune checkpoint modulator is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint modulator is an immune checkpoint enhancer or stimulator.

[00126] In certain embodiments of the methods described herein, a method of enhancing treatment for a subject who is being treated with an immune checkpoint modulator comprises administering to the subject a therapeutically effective amount of a RSPO or LGR antagonist, such as an anti-RSPO or anti-LGR antibody or a soluble LGR receptor. In some embodiments, the immune checkpoint modulator is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is a PD-1 antagonist. In some embodiments, the immune checkpoint inhibitor is an antibody that specifically binds PD-1. In some embodiments, the immune checkpoint inhibitor is a PD-L 1 antagonist. In some embodiments, immune checkpoint inhibitor is an antibody that specifically binds PD-L1. In some embodiments, the immune checkpoint inhibitor is a CTLA-4 antagonist. In some embodiments, the immune checkpoint inhibitor is an antibody that specifically binds CTLA-4.

[00127] In some embodiments, the method of inhibiting tumor growth comprises contacting the tumor or tumor cells with a RSPO or LGR antagonist, such as an anti-RSPO or anti-LGR antibody or a soluble LGR receptor and an immunotherapeutic agent in vivo. In certain embodiments, contacting a tumor or tumor cell with a RSPO or LGR antagonist and an immunotherapeutic agent is undertaken in an animal model. For example, a RSPO or LGR antagonist and an immunotherapeutic agent may be administered to mice which have tumors. In some embodiments, a RSPO or LGR antagonist and an immunotherapeutic agent increases, promotes, and/or enhances the activity of immune cells in the mice. In some embodiments, a RSPO or LGR antagonist and an immunotherapeutic agent are administered to an animal to inhibit growth of tumors. In some embodiments, a RSPO or LGR antagonist and an immunotherapeutic agent are administered at the same time or shortly after introduction of tumor cells into the animal (preventative model). In some embodiments, a RSPO or LGR antagonist and an immunotherapeutic agent are administered after the tumor cells have become established and grown to a tumor of specific size (therapeutic model).

[00128] In certain embodiments, a method of inhibiting growth of a tumor comprises administering to a subject a therapeutically effective amount of a RSPO or LGR antagonist, such as an anti-RSPO or anti-LGR antibody or a soluble LGR receptor and a therapeutically effective amount of an immunotherapeutic agent. In certain embodiments, the subject is a human. In certain embodiments, the subject has a tumor or has had a tumor which was removed. In certain embodiments, the tumor comprises cancer stem cells. In certain embodiments, the frequency of cancer stem cells in the tumor is reduced by administration of the RSPO or LGR antagonist.

[00129] The invention also provides a method of reducing or preventing metastasis in a subject comprising administering to the subject a therapeutically effective amount of a RSPO or LGR antagonist (e.g., an anti-RSPO antibody, anti-LGR antibody, or soluble LGR receptor) and a therapeutically effective amount of an immunotherapeutic agent. In some embodiments, the reduction or prevention of metastasis comprises inhibiting invasiveness of a tumor. In certain embodiments, the subject is a human. In certain embodiments, the subject has a tumor or has had a tumor removed.

[00130] In addition, the invention provides a method of reducing the tumorigenicity of a tumor in a subject, comprising administering to a subject a therapeutically effective amount of a RSPO or LGR antagonist (e.g., an anti-RSPO antibody, anti-LGR antibody, or soluble LGR receptor) and a therapeutically effective amount of an immunotherapeutic agent. In certain embodiments, the tumor comprises cancer stem cells. In some embodiments, the tumorigenicity of a tumor is reduced by reducing the frequency of cancer stem cells in the tumor. In certain embodiments, the frequency of cancer stem cells in the tumor is reduced by administration of the RSPO or LGR antagonist and the immunotherapeutic agent. In some embodiments, the tumorigenicity of the tumor is reduced by inducing apoptosis of the tumor cells. In some embodiments, the tumorigenicity of the tumor is reduced by increasing apoptosis of the tumor cells.

[00131] The invention also provides a method of reducing cancer stem cell frequency in a tumor comprising cancer stem cells, the method comprising administering to a subject a therapeutically effective amount of a RSPO or LGR antagonist (e.g., an anti-RSPO antibody, anti-LGR antibody, or soluble LGR receptor)and a therapeutically effective amount of an immunotherapeutic agent. In certain embodiments, the RSPO or LGR antagonist in combination with an immunotherapeutic agent is capable of reducing the tumorigenicity of a tumor comprising cancer stem cells in an animal model, such as a mouse model. In certain embodiments, the number or frequency of cancer stem cells in a treated tumor is reduced by at least about two-fold, about three-fold, about five-fold, about ten-fold, about 50-fold, about 100-fold, or about 1000-fold as compared to the number or frequency of cancer stem cells in an untreated tumor. In certain embodiments, the reduction in the number or frequency of cancer stem cells is determined by limiting dilution assay using an animal model. [00132] In some embodiments of the methods described herein the cancer is a cancer selected from the group consisting of lung cancer, pancreatic cancer, breast cancer, colon cancer, colorectal cancer, melanoma, gastrointestinal cancer, gastric cancer, renal cancer, ovarian cancer, liver cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, neuroblastoma, glioma, glioblastoma, glioblastoma multiforme, cervical cancer, stomach cancer, bladder cancer, head and neck cancer, and hepatoma. In some embodiments, the cancer is lung cancer. In some embodiments, the cancer is ovarian cancer.

[00133] In some embodiments, the RSPO or LGR antagonist is an RSPO-binding agent. In some embodiments, the RSPO or LGR antagonist is an 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 the antibody 131R10. In some embodiments, the RSPO or LGR antagonist is a soluble receptor. In some embodiments, the RSPO or LGR antagonist is an LGR-Fc soluble receptor. In some embodiments, the RSPO or LGR antagonist is an LGR5-Fc soluble receptor. In certain embodiments, the LGR5-Fc soluble receptor comprises amino acid sequence of SEQ ID NO: 57.

[00134] 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. In some embodiments, the antibody specifically binds at least one human RSP03.

[00135] In some embodiments, the antibody specifically binds at least human RSPO 1. In some embodiments, the antibody includes (a) a heavy chain CDR1 including TGYTMH (SEQ ID NO:5), a heavy chain CDR2 including GINPNNGGTTYNQNFKG (SEQ ID NO:6), and a heavy chain CDR3 including KEFSDGYYFFAY (SEQ ID NO:7); and/or (b) a light chain CDR1 including

KASQDVIFAVA (SEQ ID NO: 8), a light chain CDR2 including WASTRHT (SEQ ID NO:9), and a light chain CDR3 including QQHYSTPW (SEQ ID NO: 10). In some embodiments, the antibody includes a heavy chain variable region including an amino acid sequence at least 80%, 85%, 90%, 90%, 95%, or 100% identical to SEQ ID NO: 11 or 44 (e.g., including one, two or three of the heavy chain CDRs mentioned above), and a light chain variable region including an amino acid sequence at least 80%, 85%, 90%, 90%, 95%, or 100% identical to SEQ ID NO: 12 or 45 (e.g., including one, two, or three of the light chain CDRs mentioned above). In some embodiments, the antibody includes a heavy chain variable region including SEQ ID NO: 1 1 and a light chain variable region including SEQ ID NO: 12.

[00136] In some embodiments, the antibody specifically binds at least human RSP02. In some embodiments, the antibody includes (a) a heavy chain CDR1 including SSYAMS (SEQ ID NO: 17), a heavy chain CDR2 including SISSGGSTYYPDSVKG (SEQ ID NO: 18), and a heavy chain CDR3 including RGGDPGVYNGDYEDAMDY (SEQ ID NO: 19); and (b) a light chain CDR1 including KASQDVSSAVA (SEQ ID NO:20), a light chain CDR2 including WASTRHT (SEQ ID NO:21), and a light chain CDR3 including QQHYSTP (SEQ ID NO:22). In some embodiments, the antibody includes a heavy chain variable region including an amino acid sequence at least 80%, 85%, 90%, 90%, 95%, or 100% identical to SEQ ID NO:23 (e.g., including one, two, or three of the heavy chain CDRs mentioned above)and a light chain variable region including an amino acid sequence at least 80%, 85%, 90%, 90%, 95%, or 100% identical to SEQ ID NO:24 (e.g., including one, two, or three of the light CDRs mentioned above).

[00137] In some embodiments, the antibody specifically binds at least human RSP03. In some embodiments, the antibody includes (a) a heavy chain CDR1 including DYSIH (SEQ ID NO:29), a heavy chain CDR2 including YIYPSNGDSGYNQKFK (SEQ ID NO:30), and a heavy chain CDR3 including TYFANNFD (SEQ ID NO:31) or ATYF ANNTD Y( SEQ ID NO:32); and (b) a light chain CDR1 including KASQSVDYDGDSYMN (SEQ ID NO:33), a light chain CDR2 including

AASNLES (SEQ ID NO:34) or AAS (SEQ ID NO:35), and a light chain CDR3 including

[00138] In some embodiments, the RSPO or LGR antagonist is an antibody that specifically binds at least one human LGR protein (e.g., LGR4, LGR5, and LGR6). In some embodiments, the antibody specifically binds at least human LGR5. In certain embodiments, the antibody includes (a) 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 (b) 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 some embodiments, the antibody includes 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.

[00139] In some embodiments, the antibody is a monoclonal antibody, a recombinant antibody, a chimeric antibody, a humanized antibody, a human antibody, or an antibody fragment comprising an antigen-binding site. In some embodiments, the antibody is a monospecific antibody or a bispecific antibody. In some embodiments, the antibody is an IgGl antibody or an IgG2 antibody. In some embodiments, the RSPO or LGR antagonist is the antibody 131R10 or an antibody that contains the 131R10 CDR regions.

[00140] In some embodiments, the RSPO or LGR antagonist is a soluble receptor including an extracellular domain of a human LGR protein (e.g., LGR5), where the extracellular domain is capable of binding a human RSPO protein. In some embodiments, the extracellular domain of a human LGR protein includes amino acids 22-564 of human LGR5 (SEQ ID NO:56). In some embodiments, the soluble receptor includes 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 includes a human Fc region (e.g., includes SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, or SEQ ID NO:62, or an amino acid sequence at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, or SEQ ID NO:62).

[00141] In certain embodiments of any of the methods described herein, the RSPO or LGR antagonist is an antibody that specifically binds at least one human RSPO protein and the immunotherapeutic agent is a PD-1 antagonist. In some embodiments, the RSPO or LGR antagonist is an antibody that specifically binds at least one human RSPO protein and the immunotherapeutic agent is a PD-L 1 antagonist. In some embodiments, the RSPO or LGR antagonist is an antibody that specifically binds at least one human RSPO protein and the immunotherapeutic agent is a PD-L2 antagonist. In some embodiments, the RSPO or LGR antagonist is an antibody that specifically binds human at least one human RSPO protein and the immunotherapeutic agent is a CTLA-4 antagonist. In some

embodiments, the RSPO or LGR antagonist is an antibody that specifically binds at least one human RSPO protein and the immunotherapeutic agent is a CD80 antagonist. In some embodiments, the

RSPO or LGR antagonist is an antibody that specifically binds at least one human RSPO protein and the immunotherapeutic agent is a CD86 antagonist. In some embodiments, the RSPO or LGR antagonist is an antibody that specifically binds at least one human RSPO protein and the

immunotherapeutic agent is a KIR antagonist. In some embodiments, the RSPO or LGR antagonist is an antibody that specifically binds at least one human RSPO protein and the immunotherapeutic agent is a Tim-3 antagonist. In some embodiments, the RSPO or LGR antagonist is an antibody that specifically binds at least one human RSPO protein and the immunotherapeutic agent is a LAG3 antagonist. In some embodiments, the RSPO or LGR antagonist is an antibody that specifically binds at least one human RSPO protein and the immunotherapeutic agent is a TIGIT antagonist. In some embodiments, the RSPO or LGR antagonist is an antibody that specifically binds at least one human RSPO protein and the immunotherapeutic agent is a CD96 antagonist. In some embodiments, the RSPO or LGR antagonist is an antibody that specifically binds at least one human RSPO protein and the immunotherapeutic agent is an IDOl antagonist. In some embodiments, the RSPO or LGR antagonist is an antibody that specifically binds at least one human RSPO protein and the

immunotherapeutic agent is a CD28 agonist. In some embodiments, the RSPO or LGR antagonist is an antibody that specifically binds at least one human RSPO protein and the immunotherapeutic agent is a 4-1BB agonist. In some embodiments, the RSPO or LGR antagonist is an antibody that specifically binds at least one human RSPO protein and the immunotherapeutic agent is an OX40 agonist. In some embodiments, the RSPO or LGR antagonist is an antibody that specifically binds at least one human RSPO protein and the immunotherapeutic agent is a CD27 agonist. In some embodiments, the RSPO or LGR antagonist is an antibody that specifically binds at least one human RSPO protein and the immunotherapeutic agent is a CD80 agonist. In some embodiments, the RSPO or LGR antagonist is an antibody that specifically binds at least one human RSPO protein and the immunotherapeutic agent is a CD86 agonist. In some embodiments, the RSPO or LGR antagonist is an antibody that specifically binds at least one human RSPO protein and the immunotherapeutic agent is a CD40 agonist. In some embodiments, the RSPO or LGR antagonist is an antibody that specifically binds at least one human RSPO protein and the immunotherapeutic agent is a GITR agonist. In some embodiments, the RSPO or LGR antagonist is an antibody that specifically binds at least one human RSPO protein and the immunotherapeutic agent is a cytokine. In some

embodiments, the RSPO or LGR antagonist is an antibody that specifically binds at least one human RSPO protein and the immunotherapeutic agent is an interferon. In some embodiments, the RSPO or LGR antagonist is an antibody that specifically binds at least one human RSPO protein and the immunotherapeutic agent is a lymphokine. In any of these embodiments, the RSPO protein is RPS03.

[00142] The present invention further provides compositions comprising RSPO or LGR antagonists and compositions comprising immunotherapeutic agents. In some embodiments, a composition comprises a RSPO or LGR antagonist described herein. In some embodiments, a composition comprises an antibody that specifically binds at least one human RSPO or LGR protein described herein. In some embodiments, the composition comprises a soluble receptor comprising the extracellular domain of human LGR protein (e.g., human LGR5) described herein. In some embodiments, a composition comprises an immunotherapeutic agent described herein. In some embodiments, a composition is a pharmaceutical composition comprising a RSPO or LGR antagonist and a pharmaceutically acceptable vehicle. In some embodiments, a composition is a pharmaceutical composition comprising an immunotherapeutic agent and a pharmaceutically acceptable vehicle. The pharmaceutical compositions find use in modulating immune responses in human patients, particularly immune responses to tumors. The pharmaceutical compositions find use in inhibiting tumor cell growth and treating cancer in human patients. The pharmaceutical compositions find use in any of the methods described herein. In some embodiments, a RSPO or LGR antagonist described herein finds use in the manufacture of a medicament for the treatment of cancer in combination with at least one immunotherapeutic agent. In some embodiments, a RSPO or LGR antagonist described herein finds use in the manufacture of a medicament for the treatment of cancer in combination with at least one immunotherapeutic agent.

[00143] Formulations and/or pharmaceutical compositions are prepared for storage and use by combining a therapeutic agent of the present invention with a pharmaceutically acceptable carrier, excipient, and/or stabilizer as a sterile lyophilized powder, aqueous solution, etc. (Remington: The Science and Practice of Pharmacy, 22nd Edition, 2012, Pharmaceutical Press, London). 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.

[00144] Suitable carriers, excipients, or stabilizers comprise nontoxic buffers such as phosphate, citrate, and other organic acids; salts such as sodium chloride; antioxidants including ascorbic acid and methionine; preservatives (e.g. 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 (such as 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 (e.g. Zn-protein complexes); and/or non-ionic surfactants such as polysorbate (TWEEN) or polyethylene glycol (PEG).

[00145] 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 for oral, parenteral, or rectal administration or for administration by inhalation. In solid compositions such as tablets the principal active ingredient is mixed with a pharmaceutical carrier. As described herein, pharmaceutical carriers are considered to be inactive ingredients of a formulation or composition. Conventional tableting ingredients include corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, and other diluents (e.g. water) 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 the type described above. The tablets, pills, etc., of the novel 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, including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol and cellulose acetate.

[00146] Pharmaceutical formulations may include the RSPO or LGR antagonists and/or the immunotherapeutic agents of the present invention complexed with liposomes. Liposomes can be generated by the reverse phase evaporation with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidyl ethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter.

[00147] The RSPO or LGR antagonists and/or immunotherapeutic agents 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- (methylmethacylate) 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, 22nd Edition, 2012, Pharmaceutical Press, London.

[00148] In addition, sustained-release preparations comprising RSPO or LGR antagonists and/or immunotherapeutic agents can be prepared. Suitable examples of sustained-release preparations include semi-permeable matrices of solid hydrophobic polymers containing the agent, which matrices are in the form of shaped articles (e.g., films or microcapsules). Examples of sustained-release matrices include polyesters, hydrogels such as poly(2-hydroxyethyl-methacrylate) or

poly(vinylalcohol), 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.

[00149] The RSPO or LGR antagonists and immunotherapeutic agents are administered as appropriate pharmaceutical compositions to a human patient according to known methods. The pharmaceutical compositions can be administered in any number of ways for either local or systemic treatment.

Suitable methods of administration include, but are not limited to, intravenous (administration as a bolus or by continuous infusion over a period of time), intraarterial, intramuscular (injection or infusion), intratumoral, intraperitoneal, intracerobrospinal, subcutaneous, intra-articular, intrasynovial, intracranial (e.g., intrathecal or intraventricular), or oral. In additional, administration can be topical, (e.g., transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders) or pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal). [00150] For the treatment of a disease, the appropriate dosage(s) of a RSPO or LGR antagonist in combination with an immunotherapeutic agent 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 inhibitors are 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 or LGR antagonist can be administered one time or as a series of treatments spread over several days to several months, or until a cure is effected or a diminution of the disease state is achieved (e.g., reduction in tumor size). The immunotherapeutic agent can be administered one time or as a series of treatments spread over several days to several months, or until a cure is effected or a diminution of the disease state is achieved (e.g., reduction in tumor size). Optimal dosing schedules for each agent 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 agent. The administering physician can determine optimum dosages, dosing methodologies, and repetition rates.

[00151] In some embodiments, combined administration includes co-administration in a single pharmaceutical formulation. In some embodiments, combined administration includes using separate formulations and consecutive administration in either order but generally within a time period such that all active agents can exert their biological activities simultaneously. In some embodiments, combined administration includes using separate formulations and a staggered dosing regimen. In some embodiments, combined administration includes using separate formulations and administration in a specific order. In some embodiments, combined administration includes using separate formulations and administration of the agents in a specific order and in a staggered dosing regimen.

[00152] In certain embodiments, dosage of a RSPO or LGR antagonist is from about 0.0 ^g to about lOOmg/kg of body weight, from about O. ^g to about lOOmg/kg of body weight, from about ^g to about lOOmg/kg of body weight, from about lmg to about lOOmg/kg of body weight, about lmg to about 80mg/kg of body weight from about lOmg to about lOOmg/kg of body weight, from about lOmg to about 75mg/kg of body weight, or from about lOmg to about 50mg/kg of body weight. In certain embodiments, the dosage of the RSPO or LGR antagonist is from about 0. lmg to about 20mg/kg of body weight. In some embodiments, the RSPO or LGR antagonist is administered to the subject at a dosage of about 2mg/kg to about 15mg/kg. In some embodiments, the RSPO or LGR antagonist is administered to the subject at a dosage of about 5mg/kg to about 15mg/kg. In certain embodiments, the RSPO or LGR antagonist is administered once or more daily, weekly, monthly, or yearly. In certain embodiments, the RSPO or LGR antagonist is administered once every week, once every two weeks, once every three weeks, or once every four weeks.

[00153] In certain embodiments, dosage of an immunotherapeutic agent is from about O.O^g to about lOOmg/kg of body weight, from about O. ^g to about lOOmg/kg of body weight, from about ^g to about lOOmg/kg of body weight, from about lmg to about lOOmg/kg of body weight, about lmg to about 80mg/kg of body weight from about lOmg to about lOOmg/kg of body weight, from about lOmg to about 75mg/kg of body weight, or from about lOmg to about 50mg/kg of body weight. In certain embodiments, the dosage of an immunotherapeutic agent is from about 0. lmg to about 20mg/kg of body weight. In some embodiments, an immunotherapeutic agent is administered to the subject at a dosage of about 2mg/kg to about 15mg/kg. In some embodiments, the RSPO or LGR antagonist is administered to the subject at a dosage of about 5mg/kg to about 15mg/kg. In certain embodiments, an immunotherapeutic agent is administered once or more daily, weekly, monthly, or yearly. In certain embodiments, an immunotherapeutic agent is administered once every week, once every two weeks, once every three weeks, or once every four weeks.

[00154] In some embodiments, dosage of an immunotherapeutic agent is determined by what is considered "standard-of-care" for a particular agent by those of skill in the art (e.g., treating physicians).

[00155] In some embodiments, an inhibitor may be administered at an initial higher "loading" dose, followed by one or more lower doses. In some embodiments, the frequency of administration may also change. In some embodiments, a dosing regimen may comprise administering an initial dose, followed by additional doses (or "maintenance" doses) once a week, once every two weeks, once every three weeks, or once every month. For example, a dosing regimen may comprise administering an initial loading dose, followed by a weekly maintenance dose of, for example, one-half of the initial dose. Or a dosing regimen may comprise administering an initial loading dose, followed by maintenance doses of, for example one-half of the initial dose every other week. Or a dosing regimen may comprise administering three initial doses for 3 weeks, followed by maintenance doses of, for example, the same amount every other week.

[00156] 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.

[00157] The present invention provides methods of treating cancer in a subject comprising using a dosing strategy for administering two or more agents that may reduce side effects and/or toxicities associated with administration of a RSPO or LGR antagonist and/or an immunotherapeutic agent. In some embodiments, a method for treating cancer in a human subject comprises administering to the subject a therapeutically effective dose of a RSPO or LGR antagonist in combination with a therapeutically effective dose of an immunotherapeutic agent, where one or both of the inhibitors are administered according to an intermittent dosing strategy. In some embodiments, the intermittent dosing strategy comprises administering an initial dose of a RSPO or LGR antagonist to the subject, and administering subsequent doses of the RSPO or LGR antagonist about once every 2 weeks. In some embodiments, the intermittent dosing strategy comprises administering an initial dose of a RSPO or LGR antagonist to the subject, and administering subsequent doses of the RSPO or LGR antagonist about once every 3 weeks. In some embodiments, the intermittent dosing strategy comprises administering an initial dose of a RSPO or LGR antagonist to the subject, and

administering subsequent doses of the RSPO or LGR antagonist about once every 4 weeks. In some embodiments, the RSPO or LGR antagonist is administered using an intermittent dosing strategy and the immunotherapeutic agent is administered weekly or every week for 3 weeks out of 4.

