USO09598497B2

(12) United States Patent (10) Patent No.: US 9,598.497 B2 Gurney et al. (45) Date of Patent: Mar. 21, 2017

(54) RSPO3 BINDING AGENTS AND USES 7,319,141 B2 1/2008 Tang et al. THEREOF 7,320,880 B2 1/2008 Nishikawa et al. 7,411,052 B2 8/2008 Tang 7,439,332 B2 10/2008 Nishikawa (71) Applicant: OncoMed Pharmaceuticals, Inc., 7,498.416 B2 3/2009 Yayon et al. Redwood City, CA (US) 7,541,431 B2 6/2009 Yoon et al. 7,723, 112 B2 5, 2010 Clarke et al. (72) Inventors: Austin L. Gurney, San Francisco, CA 7,951,381 B2 5/2011 Funk et al. (US); Christopher J. Bond, San 8,088,374 B2 1/2012 Niehrs et al. Mateo, CA (US) 8, 158,757 B2 4/2012 Gurney et al. s 8, 158,758 B2 4/2012 Gurney 8,540,989 B2 9/2013 Gurney (73) Assignee: OncoMed Pharmaceuticals, Inc., 8,628,774 B2 1/2014 Gurney et al. Redwood City, CA (US) 8,802,097 B2 8/2014 Gurney et al. 8,883,736 B2 11/2014 Gurney c - r 9,040,044 B2 5/2015 Gurney et al. (*) Notice: patentSt. 1s extendedE. site or adjusted th still under 9,109,0249,109,025 B2 8/2015 Gurney et al. U.S.C. 154(b) by 0 days. 9,181,333 B2 11/2015 Gurney et al. 2002fOO65394 A1 5, 2002 Jacobs et al. (21) Appl. No.: 14/870,145 2003/00222 17 A1 1/2003 Ceccardi et al. 2003. O100741 A1 5/2003 Muller et al. 1-1. 2004/O197778 A1 10, 2004 Morris et al. (22) Filed: Sep. 30, 2015 2005/0054829 A1 3/2005 Wiley et al. O O 2005. O1301.45 A1 6/2005 Yue et al. (65) Prior Publication Data 2005/0142600 A1 6/2005 Warren et al. 2005/0232927 A1 10, 2005 Clarke et al. US 2016/0108130 A1 Apr. 21, 2016 2005/0256036 A1 1 1/2005 Boyle et al.

Relatedelated U.S. Application DatUata (Continued) (62) Division of application No. 13/940,834, filed on Jul. FOREIGN PATENT DOCUMENTS 12, 2013, now Pat. No. 9, 181,333. CA 2691378 A1 1, 2009 (60) Provisional application No. 61/826,747, filed on May DE 1033982O d 1 ti 23, 2013, provisional application No. 61/789,156, (Continued) filed on Mar. 15, 2013, provisional application No. 61/753,184, filed on Jan. 16, 2013, provisional OTHER PUBLICATIONS application No. 61/671,421, filed on Jul. 13, 2012. Clevers et al., Cell 2006; 127: 469-480.* Al-Haj. M., et al., “Prospective Identification of Tumorigenic (51) Int. Cl. Breast Cancer Cells.” Proceedings of the National Academy of A 6LX 39/395 (2006.01) Sciences 100(7):3983-3988, The National Academy of Sciences, C07K 6/30 (2006.01) Rmagro, sal J.C. (e. and Fransson, J., “H“Humanization o f Antiboditibodies.” C07K 6/8 (2006.01) Frontiers in Bioscience 13:1619-1633, Frontiers in Bioscience A6 IK 45/06 (2006.01) Publications, United States (2008). CI2O I/68 (2006.01) Anonymous, “Human R-Spondin 2 Antibody. Antigen Affinity C07K 6/24 ( 2006.O1 ) purified Polyclonal Goat IgG, Catalog No. AF3266,” R&D Systems, A61 K 39/00 (2006.01) Tools for Cell Biology Research, accessed at http://www. (52) U.S. Cl rindsystems.com.pdf AF3266.pdf, accessed on Jun. 23, 2010, 1 AV e. we page. CPC ...... C07K 16/30 (2013.01); A61K 39/39533 Aubele, M. and Werner, M., “Heterogeneity in Breast Cancer and (2013.01); A61K 39/39558 (2013.01); A61 K the Problem of Relevance of Findings,” Analytical Cellular Pathol 45/06 (2013.01); C07K 16/18 (2013.01); C07K ogy 19(2):53-58, IOS Press, Netherlands (1999). I6/24 (2013.01); C12O I/6886 (2013.01); (Continued) A61 K 2039/505 (2013.01); C07K 231 7/24 Primary Examiner — Jessica H Roark (2013.01); C07K 23.17/34 (2013.01); C07K (74) Attorney, Agent, or Firm — Sterne, Kessler, 2,939;(2013.01); C12OR. 2600/158 C.3% (2013.01) (57)Goldstein and Fox, PLLC.