[00158] 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). Combination therapy may decrease the likelihood that resistant cancer cells will develop. Combination therapy comprising an

immunotherapeutic agent may allow one agent to enhance the immune response to a tumor or tumor cells while the other agent may be effective at killing tumor cells more directly.

[00159] In some embodiments, the combination of a RSPO or LGR antagonist and an

immunotherapeutic agent results in additive or synergetic results. In some embodiments, the combination therapy results in an increase in the therapeutic index of the RSPO or LGR antagonist. In some embodiments, the combination therapy results in an increase in the therapeutic index of the immunotherapeutic agent. In some embodiments, the combination therapy results in a decrease in the toxicity and/or side effects of the RSPO or LGR antagonist. In some embodiments, the combination therapy results in a decrease in the toxicity and/or side effects of the immunotherapeutic agent.

[00160] The treating physician can estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues. The progress of therapy can be monitored by conventional techniques and assays.

[00161] In certain embodiments, in addition to administering a RSPO or LGR antagonist in combination with an immunotherapeutic agent, treatment methods may further comprise

administering at least one additional therapeutic agent prior to, concurrently with, and/or subsequently to administration of the RSPO or LGR antagonist and/or the immunotherapeutic agent.

[00162] In some embodiments, the additional therapeutic agent(s) will be administered substantially simultaneously or concurrently with the RSPO or LGR antagonist or the immunotherapeutic agent.

For example, a subject may be given the RSPO or LGR antagonist and the immunotherapeutic agent while undergoing a course of treatment with the additional therapeutic agent (e.g., additional chemotherapeutic agent). In certain embodiments, the RSPO or LGR antagonist and the immunotherapeutic agent will be administered within 1 year of the treatment with the additional therapeutic agent. In certain alternative embodiments, the RSPO or LGR antagonist and the immunotherapeutic agent will be administered within 10, 8, 6, 4, or 2 months of any treatment with the additional therapeutic agent. In certain other embodiments, the RSPO or LGR antagonist and the immunotherapeutic agent will be administered within 4, 3, 2, or 1 week of any treatment with the additional therapeutic agent. In some embodiments, the RSPO or LGR antagonist and the immunotherapeutic agent will be administered within 5, 4, 3, 2, or 1 days of any treatment with the additional therapeutic agent. It will further be appreciated that the agents or treatment may be administered to the subject within a matter of hours or minutes (i.e., substantially simultaneously) with the RSPO or LGR antagonist or the immunotherapeutic agent.

[00163] Therapeutic agents that may be administered in combination with a RSPO or LGR antagonist and an immunotherapeutic agent include chemotherapeutic agents. Thus, in some embodiments, the method or treatment involves the administration of a RSPO or LGR antagonist and

immunotherapeutic agent of the present invention in combination with a chemotherapeutic agent or cocktail of multiple different chemotherapeutic agents. Treatment with a RSPO or LGR antagonist and immunotherapeutic agent can occur prior to, concurrently with, or subsequent to administration of chemotherapies. Preparation and dosing schedules for such chemotherapeutic agents can be used according to manufacturers' instructions or as determined empirically by the skilled practitioner. Preparation and dosing schedules for such chemotherapy are also described in Chemotherapy Service, 1992, M. C. Perry, Editor, Williams & Wilkins, Baltimore, MD.

[00164] Chemotherapeutic agents useful in the instant invention include, but are not limited to, alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine,

trietylenephosphoramide, triethylenethiophosphaoramide and trimethylolomelamime; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, calicheamicin, carabicin, caminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone;

aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK.; razoxane;

sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; platinum; etoposide (VP-16);

ifosfamide; mitomycin C; mitoxantrone; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT1 1 ; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; esperamicins; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Chemotherapeutic agents also include anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY1 17018, onapristone, and toremifene; and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[00165] In certain embodiments, the chemotherapeutic agent is a topoisomerase inhibitor.

Topoisomerase inhibitors are chemotherapy agents that interfere with the action of a topoisomerase enzyme (e.g., topoisomerase I or II). Topoisomerase inhibitors include, but are not limited to, doxorubicin HC1, daunorubicin citrate, mitoxantrone HC1, actinomycin D, etoposide, topotecan HC1, teniposide (VM-26), and irinotecan.

[00166] In certain embodiments, the chemotherapeutic agent is an anti -metabolite. An anti -metabolite is a chemical with a structure that is similar to a metabolite required for normal biochemical reactions, yet different enough to interfere with one or more normal functions of cells, such as cell division. Anti-metabolites include, but are not limited to, gemcitabine, fluorouracil, capecitabine, methotrexate sodium, ralitrexed, pemetrexed, tegafur, cytosine arabinoside, thioguanine, 5-azacytidine, 6- mercaptopurine, azathioprine, 6-thioguanine, pentostatin, fludarabine phosphate, and cladribine, as well as pharmaceutically acceptable salts, acids, or derivatives of any of these.

[00167] In certain embodiments, the chemotherapeutic agent is an antimitotic agent, including, but not limited to, agents that bind tubulin. In some embodiments, the agent is a taxane. In certain embodiments, the agent is paclitaxel or docetaxel, or a pharmaceutically acceptable salt, acid, or derivative of paclitaxel or docetaxel. In certain embodiments, the agent is paclitaxel (TAXOL), docetaxel (TAXOTERE), albumin-bound paclitaxel (ABRAXANE^®), DHA-paclitaxel, or PG- paclitaxel. In certain alternative embodiments, the antimitotic agent comprises a vinca alkaloid, such as vincristine, binblastine, vinorelbine, or vindesine, or pharmaceutically acceptable salts, acids, or derivatives thereof. In some embodiments, the antimitotic agent is an inhibitor of kinesin Eg5 or an inhibitor of a mitotic kinase such as Aurora A or Plkl .

[00168] In some embodiments, an additional therapeutic agent that may be administered in combination with a RSPO or LGR antagonist and an immunotherapeutic agent comprises an agent such as a small molecule. For example, treatment can involve the combined administration of a RSPO or LGR antagonist and an immunotherapeutic agent with a small molecule that acts as an inhibitor against tumor-associated antigens including, but not limited to, EGFR, HER2 (ErbB2), and/or VEGF. In some embodiments, a RSPO or LGR antagonist and an immunotherapeutic agent are administered in combination with a protein kinase inhibitor selected from the group consisting of: gefitinib (IRESSA), erlotinib (TARCEVA), sunitinib (SUTENT), lapatanib, vandetanib (ZACTIMA), AEE788, CI-1033, cediranib (RECENTIN), sorafenib (NEXAVAR), and pazopanib (GW786034B). In some embodiments, an additional therapeutic agent comprises an mTOR inhibitor.

[00169] In some embodiments, an additional therapeutic agent comprises a biological molecule, such as an antibody. For example, treatment can involve the combined administration of a RSPO or LGR antagonist and an immunotherapeutic agent with antibodies against tumor-associated antigens including, but not limited to, antibodies that bind EGFR, HER2/ErbB2, and/or VEGF. In certain embodiments, the additional therapeutic agent is an antibody specific for a cancer stem cell marker. In certain embodiments, the additional therapeutic agent is an antibody that inhibits a cancer stem cell pathway. In certain embodiments, the additional therapeutic agent is an antibody that is an angiogenesis inhibitor (e.g., an anti-VEGF or VEGF receptor antibody). In certain embodiments, the additional therapeutic agent is bevacizumab (AVASTIN), ramucirumab, trastuzumab (HERCEPTIN), pertuzumab (OMNITARG), panitumumab (VECTIBIX), nimotuzumab, zalutumumab, or cetuximab (ERBITUX).

[00170] Furthermore, treatment can involve the combined administration of a RSPO or LGR antagonist and an immunotherapeutic agent with other biologic molecules, such as one or more cytokines (e.g., lymphokines, interleukins, tumor necrosis factors, and/or growth factors) or can be accompanied by surgical removal of tumors, removal of cancer cells, or any other therapy deemed necessary by a treating physician.

[00171] In some embodiments, treatment can involve the combined administration of a RSPO or LGR antagonist and an immunotherapeutic agent with a growth factor selected from the group consisting of, but not limited to: adrenomedullin (AM), angiopoietin (Ang), BMPs, BDNF, EGF, erythropoietin (EPO), FGF, GDNF, G-CSF, GM-CSF, GDF9, HGF, HDGF, IGF, migration-stimulating factor, myostatin (GDF-8), NGF, neurotrophins, PDGF, thrombopoietin, TGF-a, TGF-β, TNF-a, VEGF, P1GF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-12, IL-15, and IL-18.

[00172] In certain embodiments, treatment can involve the combined administration of a RSPO or LGR antagonist and an immunotherapeutic agent with radiation therapy. Treatment with a RSPO or LGR antagonist and an immunotherapeutic agent can occur prior to, concurrently with, or subsequent to administration of radiation therapy. Dosing schedules for such radiation therapy can be determined by the skilled medical practitioner.

III. RSPO and LGR antagonists

[00173] Described herein are methods, including, for example, methods of inhibiting tumor growth or treating cancer, comprising administering an RSPO or LGR antagonist in combination with an immunotherapeutic .

[00174] In certain embodiments, the RSPO or LGR antagonist binds one or more RSPO or LGR proteins. In certain embodiments, the RSPO or LGR antagonist binds one or more extracellular region(s) of LGR. In certain embodiments, the RSPO or LGR antagonist inhibits β-catenin signaling. In certain embodiments, the RSPO or LGR antagonist modulates Wnt mediated β-catenin signaling.

[00175] In certain embodiments, the RSPO or LGR antagonist binds one or more human RSPO proteins. These agents are referred to herein as "RSPO-binding agents". Non-limiting examples of RSPO-binding agents can be found in U.S. Patent Nos. 8158758, 8158757, 8802097, 8088374, and U.S. Patent Publication Nos. 2014/0017253, 2014/0134703, 2013/0337533, 2014/0186917,

2012/0263730, 2012/0039912, 2009/0220495, 2012/0088727, and 2014/0056894, each of which are hereby incorporated by reference herein in their entirety for all purposes.

[00176] 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 ("RSPOl -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: 1 (RSPO l), SEQ ID NO:2 (RSP02), SEQ ID NO:3 (RSP03), and SEQ ID NO:4 (RSP04).

[00177] In certain embodiments, the antigen-binding site of an 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 an 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., RSPO 1) 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.

[00178] In certain embodiments, 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 RSPO l (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: 1). In certain embodiments, the RSPO-binding agent is an antibody that specifically binds within amino acids 147-207 of human RSPOl (SEQ ID NO: 1). In certain embodiments, the RSPO-binding agent binds a furin-like cysteine-rich domain of RSPOl. In some embodiments, the RSPO-binding agent binds at least one amino acid within a furin- like cysteine-rich domain of RSPO l . In some embodiments, the RSPO-binding agent binds the thrombospondin domain of RSPO 1. In some embodiments, the RSPO-binding agent binds at least one amino acid within the thrombospondin domain of RSPO 1.

[00179] 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). hi 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). hi certain embodiments, the RSPO-binding agent binds a furin-like cysteine-rich domain of RSP02. hi 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.

[00180] 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). hi 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.

[00181] In certain embodiments, the RSPO-binding agent or antibody binds at least one RSPO protein with a dissociation constant (KD) of about ΙμΜ or less, about lOOnM or less, about 40nM or less, about 20nM or less, about ΙΟηΜ or less, about InM or less, or about O. lnM or less. In certain embodiments, an RSPO-binding agent or antibody binds at least one RSPO protein with a dissociation constant (KD) of about ΙμΜ or less, about lOOnM or less, about 40nM or less, about 20nM or less, about ΙΟηΜ or less, about InM or less, or about 0. InM or less. In some embodiments, an RSPO- binding agent or antibody binds at least one RSPO protein with a KD of about 20nM or less. In some embodiments, an RSPO-binding agent or antibody binds at least one RSPO protein with a KD of about ΙΟηΜ or less. In some embodiments, an RSPO-binding agent or antibody binds at least one RSPO protein with a KD of about InM or less. In some embodiments, an RSPO-binding agent or antibody binds at least one RSPO protein with a KD of about 0.5nM or less. In some embodiments, an RSPO-binding agent or antibody binds at least one RSPO protein with a KD of about O. lnM or less. In certain embodiments, an RSPO-binding agent or antibody described herein binds at least two

RSPO proteins. In some embodiments, the RSPO-binding agent binds both human RSPO and mouse RSPO with a KD of about ΙΟηΜ or less. In some embodiments, an RSPO-binding agent binds both human RSPO and mouse RSPO with a KD of about InM or less. In some embodiments, an RSPO- binding agent binds both human RSPO and mouse RSPO with a KD of about O. lnM or less. In some embodiments, the dissociation constant of a binding agent (e.g., an antibody) to an RSPO protein is the dissociation constant determined using an RSPO fusion protein comprising at least a portion of the RSPO protein immobilized on a Biacore chip. In some embodiments, the dissociation constant of a binding agent (e.g., an antibody) to an RSPO protein is the dissociation constant determined using the binding agent captured by an anti-human IgG antibody on a Biacore chip and an RSPO protein.

[00182] In certain embodiments, the RSPO-binding agent (e.g., an antibody) binds to at least one human RSPO protein with a half maximal effective concentration (EC50) of about ΙμΜ or less, about lOOnM or less, about 40nM or less, about 20nM or less, about ΙΟηΜ or less, about InM or less, or about 0. InM or less. In certain embodiments, an RSPO-binding agent (e.g., an antibody) binds to at least one human RSPO with a half maximal effective concentration (EC50) of about ΙμΜ or less, about lOOnM or less, about 40nM or less, about 20nM or less, about ΙΟηΜ or less, about InM or less, or about 0. InM or less. [00183] In certain embodiments, the RSPO-binding agent is an RSPO l-binding agent (e.g., an antibody) that specifically binds human RSPO l, wherein the RSPOl-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

[00184] In certain embodiments, the RSPO-binding agent is an RSPO l-binding agent (e.g., an antibody) that specifically binds human RSPO 1 , wherein the RSPO 1 -binding agent 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). In some embodiments, the RSPO 1 -binding agent further 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 some embodiments, the RSPOl-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).

[00185] In certain embodiments, the RSPO-binding agent is an RSPO l-binding agent (e.g., an antibody or bispecific antibody) that specifically binds human RSPO 1 , wherein the RSPO 1 -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.

[00186] In certain embodiments, the RSPO-binding agent is an RSPO l-binding agent (e.g., an antibody) that specifically binds RSPOl, wherein the RSPOl-binding agent comprises a heavy chain variable region having at least about 80% sequence identity to SEQ ID NO: 11 and/or a light chain variable region having at least 80% sequence identity to SEQ ID NO: 12. In certain embodiments, the RSPOl-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: 11. In certain embodiments, the RSPO l-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: 12. In certain embodiments, the RSPOl-binding agent comprises a heavy chain variable region having at least about 95% sequence identity to SEQ ID NO: 11 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: 11, and/or a light chain variable region comprising SEQ ID NO: 12. In certain embodiments, the RSPOl-binding agent comprises a heavy chain variable region comprising SEQ ID NO: 11 and a light chain variable region comprising SEQ ID NO: 12. In certain embodiments, the RSPOl-binding agent comprises a heavy chain variable region consisting of SEQ ID NO: 11 and a light chain variable region consisting of SEQ ID NO: 12.

[00187] In certain embodiments, the RSPO-binding agent is an RSPO l-binding agent (e.g., an antibody) that specifically binds RSPOl, wherein the RSPOl-binding agent comprises a heavy chain variable region having at least about 80% sequence identity to SEQ ID NO:44 and/or a light chain variable region having at least 80% sequence identity to SEQ ID NO:45. In certain embodiments, the RSPOl-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: 44. In certain embodiments, the RSPO l-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:45. In certain embodiments, the RSPOl-binding agent comprises a heavy chain variable region having at least about 95% sequence identity to SEQ ID NO: 44 and/or a light chain variable region having at least about 95% sequence identity to SEQ ID NO: 45. In certain embodiments, the RSPO l-binding agent comprises a heavy chain variable region comprising SEQ ID NO:44 and/or a light chain variable region comprising SEQ ID NO:45. In certain embodiments, the RSPOl-binding agent comprises a heavy chain variable region comprising SEQ ID NO:44 and a light chain variable region comprising SEQ ID NO:45. In certain embodiments, the RSPOl-binding agent comprises a heavy chain variable region consisting of SEQ ID NO:44 and a light chain variable region consisting of SEQ ID NO:45.

[00188] In certain embodiments, the RSPO-binding agent is an RSPO 1-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 RSPOl-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 RSPOl-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.

[00189] In certain embodiments, the RSPO-binding agent is an RSPO 1-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:46 or SEQ ID NO:47; and/or (b) a light chain having at least 90% sequence identity to SEQ ID NO:48 or SEQ ID NO:49. In some embodiments, the RSPOl-binding agent comprises: (a) a heavy chain having at least 95% sequence identity to SEQ ID NO:46 or SEQ ID NO:47; and/or (b) a light chain having at least 95% sequence identity to SEQ ID NO:48 or SEQ ID NO:49. In some embodiments, the RSPOl-binding agent comprises a heavy chain comprising SEQ ID NO:47 and/or a light chain comprising SEQ ID NO:49. In some embodiments, the RSPO l-binding agent comprises a heavy chain comprising SEQ ID NO:47 and a light chain comprising SEQ ID NO: 49.

[00190] In certain embodiments, an RSPOl-binding agent comprises the heavy chain variable region and light chain variable region of antibody h89M5-H8L5. In certain embodiments, an RSPOl-binding agent comprises the heavy chain and light chain of antibody h89M5-H8L5 (with or without the leader sequence). In certain embodiments, an RSPOl-binding agent is antibody h89M5-H8L5. In certain embodiments, an RSPO l-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-RSPO 1 antibody is h89M5-H8L5.

[00191] In certain embodiments, an RSPOl-binding agent comprises the heavy chain variable region and light chain variable region of antibody h89M5-H2L2. In certain embodiments, an RSPOl-binding agent comprises the heavy chain and light chain of antibody h89M5-H2L2 (with or without the leader sequence). In certain embodiments, an RSPOl-binding agent is antibody h89M5-H2L2. In certain embodiments, an RSPO 1 -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-RSPO 1 antibody is h89M5-H2L2.

[00192] In certain embodiments, an 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, 2011 and assigned ATCC deposit designation number PTA-11970.

[00193] 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 l-binding agent comprises a heavy chain variable region encoded by the plasmid deposited with ATCC and designated PTA-121494. In some embodiments, the RSPOl-binding agent comprises a light chain variable region encoded by the plasmid deposited with ATCC and designated PTA-121495. In some embodiments, the RSPOl-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 RSPOl-binding agent comprises a heavy chain encoded by the plasmid deposited with ATCC and designated PTA-121494. In some embodiments, the RSPOl-binding agent comprises a light chain encoded by the plasmid deposited with ATCC and designated PTA-121495. In some embodiments, the RSPOl-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.

[00194] In certain embodiments, an RSPOl-binding agent comprises, consists essentially of, or consists of, antibody h89M5-H8L5. In certain embodiments, an RSPOl-binding agent comprises, consists essentially of, or consists of, a variant of antibody 89M5. In certain embodiments, an RSPOl- binding agent comprises, consists essentially of, or consists of, a variant of antibody h89M5-H8L5. [00195] In certain embodiments, an RSPOl -binding agent comprises, consists essentially of, or consists of, antibody h89M5-H2L2. In certain embodiments, an RSPO l-binding agent comprises, consists essentially of, or consists of, a variant of antibody 89M5. In certain embodiments, an RSPOl- binding agent comprises, consists essentially of, or consists of, a variant of antibody h89M5-H2L2.

[00196] In certain embodiments of the methods described herein, the RSPO-binding agent is an

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).

[00197] In certain embodiments, the RSPO-binding agent is an RSP02-binding agent (e.g., an antibody) that specifically binds human RSP02, wherein the RSP02-binding agent comprises a heavy chain CDRl comprising SSYAMS (SEQ ID NO: 17), a heavy chain CDR2 comprising

SISSGGSTYYPDSVKG (SEQ ID NO: 18), and a heavy chain CDR3 comprising

RGGDPGVYNGDYEDAMDY (SEQ ID NO: 19). In some embodiments, the RSP02-binding agent further comprises a light chain CDRl comprising KASQDVSSAVA (SEQ ID NO:20), a light chain CDR2 comprising WASTRHT (SEQ ID NO:21), and a light chain CDR3 comprising QQHYSTP (SEQ ID NO:22). In some embodiments, the RSP02-binding agent comprises a light chain CDRl comprising KASQDVSSAVA (SEQ ID NO:20), a light chain CDR2 comprising WASTRHT (SEQ ID NO:21), and a light chain CDR3 comprising QQHYSTP (SEQ ID NO:22). In certain

embodiments, the RSP02-binding agent comprises: (a) a heavy chain CDRl comprising SSYAMS (SEQ ID NO: 17), a heavy chain CDR2 comprising SISSGGSTYYPDSVKG (SEQ ID NO: 18), and a heavy chain CDR3 comprising RGGDPGVYNGDYEDAMDY (SEQ ID NO: 19); and (b) a light chain CDRl comprising KASQDVSSAVA (SEQ ID NO:20), a light chain CDR2 comprising WASTRHT (SEQ ID NO:21), and a light chain CDR3 comprising QQHYSTP (SEQ ID NO:22).

[00198] In certain embodiments, the RSPO-binding agent is an 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 CDRl comprising SSYAMS (SEQ ID NO: 17) or a variant thereof comprising 1, 2, 3, or 4 amino acid substitutions; (b) a heavy chain CDR2 comprising

SISSGGSTYYPDSVKG (SEQ ID NO: 18) or a variant thereof comprising 1, 2, 3, or 4 amino acid substitutions; (c) a heavy chain CDR3 comprising RGGDPGVYNGDYEDAMDY (SEQ ID NO: 19) or a variant thereof comprising 1, 2, 3, or 4 amino acid substitutions; (d) a light chain CDRl comprising KASQDVSSAVA (SEQ ID NO:20) or a variant thereof comprising 1, 2, 3, or 4 amino acid substitutions; (e) a light chain CDR2 comprising WASTRHT (SEQ ID NO:21) or a variant thereof comprising 1, 2, 3, or 4 amino acid substitutions; and (f) a light chain CDR3 comprising QQHYSTP (SEQ ID NO:22) 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. [00199] In certain embodiments, the RSPO-binding agent is an 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:23 and/or a light chain variable region having at least 80% sequence identity to SEQ ID NO:24 or SEQ ID NO:50. In certain embodiments, the RSP02-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:23. 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:24. 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:50. In certain embodiments, the RSP02-binding agent comprises a heavy chain variable region having at least about 95% sequence identity to SEQ ID NO: 23 and/or a light chain variable region having at least about 95% sequence identity to SEQ ID NO: 24 or SEQ ID NO: 50. In certain embodiments, the RSP02-binding agent comprises a heavy chain variable region comprising SEQ ID NO:23 and/or a light chain variable region comprising SEQ ID NO:24 or SEQ ID NO:50. In certain embodiments, the RSP02-binding agent comprises a heavy chain variable region comprising SEQ ID NO:23 and a light chain variable region comprising SEQ ID NO:24. In certain embodiments, the RSP02-binding agent comprises a heavy chain variable region comprising SEQ ID NO:23 and a light chain variable region comprising SEQ ID NO: 50. In certain embodiments, the RSP02-binding agent comprises a heavy chain variable region consisting of SEQ ID NO: 23 and a light chain variable region consisting of SEQ ID NO:24. In certain embodiments, the RSP02-binding agent comprises a heavy chain variable region consisting of SEQ ID NO: 23 and a light chain variable region consisting of SEQ ID NO: 50.

[00200] In certain embodiments, the RSPO-binding agent is an 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:25 or SEQ ID NO:26; 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:25 or SEQ ID NO:26; and/or (b) a light chain having at least 95% sequence identity to SEQ ID NO:27 or SEQ ID NO:28. In some embodiments, the RSP02-binding agent comprises a heavy chain comprising SEQ ID NO:26 and/or a light chain comprising SEQ ID NO:28. In some embodiments, the RSP02-binding agent comprises a heavy chain comprising SEQ ID NO:26 and a light chain comprising SEQ ID NO:28.

[00201] In certain embodiments, the RSPO-binding agent is an 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:25 or SEQ ID NO:26; and/or (b) a light chain having at least 90% sequence identity to SEQ ID NO:51 or SEQ ID NO:52. In some embodiments, the RSP02-binding agent comprises: (a) a heavy chain having at least 95% sequence identity to SEQ ID NO:25 or SEQ ID NO:26; and/or (b) a light chain having at least 95% sequence identity to SEQ ID NO:51 or SEQ ID NO:52. In some embodiments, the RSP02-binding agent comprises a heavy chain comprising SEQ ID NO:26 and/or a light chain comprising SEQ ID NO:52. In some embodiments, the RSP02-binding agent comprises a heavy chain comprising SEQ ID NO:26 and a light chain comprising SEQ ID NO:52.

[00202] In certain embodiments, an RSP02-binding agent comprises the heavy chain variable region and light chain variable region of antibody hl30M23-HlL6. In certain embodiments, an RSP02- binding agent comprises the heavy chain and light chain of antibody hl30M23-HlL6 (with or without the leader sequence). In certain embodiments, an RSP02-binding agent is antibody hl30M23-HlL6. In certain embodiments, an 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.

[00203] In certain embodiments, an RSP02-binding agent comprises the heavy chain variable region and light chain variable region of antibody hl30M23-HlL2. In certain embodiments, an RSP02- binding agent comprises the heavy chain and light chain of antibody hl30M23-HlL2 (with or without the leader sequence). In certain embodiments, an RSP02-binding agent is antibody hl30M23-HlL2. In certain embodiments, an 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.

[00204] In certain embodiments of the methods described herein, an 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, 2011 and assigned ATCC deposit designation number PTA-12021.

[00205] In certain embodiments, an RSP02-binding agent comprises, consists essentially of, or consists of, antibody hl30M23-HlL6. In certain embodiments, an RSP02-binding agent comprises, consists essentially of, or consists of, a variant of antibody 130M23. In certain embodiments, an RSP02-binding agent comprises, consists essentially of, or consists of, a variant of antibody hl30M23-HlL6.

[00206] In certain embodiments, an RSP02-binding agent comprises, consists essentially of, or consists of, antibody hl30M23-HlL2. In certain embodiments, an RSP02-binding agent comprises, consists essentially of, or consists of, a variant of antibody 130M23. In certain embodiments, an RSP02 -binding agent comprises, consists essentially of, or consists of, a variant of antibody hl30M23-HlL2. [00207] In certain embodiments of the methods described herein, the RSPO-binding agent is an RSP03-binding agent (e.g., an antibody) that specifically binds human RSP03, wherein the RSP03- binding agent (e.g., an antibody) comprises one, two, three, four, five, and/or six of the CDRs of antibody 131R10 (see Table 1 herein).

[00208] In certain embodiments, the RSPO-binding agent is an RSP03-binding agent (e.g., an antibody) that specifically binds human RSP03, wherein the RSP03-binding agent comprises a heavy chain CDR1 comprising DYSIH (SEQ ID NO:29), a heavy chain CDR2 comprising

YIYPSNGDSGYNQKFK (SEQ ID NO:30), and a heavy chain CDR3 comprising TYFANNFD (SEQ ID NO:31) or ATYFANNFDY (SEQ ID NO:32). In some embodiments, the RSP03-binding agent further comprises a light chain CDR1 comprising KASQSVDYDGDSYMN (SEQ ID NO:33), a light chain CDR2 comprising AASNLES (SEQ ID NO:34) or AAS (SEQ ID NO:35), and a light chain CDR3 comprising QQSNEDPLT (SEQ ID NO:36) or QQSNEDPLTF (SEQ ID NO:37). In some embodiments, the RSP03-binding agent comprises a light chain CDR1 comprising

KASQSVDYDGDSYMN (SEQ ID NO:33), a light chain CDR2 comprising AASNLES (SEQ ID NO:34) or AAS (SEQ ID NO:35), and a light chain CDR3 comprising QQSNEDPLT (SEQ ID NO:36) or QQSNEDPLTF (SEQ ID NO:37). In certain embodiments, the RSP03-binding agent comprises: (a) a heavy chain CDR1 comprising DYSIH (SEQ ID NO:29), a heavy chain CDR2 comprising YIYPSNGDSGYNQKFK (SEQ ID NO:30), and a heavy chain CDR3 comprising TYFANNFD (SEQ ID NO:31); and (b) a light chain CDR1 comprising KASQSVDYDGDSYMN (SEQ ID NO:33), a light chain CDR2 comprising AASNLES (SEQ ID NO:34), and a light chain CDR3 comprising QQSNEDPLT (SEQ ID NO:36).

[00209] In certain embodiments, the RSPO-binding agent is an RSP03-binding agent (e.g., an antibody or bispecific antibody) that specifically binds human RSP03, wherein the RSP03-binding agent comprises: (a) a heavy chain CDR1 comprising DYSIH (SEQ ID NO:29) or a variant thereof comprising 1, 2, 3, or 4 amino acid substitutions; (b) a heavy chain CDR2 comprising

YIYPSNGDSGYNQKFK (SEQ ID NO: 30) or a variant thereof comprising 1, 2, 3, or 4 amino acid substitutions; (c) a heavy chain CDR3 comprising TYFANNFD (SEQ ID NO:31), ATYFANNFDY (SEQ ID NO:32), or a variant thereof comprising 1, 2, 3, or 4 amino acid substitutions; (d) a light chain CDR1 comprising KASQSVDYDGDSYMN (SEQ ID NO:33) or a variant thereof comprising 1, 2, 3, or 4 amino acid substitutions; (e) a light chain CDR2 comprising AASNLES (SEQ ID NO:34), AAS (SEQ ID NO:35), or a variant thereof comprising 1, 2, 3, or 4 amino acid substitutions; and (f a light chain CDR3 comprising QQSNEDPLT (SEQ ID NO:36), QQSNEDPLTF (SEQ ID NO:37), 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. [00210] In certain embodiments, the RSPO-binding agent is an RSP03-binding agent (e.g., an antibody) that specifically binds RSP03, wherein the RSP03-binding agent comprises a heavy chain variable region having at least about 80% sequence identity to SEQ ID NO:38 and/or a light chain variable region having at least 80% sequence identity to SEQ ID NO:39. 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:38. In certain embodiments, the RSP03-binding agent comprises a light chain variable region having at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% sequence identity to SEQ ID NO:39. In certain embodiments, the RSP03-binding agent comprises a heavy chain variable region having at least about 95% sequence identity to SEQ ID NO:38 and/or a light chain variable region having at least about 95% sequence identity to SEQ ID NO:39. In certain embodiments, the RSP03-binding agent comprises a heavy chain variable region comprising SEQ ID NO:38, and/or a light chain variable region comprising SEQ ID NO:39. In certain embodiments, the RSP03-binding agent comprises a heavy chain variable region comprising SEQ ID NO:38 and a light chain variable region comprising SEQ ID NO:39. In certain embodiments, the RSP03-binding agent comprises a heavy chain variable region consisting of SEQ ID NO:38 and a light chain variable region consisting of SEQ ID NO:39.

[00211] In certain embodiments, the RSPO-binding agent is an RSP03-binding agent (e.g., an antibody) that specifically binds RSP03, wherein the RSP03-binding agent comprises: (a) a heavy chain having at least 90% sequence identity to SEQ ID NO: 40 or SEQ ID NO: 41 ; and/or (b) a light chain having at least 90% sequence identity to SEQ ID NO: 42 or SEQ ID NO: 43. In some embodiments, the RSP03 -binding agent comprises: (a) a heavy chain having at least 95% sequence identity to SEQ ID NO: 40 or SEQ ID NO:41; and/or (b) a light chain having at least 95% sequence identity to SEQ ID NO:42 or SEQ ID NO:43. In some embodiments, the RSP03-binding agent comprises a heavy chain comprising SEQ ID NO:41 and/or a light chain comprising SEQ ID NO:43. In some embodiments, the RSP03-binding agent comprises a heavy chain comprising SEQ ID NO:41 and a light chain comprising SEQ ID NO:43.

[00212] In certain embodiments, an RSP03-binding agent comprises the heavy chain variable region and light chain variable region of antibody 131R10. In certain embodiments, an RSP03-binding agent comprises the heavy chain and light chain of antibody 131R10 (with or without the leader sequence). In certain embodiments, an RSP03 -binding agent is antibody 131R10. In certain embodiments, an RSP03 -binding agent comprises the heavy chain variable region and/or light chain variable region of antibody 131R10 in a chimeric form of the antibody. In certain embodiments, an RSP03-binding agent comprises the heavy chain CDRs and/or light chain CDRs of antibody 131R10. In some embodiments, the anti-RSP03 antibody is 131R10. [00213] Plasmids encoding the heavy chain and light chain of antibody 131R10 were deposited with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, VA, USA, under the conditions of the Budapest Treaty on June 18, 2013 and assigned ATCC deposit designation number PTA-120420 and PTA-120421. In some embodiments, the RSP03-binding agent comprises a heavy chain variable region encoded by the plasmid deposited with ATCC and designated PTA- 120420. In some embodiments, the RSP03-binding agent comprises a light chain variable region encoded by the plasmid deposited with ATCC and designated PTA-120421. In some embodiments, the RSP03-binding agent comprises a heavy chain variable region encoded by the plasmid deposited with ATCC and designated PTA-120420 and a light chain variable region encoded by the plasmid deposited with ATCC and designated PTA-120421. In some embodiments, the RSP03 -binding agent comprises a heavy chain encoded by the plasmid deposited with ATCC and designated PTA-120420. In some embodiments, the RSP03-binding agent comprises a light chain encoded by the plasmid deposited with ATCC and designated PTA-120421. In some embodiments, the RSP03-binding agent comprises a heavy chain encoded by the plasmid deposited with ATCC and designated PTA-120420 and a light chain encoded by the plasmid deposited with ATCC and designated PTA-120421.

[00214] In certain embodiments, an RSP03-binding agent comprises, consists essentially of, or consists of, antibody 131R10. In certain embodiments, an RSP03-binding agent comprises, consists essentially of, or consists of, a variant of antibody 131R10.

[00215] Described herein are methods comprising polypeptides, including, but not limited to, antibodies that specifically bind human at least one RSPO 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.

[00216] 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 131R10 (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.

[00217] In some embodiments, the RSPO-binding agent is 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: 11 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: 11, 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: 11 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: 11 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.

[00218] In some embodiments, the RSPO-binding agent is 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:44 and/or SEQ ID NO:45. In some embodiments, the polypeptide comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:46 and/or an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:48. In some embodiments, the polypeptide comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:47 and/or an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:49. 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:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, or SEQ ID NO:49. In certain embodiments, the polypeptide comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO:44 and/or an amino acid sequence having at least about 95% sequence identity to SEQ ID NO:45. 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:48. In certain embodiments, the polypeptide comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 47 and/or an amino acid sequence having at least about 95% sequence identity to SEQ ID NO:49. In certain embodiments, the polypeptide comprises an amino acid sequence of SEQ ID NO:44 and/or an amino acid sequence of SEQ ID NO:45. 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:48. In certain embodiments, the polypeptide comprises an amino acid sequence of SEQ ID NO:47 and/or an amino acid sequence of SEQ ID NO:49.

[00219] In some embodiments, the RSPO-binding agent is 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:23 and/or SEQ ID NO:24. In some embodiments, the RSPO-binding agent is 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:23 and/or 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:25 and/or an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:27 or SEQ ID NO:51. In some embodiments, the polypeptide comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:26 and/or an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:28 or SEQ ID NO: 52. 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:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:50, SEQ ID NO:51, or SEQ ID NO:52. In certain embodiments, the polypeptide comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO:23 and/or an amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 24 or 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: 25 and/or an amino acid sequence having at least about 95% sequence identity to SEQ ID NO:27 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: 26 and/or an amino acid sequence having at least about 95% sequence identity to SEQ ID NO:28 or SEQ ID NO:52. In certain embodiments, the polypeptide comprises an amino acid sequence of SEQ ID NO:23 and/or an amino acid sequence of SEQ ID NO:24. In certain embodiments, the polypeptide comprises an amino acid sequence of SEQ ID NO:23 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:25 and/or an amino acid sequence of SEQ ID NO:27. In certain embodiments, the polypeptide comprises an amino acid sequence of SEQ ID NO: 25 and/or an amino acid sequence of SEQ ID NO: 51. In certain embodiments, the polypeptide comprises an amino acid sequence of SEQ ID NO: 26 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:26 and/or an amino acid sequence of SEQ ID NO:52.

[00220] In some embodiments, the RSPO-binding agent is a polypeptide that specifically binds a human RSP03, wherein the polypeptide comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:38 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:40 and/or an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:42. In some embodiments, the polypeptide comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:41 and/or an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:43. 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:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, or SEQ ID NO:43. In certain embodiments, the polypeptide comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO:38 and/or an amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 39. In certain embodiments, the polypeptide comprises an amino acid sequence having at least about 95% sequence identity to

SEQ ID NO:40 and/or an amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 42. In certain embodiments, the polypeptide comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO:41 and/or an amino acid sequence having at least about 95% sequence identity to SEQ ID NO:43. In certain embodiments, the polypeptide comprises an amino acid sequence of SEQ ID NO:38 and/or an amino acid sequence of SEQ ID NO: 39. In certain embodiments, the polypeptide comprises an amino acid sequence of SEQ ID NO:40 and/or an amino acid sequence of SEQ ID NO:42. In certain embodiments, the polypeptide comprises an amino acid sequence of SEQ ID NO:41 and/or an amino acid sequence of SEQ ID NO:43.

[00221] In certain embodiments, the RSPO-binding agent is an RSPO l-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 RSPO-binding agent is an 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 RSPO-binding agent is an RSP03-binding agent (e.g., antibody) that competes for specific binding to RSP03 with an antibody that comprises the CDRs of antibody 131R10.

[00222] In certain embodiments, the RSPO-binding agent is an RSPOl-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 RSPO-binding agent is an 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 RSPO-binding agent is an RSP03-binding agent (e.g., an antibody) that binds the same epitope, or essentially the same epitope on RSP03, as an antibody that comprises the CDRs of antibody 131R10.

[00223] In certain embodiments, the RSPO-binding agent is an RSPOl-binding agent (e.g., an antibody) that binds an epitope on RSPO 1 that overlaps with the epitope on RSPO 1 bound by an antibody comprising the CDRs of antibody 89M5. In certain embodiments, the RSPO-binding agent is an 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 RSPO-binding agent is an RSP03-binding agent (e.g., an antibody) that binds an epitope on RSP03 that overlaps with the epitope on RSP03 bound by an antibody comprising the CDRs of antibody 131R10.

[00224] In certain embodiments of the methods described herein, an RSPO-binding agent (e.g., an antibody) binds at least one human RSPO protein and modulates RSPO activity. In some

embodiments, the RSPO-binding agent is an RSPO antagonist and decreases RSPO activity. In some embodiments, the RSPO-binding agent is an RSPO antagonist and decreases β-catenin activity.

[00225] In certain embodiments, an RSPOl-binding agent (e.g., an antibody) binds human RSPOl and modulates RSPO l activity. In some embodiments, an RSPOl-binding agent is an RSPOl antagonist and decreases RSPOl activity. In some embodiments, an RSPOl-binding agent is an RSPOl antagonist and decreases β-catenin activity. In certain embodiments, an RSP02-binding agent (e.g., an antibody) binds human RSP02 and modulates RSP02 activity. In some embodiments, an RSP02 -binding agent is an RSP02 antagonist and decreases RSP02 activity. In some embodiments, an RSP02-binding agent is an RSP02 antagonist and decreases β-catenin activity. In certain embodiments, an RSP03-binding agent (e.g., an antibody) binds human RSP03 and modulates RSP03 activity. In some embodiments, an RSP03-binding agent is an RSP03 antagonist and decreases RSP03 activity. In some embodiments, an RSP03-binding agent is an RSP03 antagonist and decreases β-catenin activity.

[00226] In certain embodiments, the RSPO-binding agent (e.g., an antibody) is an antagonist of at least one human RSPO protein. In some embodiments, the RSPO-binding agent is an antagonist of at least one RSPO and inhibits RSPO activity. In certain embodiments, the RSPO-binding agent inhibits RSPO activity by at least about 10%, at least about 20%, at least about 30%, at least about 50%, at least about 75%, at least about 90%, or about 100%. In some embodiments, the RSPO-binding agent inhibits activity of one, two, three, or four RSPO proteins. In some embodiments, the RSPO-binding agent inhibits activity of human RSPOl, RSP02, RSP03, and/or RSP04.

[00227] In certain embodiments, the RSPO-binding agent (e.g., antibody) is an antagonist of at least one human RSPO protein. In certain embodiments, the RSPO-binding agent 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.

[00228] In certain embodiments, the RSPO-binding agent (e.g., antibody) is an antagonist of β-catenin signaling. In certain embodiments, the RSPO-binding agent inhibits β-catenin signaling by at least about 10%, at least about 20%, at least about 30%, at least about 50%, at least about 75%, at least about 90%, or about 100%. [00229] In certain embodiments, the RSPO-binding agent (e.g., antibody) 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 an RSPO protein to at least one LGR protein. In some embodiments, the RSPO-binding agent inhibits binding of an RSPO protein to LGR4 (SEQ ID NO: 53), LGR5 (SEQ ID NO:54), and/or LGR6 (SEQ ID NO:55). In certain embodiments, the inhibition of binding of an RSPO-binding agent to at least one LGR protein is at least about 10%, at least about 25%, at least about 50%, at least about 75%, at least about 90%, or at least about 95%. In certain embodiments, an RSPO-binding agent that inhibits binding of at least one RSPO to at least one LGR protein further inhibits β-catenin signaling.

[00230] In certain embodiments, the RSPO-binding agent (e.g., antibody) 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 an RSPO to at least one LGR protein. In some embodiments, the RSPO-binding agent blocks binding of at least one RSPO protein to LGR4 (SEQ ID NO:53), LGR5 (SEQ ID NO:54), and/or LGR6 (SEQ ID NO:55). In certain embodiments, the blocking of binding of an RSPO-binding agent to at least one LGR protein is at least about 10%, at least about 25%, at least about 50%, at least about 75%, at least about 90%, or at least about 95%. In certain embodiments, an RSPO-binding agent that blocks binding of at least one RSPO protein to at least one LGR protein further inhibits β- catenin signaling.

[00231] In certain embodiments, the RSPO-binding agent (e.g., an antibody) inhibits β-catenin signaling. It is understood that an RSPO-binding agent that inhibits β-catenin signaling can, in certain embodiments, inhibit signaling by one or more receptors in the β-catenin signaling pathway but not necessarily inhibit signaling by all receptors. In certain alternative embodiments, β-catenin signaling by all human receptors can be inhibited. In certain embodiments, β-catenin signaling by one or more receptors selected from the group consisting of LGR4 (SEQ ID NO:53), LGR5 (SEQ ID NO:54), and/or LGR6 (SEQ ID NO:55) is inhibited. In certain embodiments, the inhibition of β-catenin signaling by an RSPO-binding agent is a reduction in the level of β-catenin signaling of at least about 10%, at least about 25%, at least about 50%, at least about 75%, at least about 90%, or at least about 95%.

[00232] In certain embodiments, the RSPO-binding agent (e.g., an antibody) inhibits activation of β- catenin. It is understood that an RSPO-binding agent that inhibits activation of β-catenin can, in certain embodiments, inhibit activation of β-catenin by one or more receptors, but not necessarily inhibit activation of β-catenin by all receptors. In certain alternative embodiments, activation of β- catenin by all human receptors can be inhibited. In certain embodiments, activation of β-catenin by one or more receptors selected from the group consisting of LGR4 (SEQ ID NO:53), LGR5 (SEQ ID NO:54), and LGR6 (SEQ ID NO:55) is inhibited. In certain embodiments, the inhibition of activation of β-catenin by an RSPO-binding agent is a reduction in the level of activation of β-catenin of at least about 10%, at least about 25%, at least about 50%, at least about 75%, at least about 90%, or at least about 95%.

[00233] In certain embodiments, the RSPO or LGR antagonists are agents that bind one or more human LGR proteins. These agents are referred to herein as "LGR-binding agents". Non-limiting examples of LGR-binding agents can be found in U.S. Patent Nos. 8158758, 8158757, 8802097, and U.S. Patent Publication Nos. 2012/0135422, 2013/0209473, 2014/0044713, each of which are hereby incorporated by reference herein in their entirety for all purposes.

[00234] In some embodiments, the LGR-binding agent binds at least one human LGR protein. In alternative embodiments, the LGR-binding agent binds two or more human LGR proteins. In some embodiments, the LGR-binding agent is an antibody. In some embodiments, the LGR-binding agent inhibits (partially or wholly) the binding of at least one RSPO protein (e.g., RSPOl, RSP02, RSP03, and/or RSP04) to at least one LGR protein (e.g., LGR4, LGR5, and/or LGR6). In certain

embodiments, the LGR-binding agent inhibits RSPO-activated LGR signaling, such as LGR5 signaling. In certain embodiments, the LGR-binding agent inhibits beta-catenin signaling.

[00235] In certain embodiments, an LGR-binding agent is an antibody, for example, an antibody that binds at least one LGR protein. Thus, the LGR-binding agent can be an antibody that specifically binds LGR5. In certain alternative embodiments, the LGR-binding agent is an antibody that specifically binds LGR4 or LGR6.

[00236] In certain embodiments, an LGR-binding agent is an antibody that specifically binds at least one human LGR protein. In certain embodiments, the antibody specifically binds at least one human LGR protein selected from the group consisting of LGR4, LGR5, and LGR6. In certain embodiments, the antibody specifically binds LGR5. In certain embodiments, the antibody specifically binds two or more human LGR proteins selected from the group consisting of LGR4, LGR5, and LGR6. In certain embodiments, the antibody that specifically binds at least one human LGR protein, also inhibits binding of at least one RSPO protein (e.g., RSPO l, RSP02, RSP03, and/or RSP04) to the at least one human LGR protein (e.g., LGR5). In certain embodiments, the antibody that specifically binds at least one human LGR protein is characterized by an ability to inhibit RSPO activation of LGR signaling and/or an ability to inhibit beta-catenin signaling. In certain embodiments, the antibody that specifically binds at least one human LGR protein is characterized by the ability to inhibit tumor growth, such as the growth of a solid tumor comprising solid tumor stem cells. For example, in some embodiments, the antibody that specifically binds at least one human LGR protein, disrupts or inhibits RSPO binding to LGR, and inhibits tumor growth. In certain alternative embodiments, the antibody that specifically binds at least one LGR protein, also disrupts RSPO activation of LGR signaling and inhibits tumor growth. In certain alternative embodiments, the antibody that specifically binds at least one LGR protein, also inhibits RSPO activation of LGR signaling and/or beta-catenin signaling and inhibits tumor growth.

[00237] In certain embodiments, an LGR-binding agent that inhibits binding of an RSPO protein to an LGR protein, inhibits at least about 25%, at least about 50 %, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the binding of the RSPO protein to an LGR protein in an in vitro or in vivo assay.

[00238] Likewise, in certain embodiments, an LGR-binding agent that inhibits (a) RSPO activation of LGR signaling and/or (b) beta-catenin signaling, inhibits at least about 25%, at least about 50 %, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the signaling in an in vitro or in vivo assay.

[00239] In certain embodiments, an LGR-binding agent is an isolated antibody that specifically binds to an extracellular domain of a human LGR protein and inhibits growth of a solid tumor comprising solid tumor stem cells. In certain embodiments, the extracellular domain comprises amino acids 22- 564 of human LGR5 (SEQ ID NO:56). In certain embodiments, the antibody is a monoclonal antibody. In certain embodiments, the antibody is a humanized or human antibody.

[00240] In certain embodiments, an LGR-binding agent is an isolated antibody that specifically binds to an extracellular domain of a human LGR protein and disrupts RSPO activation of LGR signaling. In certain embodiments, the extracellular domain comprises amino acids 22-564 of human LGR5 (SEQ ID NO:56). In certain embodiments, the antibody is a monoclonal antibody. In certain embodiments, the antibody is a humanized or human antibody.

[00241] In certain embodiments, an LGR-binding agent is monoclonal anti-LGR5 antibody 88M1. The 88M1 monoclonal antibody is produced by a hybridoma cell line deposited with the American Type Culture collection (ATCC), 10801 University Blvd, Manassas, Virginia, 20110, USA, on July 2, 2008, in accordance with the Budapest Treaty, under ATCC deposit number PTA-9342. In certain embodiments, an LGR-binding agent is an antibody that specifically binds human LGR5 and (a) comprises a heavy chain variable region that has at least about 95% sequence identity (e.g., at least about 98% or about 100% sequence identity) to the heavy chain variable region of 88M1; (b) comprises a light chain variable region that has at least about 95% (e.g., at least about 98% or about 100% sequence identity) sequence identity to the light chain variable region of 88M1; (c) comprises the heavy chain CDRs of 88M1; (d) comprises the light chain CDRs of 88M1; (e) binds to an epitope capable of binding 88M1; and/or (f) competes with 88M1 in a competitive binding assay.

[00242] In certain embodiments, the LGR5 antibody is BNC101. In certain embodiments, an LGR- binding agent is an antibody that specifically binds human LGR5 and (a) comprises a heavy chain variable region that has at least about 95% sequence identity (e.g., at least about 98% or about 100% sequence identity) to the heavy chain variable region of BNC101; (b) comprises a light chain variable region that has at least about 95% (e.g., at least about 98% or about 100% sequence identity) sequence identity to the light chain variable region of BNC101; (c) comprises the heavy chain CDRs of BNC101; (d) comprises the light chain CDRs of BNC101; (e) binds to an epitope capable of binding BNC101; and/or (f) competes with BNC101 in a competitive binding assay.

[00243] 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 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.

[00244] RSPO-binding agents and LGR-binding agents (e.g., antibodies) can be assayed for specific binding by any method known in the art. The immunoassays which can be used include, but are not limited to, competitive and non-competitive assay systems using techniques such as Biacore analysis, FACS analysis, immunofluorescence, immunocytochemistry, Western blot analysis,

radioimmunoassays, ELISAs, "sandwich" immunoassays, immunoprecipitation assays, precipitation reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, and protein A immunoassays. Such assays are 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).

[00245] For example, the specific binding of an agent (e.g., RSPO-binding agent and LGR-binding agent) to a human RSPO protein or human LGR protein can be determined using ELISA. An ELISA assay comprises preparing antigen, coating wells of a 96 well microtiter plate with antigen, adding the RSPO-binding agent or LGR-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 or LGR-binding agent is not conjugated to a detectable compound, but instead a second antibody that recognizes the RSPO-binding agent or LGR-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 or LGR-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.

[00246] In another example, the specific binding of an agent (e.g., RSPO-binding agent and LGR- binding agent) to a human RSPO protein or human LGR protein can be determined using FACS. A FACS screening assay can comprise generating a cDNA construct that expresses an antigen (e.g., LGR), optionally 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 or LGR-binding agent with the transfected cells, and incubating for a period of time. The cells bound by the RSPO- binding agent or LGR-binding agent can be identified using a secondary antibody conjugated to a detectable compound (e.g., PE-conjugated anti-Fc antibody) and a flow cytometer. One of skill in the art would be knowledgeable as to the parameters that can be modified to optimize the signal detected as well as other variations of FACS that can enhance screening (e.g., screening for blocking antibodies).

[00247] The binding affinity of an agent (e.g., RSPO-binding agent and LGR-binding 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 3H or 1251), 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.

[00248] In vivo and in vitro assays for determining whether an RSPO-binding agent or LGR-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 can be used to measure β-catenin signaling levels in vitro (Gazit et al., 1999, Oncogene, 18; 5959-66; TOPflash, Millipore, Billerica MA). The level of β- catenin signaling in the presence of one or more Wnts (e.g., Wnt(s) expressed by transfected cells or provided by Wnt-conditioned media) with or without an RSPO protein or RSPO-conditioned media in the presence of an RSPO-binding agent or LGR-binding agent is compared to the level of signaling without the RSPO-binding agent or LGR-binding agent present. In addition to the TCF/Luc reporter assay, the effect of an RSPO-binding agent or LGR-binding agent on β-catenin signaling can be measured in vitro or in vivo by measuring the effect of the agent on the level of expression of β- catenin-regulated genes, such as c-myc (He et al., 1998, Science, 281 : 1509-12), cyclin Dl (Tetsu et al, 1999, Nature, 398:422-6) and/or fibronectin (Gradl et al. 1999, Mol. Cell Biol., 19:5576-87). In certain embodiments, the effect of an RSPO-binding agent or LGR-binding agent on β-catenin signaling can also be assessed by measuring the effect of the agent on the phosphorylation state of Dishevelled- 1, Dishevelled-2, Dishevelled-3, LRP5, LRP6, and/or β-catenin.

[00249] In some embodiments, the RSPO or LGR antagonists (e.g., RSPO-binding agent and LGR- binding agent) are polyclonal antibodies. Polyclonal antibodies can be prepared by any known method. In some embodiments, polyclonal antibodies are raised by immunizing an animal (e.g., a rabbit, rat, mouse, goat, donkey) by multiple subcutaneous or intraperitoneal injections of the relevant antigen (e.g., a purified peptide fragment, full-length recombinant protein, or fusion protein). The antigen can be 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 blood and/or ascites of the immunized animal. 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.

[00250] In some embodiments, the RSPO or LGR antagonists (e.g., RSPO-binding agent and LGR- binding agent) 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 will 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.

[00251] Following immunization, lymphocytes are isolated and fused with a suitable myeloma cell line using, for example, polyethylene glycol, to form hybridoma cells that can then be selected away from unfused lymphocytes and myeloma cells. Hybridomas that produce monoclonal antibodies directed specifically against a chosen antigen can be identified by a variety of methods including, but not limited to, immunoprecipitation, immunoblotting, and in vitro binding assay (e.g., flow cytometry, FACS, ELISA, and radioimmunoassay). The hybridomas can be propagated either in in vitro culture using standard methods 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. [00252] In certain embodiments, monoclonal antibodies can be made using recombinant DNA techniques 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 genes encoding the heavy and light chains of the antibody, and their sequence is determined using conventional 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. In other embodiments, recombinant monoclonal antibodies, or fragments thereof, can be isolated from phage display libraries.

[00253] The polynucleotide(s) encoding a monoclonal antibody can be further modified in a number of different manners using recombinant DNA technology to generate alternative antibodies. In some embodiments, the constant domains of the light and heavy chains of, for example, a mouse monoclonal antibody can be substituted for those regions of, for example, a human antibody to generate a chimeric antibody, or for a non-immunoglobulin polypeptide to generate a fusion antibody. In some embodiments, the constant regions are truncated or removed to generate the desired antibody fragment of a monoclonal antibody. In some embodiments, site-directed or high-density mutagenesis of the variable region can be used to optimize specificity, affinity, etc. of a monoclonal antibody.

[00254] In some embodiments, the RSPO or LGR antagonist (e.g., RSPO-binding agent and LGR- binding agent) is a humanized antibody. Typically, humanized antibodies are human

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

[00255] In certain embodiments, the RSPO or LGR antagonist (e.g., RSPO-binding agent and LGR- binding agent) is a human antibody. Human antibodies can be directly prepared using various techniques known in the art. In some embodiments, immortalized human B lymphocytes immunized in vitro or isolated from an immunized individual that produces an antibody directed against a target antigen can be generated. 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 in the art and antibody phage libraries are commercially available. Affinity maturation strategies including, but not limited to, chain shuffling and site-directed mutagenesis, are known in the art and can be employed to generate high affinity human antibodies.

[00256] In some embodiments, human antibodies can be made in transgenic mice that contain human immunoglobulin loci. These mice are capable, upon immunization, of producing the full repertoire of human antibodies in the absence of endogenous immunoglobulin production.

[00257] In certain embodiments, the RSPO or LGR antagonist (e.g., RSPO-binding agent and LGR- binding agent) is bispecific antibodies that specifically recognize at least one human RSPO protein or at least one LGR protein. Bispecific antibodies are capable of specifically recognizing and binding at least two different epitopes. The different epitopes can either be within the same molecule (e.g., two different epitopes on human RSP03) or on different molecules (e.g., one epitope on RSP03 and a different epitope on a second protein). In some embodiments, the bispecific antibodies are monoclonal human or humanized antibodies. In some embodiments, the bispecific antibodies are intact antibodies. In some embodiments, the bispecific antibodies are antibody fragments. In certain embodiments, the antibodies are multi specific. In some embodiments, the antibodies can specifically recognize and bind a first antigen target, (e.g., a LGR protein) as well as a second antigen target, such as an effector molecule on a leukocyte (e.g., CD2, CD3, CD28, CD80 or CD86) or a Fc receptor (e.g., CD64, CD32, or CD 16) so as to focus cellular defense mechanisms to the cell expressing the first antigen target. In some embodiments, the antibodies can be used to direct cytotoxic agents to cells which express a particular target antigen. These antibodies possess an antigen-binding arm and an arm which binds a cytotoxic agent or a radionuclide chelator, such as EOTUBE, DPT A, DOTA, or TETA. Techniques for making bispecific or multispecific antibodies are known by those skilled in the art.

[00258] In certain embodiments, the RSPO or LGR antagonist (e.g., RSPO-binding agent and LGR- binding agent) is a monospecific antibody. For example, in certain embodiments, each of the one or more antigen-binding sites that an antibody contains is capable of binding (or binds) a homologous epitope on different proteins.

[00259] In certain embodiments, the RSPO or LGR antagonist is an antibody fragment comprising an antigen-binding site. Antibody fragments can have different functions or capabilities than intact antibodies; for example, antibody fragments can have increased tumor penetration. Various techniques are known for the production of antibody fragments including, but not limited to, proteolytic digestion of intact antibodies. In some embodiments, antibody fragments include a F(ab')2 fragment produced by pepsin digestion of an antibody molecule. In some embodiments, antibody fragments include a Fab fragment generated by reducing the disulfide bridges of an F(ab')2 fragment. In other embodiments, antibody fragments include a Fab fragment generated by the treatment of the antibody molecule with papain and a reducing agent. In certain embodiments, antibody fragments are produced recombinantly. In some embodiments, antibody fragments include Fv or single chain Fv (scFv) fragments. 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 an RSPO or LGR 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 or LGR antagonist 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.

[00260] It can further be desirable, especially in the case of antibody fragments, to modify an antibody in order to increase its serum half-life. This can be achieved, for example, by incorporation of a salvage receptor binding epitope into the antibody fragment by mutation of the appropriate region in the antibody fragment or by incorporating the epitope into a peptide tag that is then fused to the antibody fragment at either end or in the middle (e.g., by DNA or peptide synthesis). In some embodiments, an antibody is modified to decrease its serum half-life.

[00261] In certain embodiments, the RSPO or LGR antagonist is a heteroconjugate antibody.

Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune cells to unwanted cells. 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.

[00262] 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., a human RSPO protein or a human LGR protein). In this regard, the variable region can comprise or be derived from any type of mammal that can be induced to mount a humoral response and generate immunoglobulins against the desired tumor-associated antigen. As such, the variable region of the modified antibodies can be, for example, of human, murine, non-human primate (e.g. cynomolgus monkeys, macaques, etc.) or rabbit origin. In some embodiments, both the variable and constant regions of the modified immunoglobulins are human. In other embodiments, the variable regions of compatible antibodies (usually derived from a non-human source) can be engineered or specifically tailored to improve the binding properties or reduce the immunogenicity of the molecule. In this respect, variable regions can be humanized or otherwise altered through the inclusion of imported amino acid sequences.

[00263] In certain embodiments, the variable domains in both the heavy and light chains are altered by at least partial replacement of one or more CDRs and, if necessary, by partial framework region replacement and sequence modification and/or alteration. Although the CDRs can be derived from an antibody of the same class or even subclass as the antibody from which the framework regions are derived, it is envisaged that the CDRs will be derived preferably from an antibody from a different species. It may not be necessary to replace all of the CDRs with all of the CDRs from the donor variable region to transfer the antigen binding capacity of one variable domain to another. Rather, it can only be necessary to transfer those residues that are necessary to maintain the activity of the antigen-binding site.

[00264] Alterations to the variable region notwithstanding, those skilled in the art will appreciate that the modified antibodies 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 tumor localization and/or increased serum half-life when compared with an antibody of approximately the same immunogenicity comprising a native or unaltered constant region. In some embodiments, the constant region of the modified antibodies will comprise a human constant region. Modifications to the constant region comprise additions, deletions or substitutions of one or more amino acids in one or more domains. The modified antibodies disclosed herein can comprise alterations or modifications to one or more of the three heavy chain constant domains (CHI, CH2 or CH3) and/or to the light chain constant domain (CL). In some embodiments, one or more domains are partially or entirely deleted from the constant regions of the modified antibodies. In some embodiments, the modified antibodies will comprise domain deleted constructs or variants wherein the entire CH2 domain has been removed (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.

[00265] In some embodiments, the modified antibodies are engineered to fuse the CH3 domain directly to the hinge region of the antibody. In other embodiments, a peptide spacer is inserted between the hinge region and the modified CH2 and/or CH3 domains. For example, constructs can be expressed wherein the CH2 domain has been deleted and the remaining CH3 domain (modified or unmodified) is joined to the hinge region with a 5-20 amino acid spacer. Such a spacer can be added to ensure that the regulatory elements of the constant domain remain free and accessible or that the hinge region remains flexible. However, it should be noted that amino acid spacers can, in some cases, prove to be immunogenic and elicit an unwanted immune response against the construct.

Accordingly, in certain embodiments, any spacer added to the construct will be relatively non- immunogenic so as to maintain the desired biological qualities of the modified antibodies.

[00266] In some embodiments, the modified antibodies can have only a partial deletion of a constant domain or substitution of a few or even a single amino acid. For example, the mutation of a single amino acid in selected areas of the CH2 domain can be enough to substantially reduce Fc binding and thereby increase cancer cell localization and/or tumor penetration. Similarly, it can be desirable to simply delete the part of one or more constant region domains that control a specific effector function (e.g. complement C lq binding). Such partial deletions of the constant regions can improve selected characteristics of the antibody (serum half-life) while leaving other desirable functions associated with the subject constant region domain intact. Moreover, as alluded to above, the constant regions of the disclosed antibodies can be modified through the mutation or substitution of one or more amino acids that enhances the profile of the resulting construct. In this respect it can be possible to disrupt the activity provided by a conserved binding site (e.g., Fc binding) while substantially maintaining the configuration and immunogenic profile of the modified antibody. In certain embodiments, the modified antibodies comprise the addition of one or more amino acids to the constant region to enhance desirable characteristics such as decreasing or increasing effector function or provide for more cytotoxin or carbohydrate attachment sites.

[00267] 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, release of inflammatory mediators, placental transfer, and control of immunoglobulin production.

[00268] In certain embodiments, the RSPO or LGR antagonists are antibodies that provide for altered effector functions. These altered effector functions can affect the biological profile of the

administered antibody. For example, in some embodiments, the deletion or inactivation (through point mutations or other means) of a constant region domain can reduce Fc receptor binding of the circulating modified antibody (e.g., anti-RSPO antibody) thereby increasing cancer cell localization and/or tumor penetration. In other embodiments, the constant region modifications increase or reduce the serum half-life of the antibody. In some embodiments, the constant region is modified to eliminate disulfide linkages or oligosaccharide moieties. Modifications to the constant region can easily be made using well known biochemical or molecular engineering techniques well within the purview of the skilled artisan.

[00269] In certain embodiments, an RSPO or LGR antagonist is an antibody that does not have one or more effector functions. For instance, in some embodiments, the antibody has no ADCC activity, and/or no CDC activity. In certain embodiments, the antibody does not bind an Fc receptor, and/or complement factors. In certain embodiments, the antibody has no effector function.

[00270] Variants and equivalents which are substantially homologous to the chimeric, humanized, and human antibodies, or antibody fragments thereof, set forth herein can also be used in the methods described herein. These can contain, for example, conservative substitution mutations.

[00271] In certain embodiments, the antibodies described herein are isolated. In certain embodiments, the antibodies described herein are substantially pure.

[00272] In certain embodiments, the RSPO-binding agent is a soluble receptor. In some embodiments, the RSPO or LGR antagonist is a soluble receptor. In certain embodiments, the soluble receptor comprises the extracellular domain of an LGR protein or fragment of the extracellular domain of an LGR protein. In certain embodiments, the LGR protein is LGR5. For example, in some embodiments, the RSPO-binding agent is a fusion protein comprising a fragment of the LGR5 receptor and the Fc portion of an antibody.

[00273] In certain embodiments, the RSPO-binding agent is a soluble receptor comprising an extracellular domain of a human LGR protein that inhibits growth of a solid tumor comprising solid tumor stem cells. In certain embodiments, the extracellular domain comprises amino acids 22-564 of human LGR5 (SEQ ID NO: 56). In certain embodiments, the extracellular domain of human LGR5 is linked in-frame to a non-LGR protein sequence. In certain embodiments, the non-LGR protein is human Fc.

[00274] In certain embodiments, the RSPO-binding agent is a soluble receptor comprising an extracellular domain of a human LGR protein that inhibits RSPO activation of LGR signaling. In certain embodiments, the extracellular domain comprises amino acids 22-564 of human LGR5 (SEQ ID NO: 56). In certain embodiments, the extracellular domain of human LGR5 is linked in-frame to a non-LGR protein sequence. In certain embodiments, the non-LGR protein is human Fc. Non-limiting examples of soluble LGR receptors can be found in can be found in in U.S. Patent Nos. 8158758, and 8158757, each of which are hereby incorporated by reference herein in their entirety for all purposes.

[00275] In certain embodiments, the RSPO-binding agent is a soluble receptor comprising an extracellular domain of a human LGR protein that inhibits growth of a solid tumor comprising solid tumor stem cells. In certain embodiments, the extracellular domain comprises amino acids 22-564 of human LGR5 (SEQ ID NO: 56). In certain embodiments, the extracellular domain of human LGR5 is linked in-frame to a non-LGR protein sequence. In certain embodiments, the non-LGR protein is human Fc.

[00276] In certain embodiments, the soluble receptor comprises a variant of the aforementioned extracellular domain of a human LGR protein that comprises one or more (e.g., one, two, three, four, five, six, seven, eight, nine, ten, etc.) conservative substitutions and is capable of binding RSPO protein(s).

[00277] In certain embodiments, the soluble receptor, such as an agent comprising an extracellular domain of a human LGR protein, further comprises a non-LGR (e.g., heterologous) polypeptide. In some embodiments, a soluble receptor can include a LGR ECD linked to other non-LGR functional and structural polypeptides including, but not limited to, a human Fc region, at least one protein tag (e.g., myc, FLAG, GST, GFP), other endogenous proteins or protein fragments, or any other useful protein sequence including any linker region between a LGR ECD and a second polypeptide. In certain embodiments, the non-LGR polypeptide comprises a human Fc region. The Fc region can be obtained from any of the classes of immunoglobulin, IgG, IgA, IgM, IgD and IgE. In some embodiments, the Fc region is a human IgGl Fc region. In some embodiments, the Fc region is a human IgG2 Fc region. In some embodiments, the Fc region is a wild-type Fc region. In some embodiments, the Fc region is a mutated Fc region. In some embodiments, the Fc region is truncated at the N-terminal end by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, (e.g., in the hinge domain). In some embodiments, an amino acid in the hinge domain is changed to hinder undesirable disulfide bond formation. In some embodiments, a cysteine is replaced with a serine to hinder undesirable disulfide bond formation. In some embodiments, the Fc region is truncated at the C-terminal end by 1, 2, 3, or more amino acids. In some embodiments, the Fc region is truncated at the C-terminal end by 1 amino acid. In certain embodiments, the non-LGR polypeptide comprises SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, or SEQ ID NO:62. In certain embodiments, the non-LGR polypeptide consists essentially of SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, or SEQ ID NO: 62. In certain embodiments, the non-LGR polypeptide comprises SEQ ID NO:61. In certain embodiments, the non-LGR polypeptide consists essentially of SEQ ID NO:61.

[00278] In certain embodiments, a soluble receptor is a fusion protein comprising an extracellular domain of an LGR polypeptide capable of binding an RSPO protein and a Fc region. As used herein, a "fusion protein" is a hybrid protein expressed by a nucleic acid molecule comprising nucleotide sequences of at least two genes. In some embodiments, the C-terminus of the first polypeptide is linked to the N-terminus of the immunoglobulin Fc region. In some embodiments, the first polypeptide (e.g., an extracellular domain of an LGR polypeptide) is directly linked to the Fc region (i.e. without an intervening peptide linker). In some embodiments, the first polypeptide is linked to the Fc region via a linker. [00279] As used herein, the term "linker" refers to a linker inserted between a first polypeptide (e.g., a LGR component) and a second polypeptide (e.g., a Fc region). In some embodiments, the linker is a peptide linker. Linkers should not adversely affect the expression, secretion, or bioactivity of the polypeptide. Linkers should not be antigenic and should not elicit an immune response. Suitable linkers are known to those of skill in the art and often include mixtures of glycine and serine residues and often include amino acids that are sterically unhindered. Other amino acids that can be incorporated into useful linkers include threonine and alanine residues. Linkers can range in length, for example from 1-50 amino acids in length, 1-22 amino acids in length, 1-10 amino acids in length, 1-5 amino acids in length, or 1-3 amino acids in length. As used herein, a "linker" is an intervening peptide sequence that does not include amino acid residues from either the C-terminus of the first polypeptide (e.g., LGR component) or the N-terminus of the second polypeptide (e.g., a Fc region).

[00280] LGR proteins contain a signal sequence that directs the transport of the proteins. Signal sequences (also referred to as signal peptides or leader sequences) are generally located at the N- terminus of nascent polypeptides (e.g., amino acids 1-21 of human LGR5 (SEQ ID NO:54)). 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 outer membrane, or to the cell exterior via secretion. Most signal sequences are cleaved from the protein by a signal peptidase after the proteins are transported to the endoplasmic reticulum. The cleavage of the signal sequence from the polypeptide usually occurs at a specific site in the amino acid sequence and is dependent upon amino acid residues within the signal sequence . Although there is usually one specific cleavage site, more than one cleavage site can be recognized and/or used by a signal peptidase resulting in a non-homogenous N-terminus of the polypeptide. For example, the use of different cleavage sites within a signal sequence can result in a polypeptide expressed with different N-terminal amino acids. Accordingly, in some embodiments, the polypeptides as described herein can comprise a mixture of polypeptides with different N-termini. In some embodiments, the N-termini differ in length by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids. 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. 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.

[00281] In certain embodiments, an RSPO-binding agent (e.g., soluble receptor) comprises a Fc region of an immunoglobulin. Those skilled in the art will appreciate that some of the binding agents will comprise fusion proteins in which at least a portion of the Fc region has been deleted or otherwise altered so as to provide desired biochemical characteristics, such as increased cancer cell localization, increased tumor penetration, reduced serum half-life, or increased serum half-life, when compared with a fusion protein of approximately the same immunogenicity comprising a native or unaltered constant region. Modifications to the Fc region can include additions, deletions, or substitutions of one or more amino acids in one or more domains. The modified fusion proteins disclosed herein can comprise alterations or modifications to one or more of the two heavy chain constant domains (CH2 or CH3) or to the hinge region. In other embodiments, the entire CH2 domain can be removed (ACH2 constructs). In some embodiments, the omitted constant region domain is replaced by a short amino acid spacer (e.g., 10 residues) that provides some of the molecular flexibility typically imparted by the absent constant region domain.

[00282] In some embodiments, the modified fusion proteins are engineered to link the CH3 domain directly to the hinge region. In other embodiments, a peptide spacer is inserted between the hinge region and the modified CH2 and/or CH3 domains. For example, constructs can be expressed wherein the CH2 domain has been deleted and the remaining CH3 domain (modified or unmodified) is joined to the hinge region with a 5-20 amino acid spacer. Such a spacer can be added to ensure that the regulatory elements of the constant domain remain free and accessible or that the hinge region remains flexible. However, it should be noted that amino acid spacers can, in some cases, prove to be immunogenic and elicit an unwanted immune response against the construct. Accordingly, in certain embodiments, any spacer added to the construct will be relatively non-immunogenic so as to maintain the desired biological qualities of the fusion protein.

[00283] In some embodiments, the modified fusion proteins can have only a partial deletion of a constant domain or substitution of a few or even a single amino acid. For example, the mutation of a single amino acid in selected areas of the CH2 domain can be enough to substantially reduce Fc binding and thereby increase cancer cell localization and/or tumor penetration. Similarly, it can be desirable to simply delete that part of one or more constant region domains that control a specific effector function (e.g., complement Clq binding). Such partial deletions of the constant regions can improve selected characteristics of the binding agent (e.g., 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 fusion proteins can be modified through the mutation or substitution of one or more amino acids that enhances the profile of the resulting construct. In this respect it can be possible to disrupt the activity provided by a conserved binding site (e.g., Fc binding) while substantially maintaining the configuration and immunogenic profile of the modified fusion protein. In certain embodiments, the modified fusion proteins 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.

[00284] 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 immunoglobulin can bind to 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, release of inflammatory mediators, placental transfer, and control of immunoglobulin production.

[00285] In some embodiments, the modified fusion proteins provide for altered effector functions that, in turn, affect the biological profile of the administered agent. For example, in some embodiments, the deletion or inactivation (through point mutations or other means) of a constant region domain can reduce Fc receptor binding of the circulating modified agent, thereby increasing cancer cell localization and/or tumor penetration. In other embodiments, the constant region modifications increase or reduce the serum half-life of the agent. In some embodiments, the constant region is modified to eliminate disulfide linkages or oligosaccharide moieties.

[00286] In certain embodiments, a modified fusion protein does not have one or more effector functions normally associated with an Fc region. In some embodiments, the agent has no antibody- dependent cell-mediated cytotoxicity (ADCC) activity, and/or no complement-dependent cytotoxicity (CDC) activity. In certain embodiments, the agent does not bind to the Fc receptor and/or complement factors. In certain embodiments, the agent has no effector function.

[00287] In some embodiments, the RSPO-binding agent (e.g., a soluble receptor) described herein is modified to reduce immunogenicity. In general, immune responses against completely normal human proteins are rare when these proteins are used as therapeutics. However, although many fusion proteins comprise polypeptides sequences that are the same as the sequences found in nature, several therapeutic fusion proteins have been shown to be immunogenic in mammals. In some studies, a fusion protein comprising a linker has been found to be more immunogenic than a fusion protein that does not contain a linker. Accordingly, in some embodiments, the polypeptides are analyzed by computation methods to predict immunogenicity. In some embodiments, the polypeptides are analyzed for the presence of T-cell and/or B-cell epitopes. If any T-cell or B-cell epitopes are identified and/or predicted, modifications to these regions (e.g., amino acid substitutions) can be made to disrupt or destroy the epitopes. Various algorithms and software that can be used to predict T-cell and/or B-cell epitopes are known in the art. For example, the software programs SYFPEITHI, HLA Bind, PEPVAC, RANKPEP, DiscoTope, ElliPro, and Antibody Epitope Prediction are all publicly available. [00288] In some embodiments, the RSPO or LGR antagonists are polypeptides. The polypeptides can be recombinant polypeptides, natural polypeptides, or synthetic polypeptides comprising an antibody, or fragment thereof, that bind at least one human RSPO protein or at least one LGR protein. It will be recognized in the art that some amino acid sequences can be varied without significant effect on the structure or function of the protein. Thus, the methods described herein further encompass using variations of the polypeptides which show substantial activity or which include regions of an antibody, or fragment thereof, against a human RSPO protein or a LGR protein. In some

embodiments, amino acid sequence variations of RSPO -binding polypeptides or LGR-binding polypeptides can include deletions, insertions, inversions, repeats, and/or other types of substitutions.

[00289] The polypeptides, analogs and variants thereof, can be further modified to contain additional chemical moieties not normally part of the polypeptide. The derivatized moieties can improve the solubility, the biological half-life, and/or absorption of the polypeptide. The moieties can also reduce or eliminate any undesirable side effects of the polypeptides and variants. An overview for chemical moieties can be found in Remington: The Science and Practice of Pharmacy, 22st Edition, 2012, Pharmaceutical Press, London.

[00290] Many proteins, including antibodies and soluble receptors, 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 can be recognized and/or can be used by a signal peptidase resulting in a non-homogenous N-terminus of the polypeptide. For example, the use of different cleavage sites within a signal sequence can result in a polypeptide expressed with different N-terminal amino acids. Accordingly, in some embodiments, the polypeptides as described herein can comprise a mixture of polypeptides with different N-termini. In some embodiments, the N-termini differ in length by 1, 2, 3, 4, or 5 amino acids. In some embodiments, the polypeptide is substantially homogeneous, i.e., the polypeptides have the same N-terminus. In some embodiments, the signal sequence of the polypeptide comprises one or more (e.g., one, two, three, four, five, six, seven, eight, nine, ten, etc.) amino acid substitutions and/or deletions as compared to a "native" or "parental" signal sequence. In some embodiments, the signal sequence of the polypeptide comprises amino acid substitutions and/or deletions that allow one cleavage site to be dominant, thereby resulting in a substantially

homogeneous polypeptide with one N-terminus. In some embodiments, a signal sequence of the polypeptide affects the expression level of the polypeptide, e.g., increased expression or decreased expression.

[00291] The isolated polypeptides described herein can be produced by any suitable method known in the art. Such methods range from direct protein synthesis methods to constructing a DNA sequence encoding polypeptide sequences and expressing those sequences in a suitable host. In some embodiments, a DNA sequence is constructed using recombinant technology by isolating or synthesizing a DNA sequence encoding a wild-type protein of interest. Optionally, the sequence can be mutagenized by site-specific mutagenesis to provide functional analogs thereof.

[00292] In some embodiments, a DNA sequence encoding a polypeptide of interest can 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.

[00293] 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.

[00294] Recombinant expression vectors can be used to amplify and express DNA encoding agents (e.g., antibodies or soluble receptors), or fragments thereof, which bind 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 an RSPO- binding agent, a LGR-binding agent, an anti-RSPO antibody or fragment thereof, an anti-LGR antibody or fragment thereof, or a LGR-Fc soluble receptor 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. Structural elements intended for use in yeast expression systems can include a leader sequence enabling extracellular secretion of translated protein by a host yeast cell. 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.

[00295] 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 M13 and other filamentous single -stranded DNA phages.

[00296] Suitable host cells for expression of an RSPO-binding or LGR-binding agent (or a protein to use as an antigen) include prokaryotes, yeast cells, insect cells, or higher eukaryotic cells. 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 can also be employed. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts are known to those skilled in the art.

[00297] Various mammalian cell culture systems are used to express recombinant polypeptides. Expression of recombinant proteins in mammalian cells can be preferred because such proteins are generally correctly folded, appropriately modified, and biologically functional. Examples of suitable mammalian host cell lines include COS-7 (monkey kidney-derived), L-929 (murine fibroblast- derived), C127 (murine mammary tumor-derived), 3T3 (murine fibroblast-derived), CHO (Chinese hamster ovary-derived), HeLa (human cervical cancer-derived), BHK (hamster kidney fibroblast- derived), 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.

[00298] 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.

[00299] 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. Isolated proteins can also be physically characterized using such techniques as proteolysis, mass spectrometry (MS), nuclear magnetic resonance (NMR), high performance liquid chromatography (HPLC), and x-ray crystallography.

[00300] 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 binding agent. Some or all of the foregoing purification steps, in various combinations, can also be employed to provide a homogeneous recombinant protein.

[00301] 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.

[00302] In certain embodiments, the 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, antibodies can be used in a non-conjugated form to harness the subject's natural defense mechanisms including complement-dependent cytotoxicity and antibody dependent cellular toxicity to eliminate the malignant or cancer cells.

[00303] In some embodiments, the 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, nonbinding active fragments of diphtheria toxin, exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. In some embodiments, the cytotoxic agent is a radioisotope to produce a radioconjugate or a radioconjugated antibody. A variety of radionuclides are available for the production of radioconjugated antibodies including, but not limited to, 90Y, 1251, 1311, 1231, l l lln, 131In, 105Rh, 153Sm, 67Cu, 67Ga, 166Ho, 177Lu, 186Re, 188Re and 212BL In some embodiments, conjugates of an antibody and one or more small molecule toxins, such as a calicheamicin, maytansinoids, a trichothene, and CC1065, and the derivatives of these toxins that have toxin activity, can be produced. In certain embodiments, conjugates of an antibody and a cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyidithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p- diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as l,5-difluoro-2,4-dinitrobenzene).

[00304] In certain embodiments, the RSPO or LGR antagonist (e.g., antibody or soluble receptor) is an antagonist of at least one RSPO protein (i.e., 1, 2, 3, or 4 RSPO proteins). In certain embodiments, the RSPO or LGR antagonist inhibits activity of the RSPO protein(s) to which it binds. In certain embodiments, the RSPO or LGR antagonist inhibits at least about 10%, at least about 20%, at least about 30%, at least about 50%, at least about 75%, at least about 90%, or about 100% of the activity of the human RSPO protein(s) to which it binds. In certain embodiments, the RSPO or LGR antagonist inhibits activity of RSP03.

[00305] In certain embodiments, the RSPO or LGR antagonist (e.g., antibody or soluble receptor) inhibits binding of at least one human RSPO to an appropriate receptor. In certain embodiments, the RSPO or LGR antagonist inhibits binding of at least one human RSPO protein to one or more human LGR proteins. In some embodiments, the at least one RSPO protein is selected from the group consisting of: RSPO l, RSP02, RSP03, and RSP04. In some embodiments, the at least one RSPO protein is RSP03. In some embodiments, the one or more human LGR proteins are selected from the group consisting of: LGR4, LGR5, and LGR6. In certain embodiments, the RSPO or LGR antagonist inhibits binding of one or more RSPO proteins to LGR4, LGR5, and/or LGR6. In certain

embodiments, the inhibition of binding of a particular RSPO to a LGR protein by an RSPO or LGR antagonist is at least about 10%, at least about 25%, at least about 50%, at least about 75%, at least about 90%, or at least about 95%. In certain embodiments, an RSPO or LGR antagonist is an antibody, for example, an anti-RSPO antibody such as an anti-RSP03 antibody. In certain embodiments, an RSPO or LGR antagonist is 131R10. In certain embodiments, an RSPO or LGR antagonist is an antibody comprising the 6 CDRs of 131R10. In certain embodiments, an RSPO or LGR antagonist an anti-LGR antibody. In certain embodiments, an RSPO or LGR antagonist is a LGR-Fc soluble receptor. In certain embodiments, an RSPO or LGR antagonist is a LGR5-Fc soluble receptor. In certain embodiments, the LGR5-Fc soluble receptor comprises amino acid sequence of SEQ ID NO:57.

[00306] In certain embodiments, the RSPO or LGR antagonists (e.g., antibody or soluble receptor) described herein are antagonists of at least one human RSPO protein and inhibit RSPO activity. In certain embodiments, the RSPO or LGR antagonist inhibits RSPO activity by at least about 10%, at least about 20%, at least about 30%, at least about 50%, at least about 75%, at least about 90%, or about 100%. In some embodiments, the RSPO or LGR antagonist inhibits activity of one, two, three, or four RSPO proteins. In some embodiments, the RSPO or LGR antagonist inhibits activity of at least one human RSPO protein selected from the group consisting of: RSPOl, RSP02, RSP03, and RSP04. In some embodiments, the RSPO-binding agent binds at least RSP03. In certain

embodiments, an RSPO or LGR antagonist that inhibits human RSPO activity is an antibody. In certain embodiments, an RSPO or LGR antagonist that inhibits human RSPO activity is an anti-RSPO antibody. In certain embodiments, an RSPO or LGR antagonist that inhibits human RSPO activity is an anti-RSP03 antibody. In certain embodiments, an RSPO or LGR antagonist that inhibits human RSPO activity is 131R10. In certain embodiments, an RSPO or LGR antagonist that inhibits human RSPO activity is an antibody comprising the 6 CDRs of 131R10. In certain embodiments, an RSPO or LGR antagonist that inhibits human RSPO activity is a LGR-Fc soluble receptor. In certain embodiments, an RSPO or LGR antagonist that inhibits human RSPO activity is a LGR5-Fc soluble receptor. In certain embodiments, the LGR5-Fc soluble receptor comprises amino acid sequence of SEQ ID NO: 57.

[00307] In certain embodiments, the RSPO or LGR antagonist described herein is LGR antagonist of at least one human LGR protein. In some embodiments, the RSPO or LGR antagonist inhibits activity of at least one human LGR protein selected from the group consisting of: LGR4, LGR5, and LGR6. In certain embodiments, the RSPO or LGR antagonist is an LGR5 antagonist. In some embodiments, the LGR antagonist is an anti-LGR antibody. In certain embodiments, the LGR antagonist is anti- LGR antibody comprising the 3 heavy chain CDRs of 88M1, and/or the 3 light chain CDRs of 88M1. In some embodiments, the anti-LGR antibody comprises the heavy chain variable region of 88M1, and/or the light chain variable region of 88M1. In certain embodiments, the anti-LGR antibody comprises the 3 heavy chain CDRs of BNC101, and/or the 3 light chain CDRs of BNC101. In some embodiments, the anti-LGR antibody comprises the heavy chain variable region of BNC101, and/or the light chain variable region of BNC101.

[00308] In certain embodiments, an RSPO or LGR antagonist described herein is an antagonist of β- catenin signaling. In certain embodiments, the RSPO or LGR antagonist inhibits β-catenin signaling by at least about 10%, at least about 20%, at least about 30%, at least about 50%, at least about 75%, at least about 90%, or about 100%. In certain embodiments, an RSPO or LGR antagonist that inhibits β-catenin signaling is an antibody. In certain embodiments, an RSPO or LGR antagonist that inhibits β-catenin signaling is an anti-RSPO antibody. In certain embodiments, an RSPO or LGR antagonist that inhibits β-catenin signaling is an anti-RSP03 antibody. In certain embodiments, an RSPO or LGR antagonist that inhibits β-catenin signaling is 131R10. In certain embodiments, an RSPO or

LGR antagonist that inhibits β-catenin signaling is an antibody comprising the 6 CDRs of 131R10. In certain embodiments, an RSPO or LGR antagonist that inhibits β-catenin signaling is an anti-LGR antibody. In certain embodiments, the anti-LGR antibody comprises the 3 heavy chain CDRs of 88M1, and/or the 3 light chain CDRs of 88M1. In some embodiments, the anti-LGR antibody comprises the heavy chain variable region of 88M1, and/or the light chain variable region of 88M1. In certain embodiments, the anti-LGR antibody comprises the 3 heavy chain CDRs of BNC101, and/or the 3 light chain CDRs of BNC101. In some embodiments, the anti-LGR antibody comprises the heavy chain variable region of BNC101, and/or the light chain variable region of BNClOl .In certain embodiments, an RSPO or LGR antagonist that inhibits β-catenin signaling is a soluble receptor. In certain embodiments, the soluble receptor is a LGR-Fc soluble receptor. In certain embodiments, the soluble receptor is a LGR5-Fc soluble receptor. In certain embodiments, the LGR5-Fc soluble receptor comprises amino acid sequence of SEQ ID NO:57.

[00309] In certain embodiments, the RSPO or LGR antagonist described herein inhibits binding of at least one RSPO protein to a receptor. In certain embodiments, the RSPO or LGR antagonist inhibits binding of at least one human RSPO protein to one or more of its receptors. In some embodiments, the RSPO or LGR antagonist inhibits binding of at least one RSPO protein to at least one LGR protein. In some embodiments, the RSPO-binding agent inhibits binding of at least one RSPO protein to LGR4, LGR5, and/or LGR6. In certain embodiments, the inhibition of binding of at least one RSPO to at least one LGR protein is at least about 10%, at least about 25%, at least about 50%, at least about 75%, at least about 90%, or at least about 95%. In certain embodiments, an RSPO or LGR antagonist that inhibits binding of at least one RSPO to at least one LGR protein further inhibits β- catenin signaling. In certain embodiments, an RSPO or LGR antagonist that inhibits binding of at least one human RSPO to at least one LGR protein is an antibody. In certain embodiments, an RSPO or LGR antagonist that inhibits binding of at least one human RSPO to at least one LGR protein is an anti-LGR antibody. In certain embodiments, an RSPO or LGR antagonist that inhibits binding of at least one human RSPO to at least one LGR protein is an anti-LGR antibody comprising the 3 heavy chain CDRs of 88M1, and/or the 3 light chain CDRs of 88M1. In some embodiments, the anti-LGR antibody comprises the heavy chain variable region of 88M1, and/or the light chain variable region of 88M1. In certain embodiments, the anti-LGR antibody comprises the 3 heavy chain CDRs of BNC101, and/or the 3 light chain CDRs of BNC 101. In some embodiments, the anti-LGR antibody comprises the heavy chain variable region of BNC101, and/or the light chain variable region of

BNC101. In certain embodiments, an RSPO or LGR antagonist that inhibits binding of at least one human RSPO to at least one LGR protein is a soluble receptor. In certain embodiments, an RSPO or LGR antagonist that inhibits binding of at least one human RSPO to at least one LGR protein is a LGR-Fc soluble receptor. In certain embodiments, an RSPO or LGR antagonist that inhibits binding of at least one human RSPO to at least one LGR protein is a LGR5-Fc soluble receptor. In certain embodiments, the LGR5-Fc soluble receptor comprises amino acid sequence of SEQ ID NO:57.

[00310] In certain embodiments, the RSPO or LGR antagonist described herein inhibits activation of β-catenin. 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 an RSPO-binding agent or LGR-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%. In some embodiments, an RSPO or LGR antagonist that inhibits activation of β-catenin is an antibody. In some embodiments, an RSPO or LGR antagonist that inhibits activation of β-catenin is an anti-LGR antibody. In some embodiments, an RSPO or LGR antagonist that inhibits activation of β-catenin is an anti-LGR antibody comprising the 3 heavy chain CDRs of 88M1, and/or the 3 light chain CDRs of 88M1. In some embodiments, the anti-LGR antibody comprises the heavy chain variable region of 88M1, and/or the light chain variable region of 88M1. In certain embodiments, the anti-LGR antibody comprises the 3 heavy chain CDRs of BNC 101, and/or the 3 light chain CDRs of BNC 101. In some embodiments, the anti-LGR antibody comprises the heavy chain variable region of BNC 101, and/or the light chain variable region of

BNC 101. In some embodiments, an RSPO or LGR antagonist that inhibits activation of β-catenin is a soluble receptor. In some embodiments, an RSPO or LGR antagonist that inhibits activation of β- catenin is a LGR-Fc soluble receptor. In some embodiments, an RSPO or LGR antagonist that inhibits activation of β-catenin is a LGR5-Fc soluble receptor. In certain embodiments, the LGR5-Fc soluble receptor comprises amino acid sequence of SEQ ID NO:57. [00311] In certain embodiments, an RSPO or LGR antagonist has one or more of the following effects: inhibit proliferation of tumor cells, inhibit tumor growth, reduce the frequency of cancer stem cells in a tumor, reduce the tumorigenicity of a tumor, reduce the tumorigenicity of a tumor by reducing the frequency of cancer stem cells in the tumor, trigger cell death of tumor cells, induce cells in a tumor to differentiate, differentiate tumorigenic cells to a non-tumorigenic state, induce expression of differentiation markers in the tumor cells, prevent metastasis of tumor cells, or decrease survival of tumor cells.

[00312] In certain embodiments, an RSPO or LGR antagonist is capable of inhibiting tumor growth. In certain embodiments, an RSPO or LGR antagonist is capable of inhibiting tumor growth in vivo (e.g., in a xenograft mouse model and/or in a human having cancer). In some embodiments, the tumor is a tumor selected from the group consisting of colorectal tumor, colon tumor, pancreatic tumor, lung tumor, ovarian tumor, liver tumor, breast tumor, kidney tumor, prostate tumor, gastrointestinal tumor, melanoma, cervical tumor, bladder tumor, glioblastoma, and head and neck tumor. In certain embodiments, the tumor is a breast tumor. In certain embodiments, the tumor is an ovarian tumor. In certain embodiments, the tumor is a lung tumor. In certain embodiments, the tumor is a pancreatic tumor. In certain embodiments, the tumor is an RSPO-dependent tumor, LGR-dependent tumor, or β- catenin-dependent tumor.

[00313] In certain embodiments, an RSPO or LGR antagonist is capable of reducing the

tumorigenicity of a tumor. In certain embodiments, an RSPO or LGR antagonist is capable of reducing the tumorigenicity of a tumor comprising cancer stem cells in an animal model, such as a mouse xenograft model. In certain embodiments, the number or frequency of cancer stem cells in a tumor is reduced by at least about two-fold, about three-fold, about five-fold, about ten-fold, about 50- fold, about 100-fold, or about 1000-fold. In certain embodiments, the reduction in the number or frequency of cancer stem cells is determined by limiting dilution assay using an animal model.

Additional examples and guidance regarding the use of limiting dilution assays to determine a reduction in the number or frequency of cancer stem cells in a tumor can be found, e.g., in

International Publication Number WO 2008/042236, and U.S. Patent Publication Nos. 2008/0064049, and 2008/0178305, each of which are hereby incorporated by reference herein in their entirety for all purposes.

[00314] In certain embodiments, an RSPO or LGR antagonist is active in vivo for at least 1 hour, at least about 2 hours, at least about 5 hours, at least about 10 hours, at least about 24 hours, at least about 2 days, at least about 3 days, at least about 1 week, or at least about 2 weeks. In certain embodiments, the RSPO or LGR antagonist is an IgG (e.g., IgGl or IgG2) antibody that is active in vivo for at least 1 hour, at least about 2 hours, at least about 5 hours, at least about 10 hours, at least about 24 hours, at least about 2 days, at least about 3 days, at least about 1 week, or at least about 2 weeks. In certain embodiments, the RSPO or LGR antagonist is a fusion protein that is active in vivo for at least 1 hour, at least about 2 hours, at least about 5 hours, at least about 10 hours, at least about 24 hours, at least about 2 days, at least about 3 days, at least about 1 week, or at least about 2 weeks.

[00315] In certain embodiments, an RSPO or LGR antagonist has a circulating half-life in mice, cynomolgus monkeys, or humans of at least about 5 hours, at least about 10 hours, at least about 24 hours, at least about 2 days, at least about 3 days, at least about 1 week, or at least about 2 weeks. In certain embodiments, the RSPO or LGR antagonist is an IgG (e.g., IgGl or IgG2) antibody that has a circulating half-life in mice, cynomolgus monkeys, or humans of at least about 5 hours, at least about 10 hours, at least about 24 hours, at least about 2 days, at least about 3 days, at least about 1 week, or at least about 2 weeks. In certain embodiments, the RSPO or LGR antagonist is a fusion protein that has a circulating half-life in mice, cynomolgus monkeys, or humans of at least about 5 hours, at least about 10 hours, at least about 24 hours, at least about 2 days, at least about 3 days, at least about 1 week, or at least about 2 weeks. Methods of increasing (or decreasing) the half-life of agents such as polypeptides and antibodies are known in the art. For example, known methods of increasing the circulating half-life of IgG antibodies include the introduction of mutations in the Fc region which increase the pH-dependent binding of the antibody to the neonatal Fc receptor (FcRn). Known methods of increasing the circulating half-life of antibody fragments lacking the Fc region include such techniques as PEGylation.

IV. Immunotherapeutic agents

[00316] The present invention provides RSPO or LGR antagonists for use in combination therapy with immunotherapeutic agents for modulating immune responses, inhibiting tumor growth, and/or for the treatment of cancer. In some embodiments of the methods described herein, a

immunotherapeutic agent is selected from the group consisting of: a modulator of PD-1 activity, a modulator of PD-Ll activity, a modulator of PD-L2 activity, a modulator of CTLA-4 activity, a modulator of CD28 activity, a modulator of CD80 activity, a modulator of CD86 activity, a modulator of 4- IBB activity, an modulator of OX40 activity, a modulator of KIR activity, a modulator of Tim-3 activity, a modulator of LAG3 activity, a modulator of CD27 activity, a modulator of CD40 activity, a modulator of GITR activity, a modulator of TIGIT activity, a modulator of CD20 activity, a modulator of CD96 activity, a modulator of IDOl activity, a cytokine, a chemokine, an interferon, an interleukin, a lymphokine, a member of the tumor necrosis factor (TNF) family, and an

immunostimulatory oligonucleotide.

[00317] In some embodiments, an immunotherapeutic agent is selected from the group consisting of: a PD-1 antagonist, a PD-Ll antagonist, a PD-L2 antagonist, a CTLA-4 antagonist, a CD80 antagonist, a CD86 antagonist, a KIR antagonist, a Tim-3 antagonist, a LAG3 antagonist, a TIGIT antagonist, a CD20 antagonist, a CD96 antagonist, and/or an IDOl antagonist.

[00318] In some embodiments, the PD-1 antagonist is an antibody that specifically binds PD-1. In some embodiments, the antibody that binds PD-1 is Merck (KEYTRUDA, MK-3475; Merck), pidilizumab (CT-011; Curetech Ltd.), nivolumab (OPDIVO, BMS-936558, MDX-1106; Bristol Myer Squibb), MEDI0680 (AMP-514; AstraZenenca/Medlmmune), REGN2810 (Regeneron

Pharmaceuticals), BGB-A317 (BeiGene Ltd.), PDR-001 (Novartis), or STI-A1110 (Sorrento

Therapeutics). In some embodiments, the antibody that binds PD-1 is described in PCT Publication WO 2014/179664, for example, an antibody identified as APE2058, APE1922, APE1923, APE1924, APE 1950, or APE 1963 (Anaptysbio), or an antibody containing the CDR regions of any of these antibodies. In other embodiments, the PD-1 antagonist is a fusion protein that includes PD-L2, for example, AMP-224 (AstraZeneca/Medlmmune). In other embodiments, the PD-1 antagonist is a peptide inhibitor, for example, AU P-12 (Aurigene).

[00319] In some embodiments, the PD-L1 antagonist is an antibody that specifically binds PD-L1. In some embodiments, the antibody that binds PD-L1 is atezolizumab (RG7446, MPDL3280A;

Genentech), MEDI4736 (AstraZeneca Medlmmune), BMS-936559 (MDX-1105; Bristol Myers Squibb), avelumab (MSB0010718C; Merck KGaA), KD033 (Kadmon), the antibody portion of KD033, or STI-A1014 (Sorrento Therapeutics). In some embodiments, the antibody that binds PD- LI is described in PCT Publication WO 2014/055897, for example, Ab-14, Ab-16, Ab-30, Ab-31, Ab-42, Ab-50, Ab-52, or Ab-55, or an antibody that contains the CDR regions of any of these antibodies.

[00320] In some embodiments, the CTLA-4 antagonist is an antibody that specifically binds CTLA-4. In some embodiments, the antibody that binds CTLA-4 is ipilimumab (YERVOY) or tremelimumab (CP-675,206). In some embodiments, the CTLA-4 antagonist a CTLA-4 fusion protein, for example, KAHR-102 (Kahr Medical Ltd.).

[00321] In some embodiments, the LAG3 antagonist is an antibody that specifically binds LAG3. In some embodimetns, the antibody that binds LAG3 is IMP701 (Prima BioMed), IMP731 (Prima BioMed/GlaxoSmithKline), BMS-986016 (Bristol Myer Squibb), LAG525 (Novartis), and

[00322] In some embodiments, the KIR antagonist is an antibody that specifically binds KIR. In some embodiments, the antibody that binds KIR is lirilumab.

[00323] In some embodiments, an immunotherapeutic agent is selected from the group consisting of: a CD28 agonist, a 4-lBB agonist, an OX40 agonist, a CD27 agonist, a CD80 agonist, a CD86 agonist, a CD40 agonist, and a GITR agonist.

[00324] In some embodiments, the OX40 agonist includes OX40 ligand, or an OX40-binding portion thereof. For example, the OX40 agonist may be MEDI6383 (AstraZeneca). In some embodiments, the OX40 agonist is an antibody that specifically binds OX40. In some embodiments, the antibody that binds OX40 is MEDI6469 (AstraZeneca/Medlmmune), MEDI0562 (AstraZeneca/Medlmmune), or MOXR0916 (RG7888; Genentech). In some embodiments, the OX40 agonist is a vector (e.g., an expression vector or virus, such as an adenovirus) capable of expressing OX40 ligand. In some embodiments the OX40-expressing vector is Delta-24-RGDOX (DNAtrix) or DNX2401 (DNAtrix).

[00325] In some embodiments, the 4-1BB (CD137) agonist is a binding molecule, such as an anticalin. In some embodiments, the anticalin is PRS-343 (Pieris AG). In some embodiments, the 4- IBB agonist is an antibody that specifically binds 4-1BB. In some embodiments, antibody that binds 4-1BB is PF-2566 (PF-05082566; Pfizer) or urelumab (BMS-663513; Bristol Myer Squibb).

[00326] In some embodiments, the CD27 agonist is an antibody that specifically binds CD27. In some embodiments, the antibody that binds CD27 is varlilumab (CDX-1127; Celldex).

[00327] In some embodiments, the GITR agonist comprises GITR ligand or a GITR-binding portion thereof. In some embodiments, the GITR agonist is an antibody that specifically binds GITR. In some embodiments, the antibody that binds GITR is TRX518 (GITR, Inc.), MK-4166 (Merck), or INBRX-110 (Five Prime Therapeutics/Inhibrx).

[00328] In some embodiments, immunotherapeutic agents include, but are not limited to, cytokines such as chemokines, interferons, interleukins, lymphokines, and members of the tumor necrosis factor (TNF) family. In some embodiments, immunotherapeutic agents include immunostimulatory oligonucleotides, such as CpG dinucleotides.

[00329] In some embodiments, a immunotherapeutic agent includes, but is not limited to, anti-PD-1 antibodies, anti-PD-Ll antibodies, anti-PD-L2 antibodies, anti-CTLA-4 antibodies, anti-CD28 antibodies, anti-CD80 antibodies, anti-CD86 antibodies, anti-4-lBB antibodies, anti-OX40 antibodies, anti-KIR antibodies, anti-Tim-3 antibodies, anti-LAG3 antibodies, anti-CD27 antibodies, anti-CD40 antibodies, anti-GITR antibodies, anti-TIGIT antibodies, anti-CD20 antibodies, anti-CD96 antibodies, or anti-IDO l antibodies.

EXAMPLES

Example 1

Effect of adding anti-RSPO antibodies on immune checkpoint inhibitor therapy in melanoma

[00330] The effect of the anti-RSP03 antibody, 131R10, on melanoma cell line tumor growth was tested animals receiving the combination of anti-CTLA-4 and anti-PD-Ll antibodies. In these experiments, ten thousand B16F1 melanoma cells, which does not express RSP03, were injected subcutaneously into C57BL6J mice. On days 4, 7, and 10 post-implantation, "GVAX" tumor vaccine was administered by the injection of 2 million mitomycin C-treated cells of a B16F1 subclone stably transfected with a plasmid encoding m-GM-CSF (with GM-CSF expression confirmed by ELISA), as previously described by Curran and Allison, Cancer Res 69:7747, 2009. Anti-CTLA-4 clone 9D9 (BioXCell; West Lebanon, NH) was dosed on days 5, 8, and 12. Anti-PD-Ll clone 10F.9G2 (BioXCell) was dosed on days 5, 8, 12, 14, 19, 22, and 26, and either 1B7.11 isotype or 131R10 were administered days 5, 12, 19, and 26 following parental B16F1 cell implantation.

[00331] As shown in Figures 1A-1D, the addition of 131R10 decreased tumor growth as compared to administration of the anti-CTLA-4 and anti-PD-Ll antibodies without 131R10.

Example 2

Effect of adding anti-RSPO antibodies on cytokine secretion during immune checkpoint inhibitor therapy

[00332] Using the ELISpot cytokine secretion assay, secretion of IFN-gamma and IL-2 was measured following 131R10 treatment. In these assays, splenocytes were harvested from a minimum of four mice per group, filtered, and the red blood cells lysed and re-suspended in RPMI+10%

FBS+penn/strep at a concentration of 10⁶ cells/ml in the presence of lug/ml hgplOO peptide. Cells were plated on pre-coated ELISpot plates, incubated overnight and processed according to manufacturer's instructions (MabTech, Cincinnati, OH). Developed plates were read on a BioSys BioReader 6000-F b. The results from this assay are shown in Figure 2A (IFN-gamma) and Figure 2B (IL-2). Values shown are total optical density. As shown in these figures, IFN-gamma and IL-2 were increased by addition of 131R10 therapy.

Example 3

Effect of adding anti-RSPO antibodies on T-cell cytotoxicity during immune checkpoint inhibitor therapy

[00333] A T-cell cytotoxicity assay was also performed. Briefly, splenocytes were harvested from a minimum of four mice per group, filtered, and the red blood cells lysed and re-suspended in

RPMI+10% FBS+penn/strep at a concentration of 2 x 10⁶ cells/ml. Splenocytes were incubated with ^g/ml hgplOO peptide for nine days. On day 9 after plating splenocytes, B16F1 cells in culture were incubated with lOuM calcein AM viability dye per 10⁶ cells at 37°C for one hour. Cells were then incubated with ^g/ml hgp lOO for an additional hour at 37°C before being washed to remove excess dye. Splenocytes were re-suspended at 2.5xl0⁶ cells/ml, B 16F1 cells were re-suspended at 10⁵ cells/ml and ΙΟΟμΙ of each combined in each well of a V-bottom plate for an effectortarget ratio of 25: 1, with splenocytes from each individual mouse plated in triplicate. Labeled B 16F1 cells were incubated in 5% SDS to determine maximum lysis conditions, while labeled cells were incubated in medium alone to determine minimum lysis conditions. The plate was then centrifuged at lOOOrpm for four minutes and then incubated at 37°C for four hours. Culture supematants were then harvested and fluorescence of released calcein AM was read at 485nm. Percent specific lysis was determined by dividing the value of the experimental sample by the difference in values between maximum and minimum lysis conditions. The results from these experiments, which are shown in Figure 3, show an increase in T cell cytotoxocity from the combination of the anti-RSPO antibody with anti-CTLA-4 + anti-PD-Ll as compared to controls.

Example 4

Effect of adding anti-RSPO antibodies on T-cell tumor infiltration during immune checkpoint inhibitor therapy

[00334] The change is T-cell tumor infiltration was also measured. Here, B16F1 tumors were isolated from control antibody, anti-CTLA-4 and anti -mouse PD-L1 combination; and 131R10 + anti -mouse anti-CTLA-4 + anti-mouse PD-L1 combination antibody-treated mice. Single cell suspensions were acquired from 4-6 independently treated tumors and were stained for tumor infiltrating immune cells using fluorescently-labeled anti-CD45, anti-CD4, and anti-CD8 antibodies. Flow cytometry analysis (FACS) was performed and the relative percentage populations of CD45+/CD4+ and CD45+/CD8+ T cells are shown. As shown in Figures 4A and 4B, addition of 131R10 increased CD4⁺ and CD8⁺ T cell infiltration as compared to treatment with immune checkpoint inhibitors alone.

[00335] We also observed increases in CD45+ and CD3 T cell infiltration (data not shown).

Example 5

Effect of anti-RSPO antibodies on breast tumor cells

[00336] We tested the effect of anti-RSPO antibodies on growth of 4T1 breast tumor cells. In a first set of experiments, one million 4T1 breast tumor cells were injected subcutaneously into BALB/c mice. Mice were randomized for antibody treatment when tumor volumes reached 111mm³. 131R10 was administered once weekly by IP injection (25mg/kg). As shown in Figures 5A-5C,

administration of 131R10 resulted in decreased tumor volumes as compared to saline control.

[00337] We also measured cytokine expression level in splenocytes taken from mice. Here, ELISpot Cytokine Secretion Assays were used. Briefly, splenocytes were harvested from a minimum of four mice per group, filtered, the red blood cells lysed and re-suspended in RPMI+10% FBS+pen/strep at a concentration of 10⁶ cells/ml in the presence of 0.2ug/ml AH1 peptide. Cells were plated on pre- coated ELISpot plates, incubated overnight, and processed according to manufacturer's instructions (MabTech, Cincinnati, OH). Developed plates were read on a BioSys BioReader^® 6000-F b macroscope. As shown in Figures 6A-6D treatment with the anti-RSPO antibody increased secretion of interferon gamma (Figure 6A), IL-2 (Figure 6B), IL-10 (Figure 6C), and IL-17A (Figure 6D). Values shown are total optical density.

[00338] We also measured the number of splenic CD4+, CD8+, and Tregs in the mice. Briefly, single cell suspensions were acquired from 5-6 spleens from each treatment group and were stained for immune cells using fluorescently-labeled anti-CD45, anti-CD4, anti-CD8, anti-FoxP3, and anti-CD25 antibodies. Flow cytometry analysis (FACS) was performed and the relative percentage populations of CD4+, CD8+, and CD4+CD25+FoxP3+ Treg T cells were measured. These results are shown in Figures 7A-7C, respectively. Increases in all three classes of T cells were observed in mice receiving 13 lRlO as compared to control. Example 6

Effect of adding anti-RSPO antibodies on anti-PD 1 therapy in breast cancer

[00339] We tested the effects of anti-RSPO antibodies in combination with an anti-PD 1 antibody on breast tumor cells injected into mice. Briefly, one million 4T1 breast tumor cells were injected subcutaneously into BALB/c mice. Mice were randomized for antibody treatment when tumor volumes reached 111mm³. 131R10 was administered once weekly by IP injection (25mg/kg), 25C^g a-Pdl (BioXCell, clone RMPl-14) twice weekly and 33mg/kg docetaxel once weekly. 250ug purified mouse IgG2b (BioXCell, clone MPC-11) was administered once weekly to control mice.

[00340] As shown in Figures 8A-8D, the combination of 131R10 and anti-PDl was significantly more effective than control or anti-PDl alone.

[00341] We also measured splenocyte cytokine expression as described in Example 5 above. As shown in Figures 9A, interferon gamma for the 13 IRlO+anti-PD 1 combination appeared to decrease slightly relative to anti-PDl alone or control. As shown in Figure 9B, IL-10 levels were similar in both the 13 IRlO+anti-PD 1 as compared to anti-PD 1 alone. As shown in Figures 9C and 9D, IL-2 and IL-17A levels, respectively, for the 131R10+anti-PDl combination were increased relative both to control as well as anti-PDl alone.

[00342] We also measured T cell cytotoxicity in mice taken from the three treatment groups. Briefly, the mouse breast tumor cell line 4T1 was cultured in RPMI culture medium supplemented with 10% (v/v) fetal bovine serum (FBS), 2mM L-glutamine, and 100 U/mL penicillin at 37°C in a humidified atmosphere of 5% C0₂. Total splenocytes from were cultured for 7 days in RPMI 1640 medium supplemented with 10% FBS, 2 mM L-glutamine, 100 U/mL penicillin, and 0.5 μg/mL AH1 peptide (Anaspec). 4T1 target cells were labeled with 10 μΜ calcein AM (Life Technologies) for 1 hour at 37°C, pulsed with AH1 peptide for an additional hour at 37°C and then combined with the splenocytes at an effectortarget ratio of 12.5: 1. Following a four-hour incubation at 37°C, cell-free supernatants were harvested and calcein release was quantified on a fluorometer at an excitation of 485 nm and an emission of 535 nm. The percentage of specific cell lysis was determined as: % lysis = 100 x (ER-SR)/(MR-SR), where ER, SR, and MR represent experimental, spontaneous, and maximum calcein release, respectively. Spontaneous release is the fluorescence emitted by target cells incubated in media alone (i.e., in the absence of effector cells), while maximum release is determined by lysing target cells with an equal volume of 10% SDS.

[00343] As shown in Figure 10A, T cell cytotoxicity was increased in the 13 lRlO+anti-PDl combination relative to both control and anti-PD l alone. [00344] We also measured T cells in the tumor. In these experiments, at the termination of the experiments mice were euthanized, xenograft tumors were removed, cut into small pieces with a scalpel and minced with a sterile razor blade after removing necrotic portion and connective tissues. To obtain single cell suspension, a digestion solution containing Collagenase in MEM medium (Cambrex, East Rutherford, NJ) with a 1: 100 dilution of DNAsel (Worthington, Lakewood, NJ) was mixed with the tumor suspension and incubated for 1 hour at 37°C for enzymatic dissociation with mechanical agitation with a pipette every 15 min. The reaction was inactivated by adding equal volume of FACS buffer (HBSS medium supplemented with 2% heat-inactivated fetal bovine serum and 20mM Hepes) and filtered through a 40um mesh to remove aggregated and undigested tissues. Cells were centrifuged and re-suspended in 1 ml of ACK medium (0.15 M NH₄C1, 10 mM KHC0₃, and 0.1 mM Na₂EDTA in distilled water) on ice for 2 minutes to remove red blood cells. The reaction was terminated by adding FACS buffer and cells were centrifuged and re-suspended in FACS buffer. The cells were stained using fluorescently-labeled anti-CD45, anti-CD4 and anti- CD 8, antibodies. Flow cytometry analysis (FACS) was performed and the relative percentage populations of CD8+ and CD4+ T cells are shown, respectively, in Figures 10B and IOC. Increased number of both CD8+ and CD4+ were seen in mice receiving the combination of anti-PDl antibodies and 131R10, as compared to mice receiving either control or anti-PD 1 antibodies alone.

[00345] We also measured the number of splenic CD4+, CD8+, and Tregs in the mice. Briefly, single cell suspensions were acquired from 5-6 spleens from each treatment group and were stained for immune cells using fluorescently-labeled anti-CD45, anti-CD4, anti-CD8, anti-FoxP3, and anti-CD25 antibodies. Flow cytometry analysis (FACS) was performed and the relative percentage populations of CD4+, CD8+, and CD4+CD25+FoxP3+ Treg T cells were measured. These results are shown in Figures 10D-10F, respectively. Increases in all three classes of T cells were observed in subjects receiving the combination of anti-PDl antibodies and 131R10, as compared to mice receiving anti- PD1 antibodies alone.

Example 7

Effect of adding anti-RSPO antibodies on anti-CTLA-4 therapy in breast cancer

[00346] We tested the effects of anti-RSPO antibodies in combination with an anti-CTLA-4 antibody on breast tumor cells injected into mice. Briefly, one million 4T1 breast tumor cells were injected subcutaneously into BALB/c mice. Mice were randomized for antibody treatment when tumor volumes reached 111mm³. OMP-131R10 was administered once weekly by IP injection (25mg/kg), 100μg anti-CTLA-4 (BioXCell, clone 9D9) twice weekly and 33mg/kg docetaxel once weekly. Control 100μg purified rat IgG2a (BioXCell, clone 2A3) was administered once weekly.

[00347] As shown in Figures 1 lA-1 ID, the combination of 131R10 and anti-CTLA-4 was significantly more effective than control or anti-CTLA-4 alone. [00348] We also measured splenocyte cytokine expression as described in Example 5 above. As shown in Figures 12A, interferon gamma for the 131R10+anti-PD l combination appeared to decrease slightly relative to anti-PDl alone or control. As shown in Figures 12B and 12C, IL-10 and IL-2 levels, respectively, were similar in the 13 lRlO+anti-CTLA-4 as compared to anti-CTLA-4 alone or control. As shown in Figure 12D, IL-17A levels for the 131R10+anti-CLTA-4 combination were increased relative both to control and anti-CTLA-4 alone.

[00349] We also measured T cell cytotoxicity in mice taken from the three treatment groups following the procedure described in Example 6. As shown in Figure 13, no significant difference was observed among the three groups.

[00350] We also measured the number of splenic CD4+, CD8+, and Tregs in the mice. As described above, single cell suspensions were acquired from 5-6 spleens from each treatment group and were stained for immune cells using fluorescently-labeled anti-CD45, anti-CD4, anti-CD8, anti-FoxP3, and anti-CD25 antibodies. Flow cytometry analysis (FACS) was performed and the relative percentage populations of CD8+, CD4+, and CD4+CD25+FoxP3+ Treg T cells were measured. These results are shown in Figures 14A-14C, respectively, which show a statistically significant increase in Tregs and small increases in CD8+ and CD4+ cells in the 13 lRlO+anti-CTLA-4 group compared to other groups.

Example 8

Effect of combined anti-RSPO and anti-PD-1 therapy in colon cancer

[00351] We also tested the effect of anti-RSPO and anti-PD 1 antibodies on colon tumor growth in mice separately and in combination. Briefly, 30,000 colon tumor cells were injected subcutaneously into BALB/c mice. Mice were randomized for antibody treatment 10 days after cell implantation when tumor volumes reached approximately 100mm³. 25 mg/kg 131R10 and 30 mg/kg 5-FU was administered once weekly by IP injection as indicated. 250ug anti-PD l was administered twice weekly by IP injection.

[00352] As shown in Figure 15, the combination of 131R10 with anti-PDl resulted in a significant decrease in tumor volume as compared to the single antibody treatment groups or to the controls.

Example 9

Effect of combined anti-RSPO and anti-PD-1 therapy in a breast cancer cell line

[00353] 4T1 cells were injected subcutaneously into BALB/c mice. At a mean tumor volume of ~80mm³, mice were randomized for treatment with an anti-RSPO antibody (131R10; 25 mg/kg, Q1W starting day 1), an anti-PD-1 antibody (319R1; 25 mg/kg, Q 1W starting day 1), and/or docetaxel (33 mg/kg, Q 1W starting day 4). Changes in tumor size in this experiment are shown in Figure 16. The largest effect was observed in the mice receiving all three agents (131R10+anti-PD-l+docetaxel). [00354] Changes in frequency, infiltration, and activation of dendritic cells, as well as frequency of T regulatory cells were measured in 4T1 tumor-implanted mice. Further, selected cytokine levels (IL- 17a and IL-2) were measured as well. For these experiments, 4T1 cells were injected subcutaneously into BALB/c mice. At a mean tumor volume of -150 mm³, mice were randomized for treatment with the anti-RSP03 antibody 131R10 (25 mg/kg, day 0), an anti-PD-1 antibody (25 mg/kg, day 0), and/or docetaxel (33 mg/kg, day 3). Seven days following antibody administration, mice were euthanized and tissues harvested. For the flow cytometry experiments, tumor cells were brought to a single cell suspension and the red blood cells lysed. Tumor cells were incubated with Fc block and then stained for cell surface markers shown (antibodies purchased from Affymetrix, BD Biosciences, and BioLegend), washed, and stained with fixable viability dye. Treg samples were fixed and permeabilized with Mouse Regulatory T Cell Staining Kit 1, then stained for Foxp3, per

manufacturer's instructions (Affymetrix). All other samples were briefly fixed with

paraformaldehyde, washed, and stored in PBS until analysis on an LSR II cytometer (BD

Biosciences.) To measure cytokine levels, splenocytes were pressed through a 40 μΜ filter using a syringe plunger. Red blood cells were then lysed to form splenocyte cell suspensions. Two-hundred thousand cells per well were plated in the presence of AH1 peptide onto IL-2 and IL-17a ELISpot plates, incubated overnight, and processed per manufacturer's instructions (Mabtech). Total optical density was determined using a BlOreader ELISpot reader.

[00355] As shown in Figures 17A and 17B, treatment groups show increases in tumor dendritic cell frequency as compared to controls. Figure 17C shows increases in tumor cell infiltration of CD8a⁺ cells as compared to control, and Figure 17D shows increased tumor dendritic cell activation in treatment groups as compared to control. Figure 18A shows changes in splenic dendritic cells, and Figure 18B shows decreases in T regulatory cells in the spleen in treatment groups as compared to controls.

[00356] Changes in IL-17a and IL-2 levels were measured in control and treatment animals (Figures 19A-19D). 131R10 appeared to decrease IL-2 levels as compared to both controls (saline or anti-PD- 1+docetaxel), but did not have a consistent effect on IL-17a levels.

Example 10

Effect of combined anti-RSPO and anti-PD-1 therapy in a colon adenocarcinoma

[00357] Murine colon adenocarcinoma MC38 cells were injected subcutaneously into C57BL/6 mice At a mean tumor volume of ~110mm³, mice were randomized for treatment with an anti-RSPO antibody (131R10; 25 mg/kg, Q1W starting day 1) and/or an anti-PD-1 antibody (319R1; 25 mg/kg, Q1W starting day 1). To measure cytokine levels, splenocytes were pressed through a 40 μΜ filter using a syringe plunger, and red blood cells were lysed. Two-hundred thousand cells per well were plated in the presence of ADPGK peptide onto IL-2 and IL-17a ELISpot plates, incubated overnight, and processed per manufacturer's instructions (Mabtech). Total optical density was determined using a BlOreader ELISpot reader.

[00358] As shown in Figure 20, the greatest effect on tumor growth was observed in mice receiving the combination of the anti-RSP03 and anti-PD-1 antibodies. As shown in Figure 21, the anti- PvSP03/anti-PD 1 combination also resulted in the smallest number of tumor samples greater than 500 mm³.

[00359] Results from cytokine measurement experiments are shown in Figures 22A and 22B. No difference in IL-2 was observed between the mice receiving anti-PD-1 and those receiving anti-PD- l+anti-RPS03 was observed (Figure 22A), whereas a decrease in IL-17a was observed in mice receiving anti-PDl+anti-RPS03 as compared to mice receiving anti-PD-1 (Figure 22B).

[00360] 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.

[00361] 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.

[0264] Following are the sequences disclosed in the application:

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

MRLGLCVVALVLSWTHL I SSRGI KGKRQRRI SAEGSQACAKGCELCSEVNGCLKCS PKL FILLERNDIRQVGVCLPSCPPGY FDARNPDMNKCI KCKI EHCEAC FSHNFCTKCKEGLYL HKGRCYPACPEGSSAA GTMECSS PAQCEMSEWSPWGPCSKKQQLCGFRRGSEERTRRVL HAPVGDHAACSDTKETRRCTVRRVPCPEGQKRRKGGQGRRENANRNLARKE SKEAGAGSR RRKGQQQQQQQGTVGPL SAGPA

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

MQFRL FS FAL I ILNCMDY SHCQGNRWRRSKRASYVSNPICKGCLSCSKDNGCSRCQQKL F FFLRREGMRQYGECLHSCPSGYYGHRAPDMNRCARCRIENCDSCFSKDFCTKCKVGFYLH RGRC FDECPDGFAPLEETMECVEGCEVGHWSEWGTCSRNNRTCGFKWGLE R RQ IVKKP VKD I PCPT IAESRRCKMTMRHCPGGKRTPKAKEKRNKKKKRKLI ERAQEQHSVFLATDR ANQ

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

MHLRL I SWLFI IL FMEY IGSQNASRGRRQRRMHPNVSQGCQGGCATCSDYNGCLSCKPR L FFALERIGMKQIGVCLS SCPSGYYGTRYPDINKCTKCKADCDTC FNK FCTKCKSGFYL HLGKCLDNCPEGLEANNHTMECVS IVHCEVSEWNPWS PCTKKGKTCGFKRGTETRVRE I I QHPSAKGNLCPPTNETRKCTVQRKKCQKGERGKKGRERKRKKPNKGESKEAIPDSKSLES

SKEIPEQRENKQQQKKRKVQDKQKSVSVSTVH

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

MRAPLCLLLLVAHAVDMLALNRRKKQVGTGLGGNCTGCI ICSEENGCSTCQQRLFLFIRR EGIRQYGKCLHDCPPGYFGIRGQEVNRCKKCGATCESCFSQDFCIRCKRQFYLYKGKCLP TCPPGTLAHQNTRECQGECELGPWGGWSPCTHNGKTCGSAWGLESRVREAGRAGHEEAAT CQVLSESRKCPIQRPCPGERSPGQKKGRKDRRPRKDRKLDRRLDVRPRQPGLQP

89M5 Heavy chain CDR1 (SEQ ID NO:5)

TGYTMH

89M5 Heavy chain CDR2 (SEQ ID NO:6)

GINPNNGGTTYNQNFKG

89M5 Heavy chain CDR3 (SEQ ID NO:7)

KEFSDGYYFFAY

89M5 Light chain CDR1 (SEQ ID NO: 8)

KASQDVIFAVA

89M5 Light chain CDR2 (SEQ ID NO:9)

WASTRHT

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

QQHYSTPW h89M5-H8L5 Heavy chain variable region amino acid sequence (SEQ ID NO: 11)

EVQLVQSGAEVKKPGESLRISCKGSGYSFTGYTMHWVRQMPGKGLEWMGGINPNNGGTTY NQNFKGHVTISADKSISTAYLQWSSLKASDTAMYYCARKEFSDGYYFFAYWGQGTLVTVS

S

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

DIVMTQSPDSLAVSLGERA INCKASQDVI FAVAWYQQKPGQPPKLLIYWAS RHTGVPD RFSGSGSGTDFTL ISSLQAEDVAVYYCQQHYS PW FGGGTKVEIK

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

MDWTWRILFLVAAATGAHSEVQLVQSGAEVKKPGESLRISCKGSGYSFTGYTMHWVRQMP GKGLEWMGGINPNNGGTTYNQNFKGHVTISADKSISTAYLQWSSLKASDTAMYYCARKEF SDGYYFFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEP KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTK QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK h89M5-H8L5 Heavy chain amino acid sequence without predicted signal sequence (SEQ ID NO: 14)

EVQLVQSGAEVKKPGESLRISCKGSGYSFTGYTMHWVRQMPGKGLEWMGGINPNNGGTTY NQNFKGHVTISADKSISTAYLQWSSLKASDTAMYYCARKEFSDGYYFFAYWGQGTLVTVS SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

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

MVLQTQVFISLLLWISGAYGDIVMTQSPDSLAVSLGERATINCKASQDVI FAVAWYQQKP GQPPKLLIYWASTRHTGVPDRFSGSGSGTDFTL ISSLQAEDVAVYYCQQHYS PW FGG GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ ESVTEQDSKDSTYSLSNTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

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

DIVMTQSPDSLAVSLGERA INCKASQDVI FAVAWYQQKPGQPPKLLIYWAS RHTGVPD RFSGSGSGTDFTL ISSLQAEDVAVYYCQQHYS PW FGGGTKVEIKRTVAAPSVFI FPP SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSNTLT LSKADYEKHKVYACEVTHQGLSSPV KSFNRGEC

130M23 Heavy chain CDR1 (SEQ ID NO: 17)

SSYAMS

130M23 Heavy chain CDR2 (SEQ ID NO: 18)

SISSGGSTYYPDSVKG

130M23 Heavy chain CDR3 (SEQ ID NO: 19)

RGGDPGVYNGDYEDAMDY

130M23 Light chain CDR1 (SEQ ID NO: 20)

KASQDVSSAVA

130M23 Light chain CDR2 (SEQ ID NO:21)

WASTRHT

130M23 Light chain CDR3 (SEQ ID NO: 22)

QQHYSTP

hl30M23-HlL6 Heavy chain variable region amino acid sequence (SEQ ID NO:23)

EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSISSGGSTYYP DSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGGDPGVYNGDYEDAMDYWGQGTT VTVSS

hl30M23-HlL6 Light chain variable region amino acid sequence (SEQ ID NO:24)

DIQMTQSPSSLSASVGDRVTITCKASQDVSSAVAWYQQKPGKAPKLLIYWASTRHTGVPS RFSGSGSGTDFTL ISSLQPEDFA YYCQQHYS PW FGQGTKVEIK hl30M23-HlL6 Heavy chain amino acid sequence with predicted signal sequence underlined (SEQ ID NO:25)

MELGLRWVFLVAILEGVQCEVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMSWVRQAP GKGLEWVSSISSGGSTYYPDSVKGRFTISRDNAK SLYLQMNSLRAEDTAVYYCARGGDP GVYNGDYEDAMDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDK TVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNW YVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTIS KTKGQPREPQVYTLPPSREEMTK QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPM LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK hl30M23-HlL6 Heavy chain amino acid sequence without predicted signal sequence (SEQ ID NO:26) EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSISSGGSTYYP

DSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGGDPGVYNGDYEDAMDYWGQGTT VTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVA GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQF NSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK hl30M23-HlL6 Light chain amino acid sequence with predicted signal sequence underlined (SEQ ID NO:27)

MGIKMESQIQAFVFVFLWLSGVDGDIQMTQSPSSLSASVGDRVTITCKASQDVSSAVAWY QQKPGKAPKLLIYWASTRHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHYSTPW TFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSNTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC hl30M23-HlL6 Light chain amino acid sequence without predicted signal sequence (SEQ ID NO:28)

DIQMTQSPSSLSASVGDRVTITCKASQDVSSAVAWYQQKPGKAPKLLIYWASTRHTGVPS RFSGSGSGTDFTL ISSLQPEDFA YYCQQHYS PW FGQGTKVEIKRTVAAPSVFI FPP SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSNTLT LSKADYEKHKVYACEVTHQGLSSPV KSFNRGEC

131R10 Heavy chain CDR1 (SEQ ID NO:29)

DYSIH

131R10 Heavy chain CDR2 (SEQ ID NO:30)

YIYPSNGDSGYNQKFK13R10

131R10 Heavy chain CDR3 (SEQ ID NO:31)

TYFANNFD

131R10 Alternative Heavy chain CDR3 (SEQ ID NO:32)

A YFANNFDY

131R10 Light chain CDR1 (SEQ ID NO:33)

KASQSVDYDGDSYMN

131R10 Light chain CDR2 (SEQ ID NO:34)

AASNLES

131R10 Alternative Light chain CDR2 (SEQ ID NO:35)

AAS

131R10 Light chain CDR3 (SEQ ID NO:36)

QQSNEDPLT

131R10 Alternative Light chain CDR3 (SEQ ID NO:37)

QQSNEDPLTF

131R10 Heavy chain variable region amino acid sequence (SEQ ID NO: 38)

QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYSIHWVRQAPGQGLEWIGYIYPSNGDSGY NQKFKNRVTMTRDTSTSTAYMELSRLRSEDTAVYYCATYFA NFDYWGQGTTLTVSS

131R10 Light chain variable region amino acid sequence (SEQ ID NO:39)

DIQMTQSPSSLSASVGDRV ITCKASQSVDYDGDSYMNWYQQKPGKAPKLLIYAASNLES GVPSRFSGSGSGTDFTL ISPVQAEDFATYYCQQSNEDPLTFGAGTKLELKR

131R10 Heavy chain amino acid sequence with predicted signal sequence underlined (SEQ ID NO:40)

MKHLWFFLLLVAAPRWVLSQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYSIHWVRQAP GQGLEWIGYIYPSNGDSGYNQKFKNRVTMTRDTSTSTAYMELSRLRSEDTAVYYCATYFA NNFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

131R10 Heavy chain amino acid sequence without predicted signal sequence (SEQ ID NO:41) QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYSIHWVRQAPGQGLEWIGYIYPSNGDSGY NQKFKNRVTM RD S S AYMELSRLRSEDTAVYYCATYFAN FDYWGQGTTLTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY SLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT PPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK

131R10 Light chain amino acid sequence with predicted signal sequence underlined (SEQ ID NO:42) MKHLWFFLLLVAAPRWVLSDIQMTQSPSSLSASVGDRVTITCKASQSVDYDGDSYMNWYQ QKPGKAPKLLIYAASNLESGVPSRFSGSGSGTDFTL ISPVQAEDFA YYCQQSNEDPLT FGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV KSFNRGEC

131R10 Light chain amino acid sequence without predicted signal sequence (SEQ ID NO:43)

DIQMTQSPSSLSASVGDRVTITCKASQSVDYDGDSYMNWYQQKPGKAPKLLIYAASNLES GVPSRFSGSGSGTDFTL ISPVQAEDFATYYCQQSNEDPLTFGAGTKLELKRTVAAPSVF I FPPSDEQLKSGTASVVCLLN FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS STLTLSKADYEKHKVYACEVTHQGLSSPV KSFNRGEC

h89M5-H2L2 Heavy chain variable region amino acid sequence (SEQ ID NO:44)

QVQLVQSGAEVKKPGASVKVSCKTSGYTFTGYTMHWVRQAPGQRLEWMGGINPNNGGTTY NQNFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCARKEFSDGYYFFAYWGQGTLVTVS

S

h89M5-H2L2 Light chain variable region amino acid sequence (SEQ ID NO:45)

DIQMTQSPSSLSASVGDRV ITCKASQDVI FAVAWYQQKPGKAPKLLIYWAS RHTGVPS RFSGSGSGTDYTL ISSLQPEDFA YYCQQHYS PW FGGGTKVEIK h89M5-H2L2 Heavy chain amino acid sequence with predicted signal sequence underlined (SEQ ID NO:46)

MDWTWRILFLVAAATGAHSQVQLVQSGAEVKKPGASVKVSCKTSGYTFTGYTMHWVRQAP GQRLEWMGGINPNNGGTTYNQNFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCARKEF SDGYYFFAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVER KCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDG VEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK h89M5-H2L2 Heavy chain amino acid sequence without predicted signal sequence (SEQ ID NO:47)

QVQLVQSGAEVKKPGASVKVSCKTSGYTFTGYTMHWVRQAPGQRLEWMGGINPNNGGTTY NQNFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCARKEFSDGYYFFAYWGQGTLVTVS SASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTF RVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT PPMLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK h89M5-H2L2 Light chain amino acid sequence with predicted signal sequence underlined (SEQ ID NO:48)

MDMRVPAQLLGLLLLWLRGARCDIQMTQSPSSLSASVGDRVTITCKASQDVI FAVAWYQQ KPGKAPKLLIYWAS RHTGVPSRFSGSGSG DYTL ISSLQPEDFATYYCQQHYS PW F GGG KVEIKRTVAAPSVFI FPPSDEQLKSG ASVVCLLNNFYPREAKVQWKVDNALQSGN SQESVTEQDSKDSTYSLSNTLTLSKADYEKHKVYACEVTHQGLSSPV KSFNRGEC h89M5-H2L2 Light chain amino acid sequence without predicted signal sequence (SEQ ID NO:49)

DIQMTQSPSSLSASVGDRV ITCKASQDVI FAVAWYQQKPGKAPKLLIYWAS RHTGVPS RFSGSGSGTDYTL ISSLQPEDFA YYCQQHYS PW FGGGTKVEIKRTVAAPSVFI FPP SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSNTLT LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

hl30M23-HlL2 Light chain variable region amino acid sequence (SEQ ID NO:50)

DIQMTQSPSSLSASVGDRVTITCKASQDVSSAVAWYQQKPGKAPKLLIYWASTRHTGVPS RFSGSGSGTDFTL ISSVQAEDFATYYCQQHYSTPW FGQGTKVEIK

hl30M23-HlL2 Light chain amino acid sequence with predicted signal sequence underlined (SEQ ID NO:51)

MKYLLPTAAAGLLLLAAQPAMADIQMTQSPSSLSASVGDRVTITCKASQDVSSAVAWYQQ KPGKAPKLLIYWASTRHTGVPSRFSGSGSGTDFTLTISSVQAEDFATYYCQQHYSTPWTF GQG KVEIKRTVAAPSVFI FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN SQESVTEQDSKDSTYSLSNTLTLSKADYEKHKVYACEV HQGLSSPVTKSFNRGEC

hl30M23-HlL2 Light chain amino acid sequence without predicted signal sequence (SEQ ID NO:52)

DIQMTQSPSSLSASVGDRVTITCKASQDVSSAVAWYQQKPGKAPKLLIYWASTRHTGVPS RFSGSGSGTDFTL ISSVQAEDFATYYCQQHYSTPW FGQGTKVEIKRTVAAPSVFI FPP SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSNTLT LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Human LGR4 protein sequence (NM 018490; SEQ ID NO:53)

MPGPLGLLCFLALGLLGSAGPSGAAPPLCAAPCSCDGDRRVDCSGKGLTAVPEGLSAFTQ ALDISMNNITQLPEDAFKNFPFLEELQLAGNDLSFIHPKALSGLKELKVLTLQNNQLKTV PSEAIRGLSALQSLRLDANHITSVPEDSFEGLVQLRHLWLDDNSLTEVPVHPLSNLPTLQ ALTLALNKISSIPDFAFTNLSSLVVLHLHNNKIRSLSQHCFDGLDNLETLDLNYNNLGEF PQAIKALPSLKELGFHSNSISVIPDGAFDGNPLLRTIHLYDNPLSFVGNSAFHNLSDLHS LVIRGASMVQQFPNLTGTVHLESLTLTGTKISSIPNNLCQEQKMLRTLDLSYNNIRDLPS FNGCHALEEISLQRNQIYQIKEGTFQGLISLRILDLSRNLIHEIHSRAFATLGPITNLDV SFNELTSFPTEGLNGLNQLKLVGNFKLKEALAAKDFVNLRSLSVPYAYQCCAFWGCDSYA NLNTEDNSLQDHSVAQEKGTADAANVTSTLENEEHSQI IIHCTPSTGAFKPCEYLLGSWM IRLTVWFI FLVALFFNLLVILTTFASCTSLPSSKLFIGLISVSNLFMGIYTGILTFLDAV SWGRFAEFGIWWETGSGCKVAGFLAVFSSESAI FLLMLATVERSLSAKDIMKNGKSNHLK QFRVAALLAFLGATVAGCFPLFHRGEYSASPLCLPFPTGETPSLGFTVTLVLLNSLAFLL MAVIYTKLYCNLEKEDLSENSQSSMIKHVAWLI FTNCIFFCPVAFFSFAPLITAISISPE IMKSVTLI FFPLPACLNPVLYVFFNPKFKEDWKLLKRRVTKKSGSVSVSISSQGGCLEQD FYYDCGMYSHLQGNLTVCDCCESFLLTKPVSCKHLIKSHSCPALAVASCQRPEGYWSDCG TQSAHSDYADEEDSFVSDSSDQVQACGRACFYQSRGFPLVRYAYNLPRVKD

Human LGR5 protein sequence (SEQ ID NO: 54)

MDTSRLGVLLSLPVLLQLATGGSSPRSGVLLRGCPTHCHCEPDGRMLLRVDCSDLGLSEL PSNLSVFTSYLDLSMNNISQLLPNPLPSLRFLEELRLAGNALTYIPKGAFTGLYSLKVLM LQNNQLRHVPTEALQNLRSLQSLRLDA HISYVPPSCFSGLHSLRHLWLDDNALTEIPVQ AFRSLSALQAMTLALNKIHHIPDYAFGNLSSLVVLHLHNNRIHSLGKKCFDGLHSLETLD LNYNNLDEFPTAIRTLSNLKELGFHSNNIRSIPEKAFVGNPSLITIHFYDNPIQFVGRSA FQHLPELRTLTLNGASQITEFPDLTGTANLESLTLTGAQISSLPQTVCNQLPNLQVLDLS YNLLEDLPSFSVCQKLQKIDLRHNEIYEIKVD FQQLLSLRSLNLAWNKIAIIHPNAFST LPSLIKLDLSSNLLSSFPITGLHGLTHLKLTGNHALQSLISSENFPELKVIEMPYAYQCC AFGVCENAYKISNQWNKGDNSSMDDLHKKDAGMFQAQDERDLEDFLLDFEEDLKALHSVQ CSPSPGPFKPCEHLLDGWLIRIGVWTIAVLALTCNALVTSTVFRSPLYISPIKLLIGVIA AVNMLTGVSSAVLAGVDAF FGSFARHGAWWENGVGCHVIGFLSI FASESSVFLLTLAAL ERGFSVKYSAKFE KAPFSSLKVI ILLCALLALTMAAVPLLGGSKYGASPLCLPLPFGEP STMGYMVALILLNSLCFLMMTIAYTKLYCNLDKGDLENIWDCSMVKHIALLLFTNCILNC PVAFLSFSSLINLTFISPEVIKFILLVVVPLPACLNPLLYILFNPHFKEDLVSLRKQTYV WTRSKHPSLMSINSDDVEKQSCDSTQALVTFTSSSITYDLPPSSVPSPAYPVTESCHLSS VAFVPCL

Human LGR6 protein sequence (BC047905; SEQ ID NO:55)

MGRPRLTLVCQVSI I ISARDLSMNNLTELQPGLFHHLRFLEELRLSGNHLSHIPGQAFSG LYSLKILMLQNNQLGGIPAEALWELPSLQSLRLDANLISLVPERSFEGLSSLRHLWLDDN ALTEIPVRALNNLPALQAMTLALNRISHIPDYAFQNLTSLVVLHLHNNRIQHLGTHSFEG LHNLETLDLNYNKLQEFPVAIRTLGRLQELGFHNNNIKAIPEKAFMGNPLLQTIHFYDNP IQFVGRSAFQYLPKLHTLSLNGAMDIQEFPDLKGTTSLEILTLTRAGIRLLPSGMCQQLP RLRVLELSHNQIEELPSLHRCQKLEEIGLQHNRIWEIGADTFSQLSSLQALDLSWNAIRS IHPEAFSTLHSLVKLDLTDNQLTTLPLAGLGGLMHLKLKGNLALSQAFSKDSFPKLRILE VPYAYQCCPYGMCASFFKASGQWEAEDLHLDDEESSKRPLGLLARQAENHYDQDLDELQL EMEDSKPHPSVQCSPTPGPFKPCEYLFESWGIRLAVWAIVLLSVLCNGLVLLTVFAGGPV PLPPVKFVVGAIAGANTLTGISCGLLASVDALTFGQFSEYGARWETGLGCRATGFLAVLG SEASVLLLTLAAVQCSVSVSCVRAYGKSPSLGSVRAGVLGCLALAGLAAALPLASVGEYG ASPLCLPYAPPEGQPAALGFTVALVMMNSFCFLVVAGAYIKLYCDLPRGDFEAVWDCAMV RHVAWLIFADGLLYCPVAFLSFASMLGLFPVTPEAVKSVLLVVLPLPACLNPLLYLLFNP HFRDDLRRLRPRAGDSGPLAYAAAGELEKSSCDSTQALVAFSDVDLILEASEAGRPPGLE TYGFPSVTLISCQQPGAPRLEGSHCVEPEGNHFGNPQPSMDGELLLRAEGSTPAGGGLSG GGGFQPSGLAFASHV LGR5 ECD amino acids 22-564 (SEQ ID NO:56)

GSSPRSGVLLRGCPTHCHCEPDGRMLLRVDCSDLGLSELPSNLSVFTSYLDLSMNNISQL LPNPLPSLRFLEELRLAGNALTYIPKGAFTGLYSLKVLMLQNNQLRHVPTEALQNLRSLQ SLRLDANHISYVPPSCFSGLHSLRHLWLDDNALTEIPVQAFRSLSALQAMTLALNKIHHI PDYAFGNLSSLVVLHLHNNRIHSLGKKCFDGLHSLETLDLNYNNLDEFPTAIRTLSNLKE LGFHSNNIRSIPEKAFVGNPSLITIHFYDNPIQFVGRSAFQHLPELRTLTLNGASQITEF PDLTGTANLESLTLTGAQISSLPQTVCNQLPNLQVLDLSYNLLEDLPSFSVCQKLQKIDL RHNEIYEIKVDTFQQLLSLRSLNLAWNKIAI IHPNAFSTLPSLIKLDLSSNLLSSFPITG LHGLTHLKLTGNHALQSLISSENFPELKVIEMPYAYQCCAFGVCENAYKISNQWNKGDNS SMDDLHKKDAGMFQAQDERDLEDFLLDFEEDLKALHSVQCSPSPGPFKPCEHLLDGWLIR IGV

LGR5-Fc protein sequence (SEQ ID NO: 57)

MDTSRLGVLLSLPVLLQLATGGSSPRSGVLLRGCPTHCHCEPDGRMLLRVDCSDLGLSEL PSNLSVFTSYLDLSMNNISQLLPNPLPSLRFLEELRLAGNALTYIPKGAFTGLYSLKVLM LQNNQLRHVPTEALQNLRSLQSLRLDANHISYVPPSCFSGLHSLRHLWLDDNALTEIPVQ AFRSLSALQAMTLALNKIHHIPDYAFGNLSSLVVLHLHNNRIHSLGKKCFDGLHSLETLD LNYNNLDEFPTAIRTLSNLKELGFHSNNIRSIPEKAFVGNPSLITIHFYDNPIQFVGRSA FQHLPELRTLTLNGASQITEFPDLTGTANLESLTLTGAQISSLPQTVCNQLPNLQVLDLS YNLLEDLPSFSVCQKLQKIDLRHNEIYEIKVD FQQLLSLRSLNLAWNKIAIIHPNAFST LPSLIKLDLSSNLLSSFPITGLHGLTHLKLTGNHALQSLISSENFPELKVIEMPYAYQCC AFGVCENAYKISNQWNKGDNSSMDDLHKKDAGMFQAQDERDLEDFLLDFEEDLKALHSVQ CSPSPGPFKPCEHLLDGWLIRIGVGRADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDW LNGKEYKCKVSNKALPAPIEK ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK

Human Igd Fc region (SEQ ID NO: 58)

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSRDELTK QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Human Igd Fc region (SEQ ID NO: 59)

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSREEMTK QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Human Igd Fc region (SEQ ID NO: 60)

KSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTK QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Human Igd Fc region (SEQ ID NO:61)

EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAK KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTK QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Human IgG₂ Fc region (SEQ ID NO: 62)

CVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTFRVVSVL VVHQDWLNGKEYKCKVSNKGLPAPIEK ISKTKGQP REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Claims

WHAT IS CLAIMED IS:

1. A method of treating cancer comprising administering to a subject a therapeutically effective amount of a RSPO or LGR antagonist and a therapeutically effective amount of an

immunotherapeutic agent.

2. A method of inhibiting tumor growth in a subject, wherein the method comprises administering to a subject a therapeutically effective amount of a RSPO or LGR antagonist and an

immunotherapeutic agent.

3. A method of inhibiting the activity of regulatory T-cells (Tregs), wherein the method comprises administering to a subject a therapeutically effective amount of a RSPO or LGR antagonist and a therapeutically effective amount an immunotherapeutic agent.

4. A method of increasing T cell infiltration into a tumor, wherein the method comprises

administering to a subject a therapeutically effective amount of a RSPO or LGR antagonist and a therapeutically effective amount of an immunotherapeutic agent.

5. A method of increasing T cell cytotoxicity to a tumor, wherein the method comprises

6. A method of increasing tumor cell lysis, wherein the method comprises administering to a subject a therapeutically effective amount of a RSPO or LGR antagonist and a therapeutically effective amount of an immunotherapeutic agent.

7. A method to increase the efficacy of an immune checkpoint modulator, wherein the method comprises administering to a subject a therapeutically effective amount of a RSPO or LGR antagonist in combination with the immune checkpoint modulator.

8. A method of reducing or preventing cancer metastasis in a subject, wherein the method comprises administering to the subject a therapeutically effective amount of a RSPO or LGR antagonist and a therapeutically effective amount of an immunotherapeutic agent.

9. The method according to any one of claims 1-8, wherein the RSPO or LGR antagonist is an

antibody.

10. The method according to any one of claims 1-9, wherein the RSPO or LGR antagonist is an

antibody that specifically binds at least one RSPO protein or portion thereof.

11. The method of claim 9 or claim 10, wherein the antibody specifically binds at least one RSPO protein selected from the group consisting of human RSPOl, human RSP02, human RSP03, or human RSP04.

12. The method of claim 11, wherein the antibody specifically binds at least human RSPOl.

13. The method of claim 11, wherein the antibody 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).

14. The method of claim 11, wherein the antibody comprises a heavy chain variable region

comprising SEQ ID NO: 11 or 44, and a light chain variable region comprising SEQ ID NO: 12 or 45.

15. The method of claim 11, wherein the antibody comprises a heavy chain variable region

comprising SEQ ID NO: 11 and a light chain variable region comprising SEQ ID NO: 12.

16. The method of claim 11, wherein the antibody specifically binds at least human RSP02.

17. The method of claim 11, wherein the antibody comprises:

(a) a heavy chain CDR1 comprising SSYAMS (SEQ ID NO: 17), a heavy chain CDR2

comprising SISSGGSTYYPDSVKG (SEQ ID NO: 18), and a heavy chain CDR3 comprising RGGDPGVYNGDYEDAMDY (SEQ ID NO: 19); and

(b) a light chain CDR1 comprising KASQDVSSAVA (SEQ ID NO:20), a light chain CDR2 comprising WASTRHT (SEQ ID NO:21), and a light chain CDR3 comprising QQHYSTP (SEQ ID NO:22).

18. The method of claim 11, wherein the antibody comprises a heavy chain variable region

comprising SEQ ID NO:23 and a light chain variable region comprising SEQ ID NO:24.

19. The method of claim 11, wherein the antibody specifically binds at least human RSP03.

20. The method of claim 11, wherein the antibody comprises:

(a) a heavy chain CDR1 comprising DYSIH (SEQ ID NO:29), a heavy chain CDR2

comprising YIYPSNGDSGYNQKFK (SEQ ID NO: 30), and a heavy chain CDR3 comprising TYFANNFD (SEQ ID NO:31) or ATYFANNTDY(SEQ ID NO:32); and

(b) a light chain CDR1 comprising KASQSVDYDGDSYMN (SEQ ID NO: 33), a light chain CDR2 comprising AASNLES (SEQ ID NO:34) or AAS (SEQ ID NO:35), and a light chain CDR3 comprising QQSNEDPLT (SEQ ID NO:36) or QQSNEDPLTF (SEQ ID NO:37).

21. The method of claim 23, wherein the antibody comprises:

(a) a heavy chain CDR1 comprising DYSIH (SEQ ID NO:29), a heavy chain CDR2

comprising YIYPSNGDSGYNQKFK (SEQ ID NO: 30), and a heavy chain CDR3 comprising TYFANNFD (SEQ ID NO:31); and

(b) a light chain CDR1 comprising KASQSVDYDGDSYMN (SEQ ID NO: 33), a light chain

CDR2 comprising AASNLES (SEQ ID NO: 34), and a light chain CDR3 comprising QQSNEDPLT (SEQ ID NO:36).

22. The method of claim 20, wherein the antibody comprises a heavy chain variable region

comprising SEQ ID NO:38, and a light chain variable region comprising SEQ ID NO:39.

23. The method of claim 9, wherein the RSPO or LGR antagonist is an antibody that specifically binds at least one human LGR protein.

24. The method of claim 23, wherein the antibody specifically binds at least one human LGR protein is selected from the group consisting of: LGR4, LGR5, and LGR6.

25. The method of claim 23, wherein the antibody specifically binds at least human LGR5.

26. The method of claim 23, wherein the antibody comprises:

(a) 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

(b) the light chain CDR1, CDR2, and CDR3 sequences of the monoclonal antibody produced by the 88M1 hybridoma having the ATCC deposit number PTA-9342.

27. The method of claim 23, wherein 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.

28. The method of any one of claims 9 to 27, wherein the antibody is a monoclonal antibody, a recombinant antibody, a chimeric antibody, a humanized antibody, a human antibody, or an antibody fragment comprising an antigen-binding site.

29. The method of any one of claims 9 to 28, wherein the antibody is a monospecific antibody or a bispecific antibody.

30. The method of any one of claims 9 to 29, wherein the antibody is an IgGl antibody or an IgG2 antibody.

31. The method of claim 9, wherein the RSPO or LGR antagonist is 131R10.

32. The method of any one of claims 1 to 8, wherein the RSPO or LGR antagonist is a soluble

receptor comprising an extracellular domain of a human LGR protein, wherein the extracellular domain is capable of binding a human RSPO protein.

33. The method of claim 32, wherein the human LGR protein is LGR5.

34. The method of claim 32, wherein the extracellular domain of a human LGR protein comprises amino acids 22-564 of human LGR5 (SEQ ID NO:56).

35. The method of any one of claims 32 to 34, wherein the soluble receptor comprises a non-LGR polypeptide.

36. The method of claim 35, wherein the non-LGR polypeptide is directly linked to the extracellular domain of the human LGR protein.

37. The method of claim 35, wherein the non-LGR polypeptide is connected to the extracellular domain of the human LGR protein by a linker.

38. The method of any one of claims 35 to 37, wherein the non-LGR polypeptide comprises a human Fc region.

39. The method of any one of claims 35 to 38, wherein the non-LGR polypeptide comprises SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, or SEQ ID NO:62.

40. The method of any one of claims 1-40, wherein the RSPO or LGR antagonist enhances the

activity of the immunotherapeutic agent.

41. The method of any one of claims 1-40, wherein the immunotherapeutic agent enhances the

activity of the RSPO or LGR antagonist.

42. The method of any one of claims 1-40, wherein the RSPO or LGR antagonist and the

immunotherapeutic agent act synergistically.

43. The method of any one of claims 1-6 or 8-42, wherein the immunotherapeutic agent selected from a group consisting of: a modulator of PD-1 activity, a modulator of PD-L1 activity, a modulator of PD-L2 activity, a modulator of CTLA-4 activity, a modulator of CD28 activity, a modulator of CD80 activity, a modulator of CD86 activity, a modulator of 4-1BB activity, an modulator of OX40 activity, a modulator of KIR activity, a modulator of Tim -3 activity, a modulator of LAG3 activity, a modulator of CD27 activity, a modulator of CD40 activity, a modulator of GITR activity, a modulator of TIGIT activity, a modulator of CD20 activity, a modulator of CD96 activity, a modulator of IDO l activity, a cytokine, a chemokine, an interferon, an interleukin, a lymphokine, a member of the tumor necrosis factor (TNF) family, and an immunostimulatory oligonucleotide.

44. The method of any one of claims 1-6 or 8-43, wherein the immunotherapeutic agent is an immune checkpoint modulator.

45. The method of claim 7 or claim 44, wherein the immune checkpoint modulator is an immune checkpoint inhibitor.

46. The method of claim 45, wherein the immune checkpoint inhibitor is a PD-1 antagonist, a PD-Ll antagonist, a PD-L2 antagonist, a CTLA-4 antagonist, a CD80 antagonist, a CD86 antagonist, a KIR antagonist, a Tim-3 antagonist, a LAG3 antagonist, a TIGIT antagonist, a CD20 antagonist, a

CD96 antagonist, or a IDOl antagonist.

47. The method of claim 46, wherein the PD-1 antagonist is an antibody that specifically binds PD-1.

48. The method of claim 47, wherein the antibody that binds PD-1 is pembrolizumab (KEYTRUDA;

MK-3475), pidilizumab (CT-011), nivolumab (OPDIVO; BMS-936558), MEDI0680 (AMP-514), REGN2810, BGB-A317, PDR-001, or STI-A1110.

49. The method of claim 46, wherein the PD-1 antagonist comprises the extracellular domain of PD- L2.

50. The method of claim 49, wherein the PD-1 antagonist is AMP -224.

51. The method of claim 46, wherein the PD-1 antagonist is a peptide.

52. The method of claim 51, wherein the PD-1 antagonist is AUNP-12.

53. The method of claim 46, wherein the PD-Ll antagonist is an antibody that specifically binds PD- Ll .

54. The method of claim 53, wherein the antibody that binds PD-Ll is atezolizumab (RG7446;

MPDL3280A), MEDI4736, BMS-936559 (MDX-1105), avelumab (MSB0010718C), KD033, the antibody portion of KD033, or STI-A1014.

55. The method of claim 46, wherein the CTLA-4 antagonist is an antibody that specifically binds CTLA-4.

56. The method of claim 55, wherein the antibody that binds CTLA-4 is ipilimumab (YERVOY;

MDX-010, BMS-734016) or tremelimumab (CP-675,206; ticilimumab).

57. The method of claim 46, wherein the CTLA-4 antagonist comprises a soluble CTLA-4 receptor.

58. The method of claim 57, wherein the CTLA-4 antagonist is KAHR-102.

59. The method of claim 46, wherein the LAG3 antagonist is an antibody that specifically binds LAG3.

60. The method of claim 59, wherein the antibody that binds LAG3 is IMP701, BMS-986016,

LAG525, GSK2831781, or IMP731.

61. The method of claim 46, wherein the LAG3 antagonist comprises a soluble LAG3 receptor.

62. The method of claim 61, wherein the LAG3 antagonist is IMP321.

63. The method of claim 46, wherein the Tim -3 antagonist is an antibody that binds Tim-3.

64. The method of claim 46, wherein the TIGIT antagonist is an antibody that binds TIGIT.

65. The method of claim 46, wherein the KIR antagonist is an antibody that specifically binds KIR.

66. The method of claim 65, wherein tlie antibody that binds KIR is lirilumab.

67. The method of claim 45, wherein the immune checkpoint modulator is an immune checkpoint enhancer or stimulator.

68. The method of claim 67, wherein the immune checkpoint enhancer or stimulator is a CD28

agonist, a 4-lBB agonist, an OX40 agonist, a CD27 agonist, a CD80 agonist, a CD86 agonist, a CD40 agonist, or a GITR agonist.

69. Tlie method of claim 68, wherein tlie OX40 agonist comprises OX40 ligand, or an OX40-binding portion thereof.

70. The method of claim 69, wherein the OX40 agonist is MEDI6383.

71. The method of claim 68, wherein the OX40 agonist is an antibody that specifically binds OX40.

72. The method of claim 71, wherein the antibody that binds OX40 is MEDI6469, MEDI0562, or MOXR0916 (RG7888).

73. The method of claim 68, wherein tlie OX40 agonist is a vector capable of expressing OX40 ligand.

74. The method of claim 68, wherein the OX40 agonist is Delta-24-RGDOX or DNX2401.

75. The method of claim 68, wherein the 4-lBB agonist is PRS-343.

76. The method of claim 68, wherein the 4-lBB agonist is an antibody that specifically binds 4-lBB.

77. The method of claim 76, wherein the antibody that binds 4-lBB is PF-2566 (PF-05082566) or urelumab (BMS-663513).

78. The method of claim 68, wherein the CD27 agonist is an antibody that specifically binds CD27.

79. The method of claim 78, wherein the antibody that binds CD27 is varlilumab (CDX-1 127).

80. The method of claim 68, wherein the GITR agonist comprises GITR ligand or a GITR-binding portion thereof.

81. The method of claim 68, wherein the GITR agonist is an antibody that specifically binds GITR.

82. The method of claim 81 , wherein the antibody that binds GITR is TRX518, MK-4166, or

INBRX-110.

83. The method of any one of claims 1-43, wherein the immunotherapeutic agent is a cytokine.

84. The method of claim 83, wherein the cytokine is a chemokine, an interferon, an interleukin, lymphokine, or a member of the tumor necrosis factor family.

85. The method of claim 83, wherein the cytokine is IL-2, IL 15, or interferon-gamma.

86. The method of any one of claims 1 and 8-85, wherein the cancer is selected from the group

consisting of lung cancer, pancreatic cancer, breast cancer, colon cancer, colorectal cancer, melanoma, gastrointestinal cancer, gastric cancer, renal cancer, ovarian cancer, liver cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, neuroblastoma, glioma, glioblastoma, glioblastoma multiforme, cervical cancer, stomach cancer, bladder cancer, head and neck cancer, and hepatoma.

87. The method of any one of claims 2, 4-6, and 9-85, wherein the tumor is selected from the group consisting of lung tumor, pancreatic tumor, breast tumor, colon tumor, colorectal tumor, melanoma, gastrointestinal tumor, gastric tumor, renal tumor, ovarian tumor, liver tumor, endometrial tumor, kidney tumor, prostate tumor, thyroid tumor, neuroblastoma, glioma, glioblastoma, glioblastoma multiforme, cervical tumor, stomach tumor, bladder tumor, head and neck tumor, and hepatoma.

88. The method of any one of claims 1, 2, 4-6, and 8-87, wherein the subject's cancer or tumor does not respond to an immune checkpoint inhibitor.

89. The method of any one of claims 1, 2, 4-6, and 8-87, wherein the subject's cancer or tumor has progressed following an initial response to an immune checkpoint inhibitor.

90. The method of claim 88 or 89, where the immune checkpoint inhibitor is PD-1 antagonist or PD- Ll antagonist therapy.

91. The method of any one of claims 1-90, wherein the subject is a human.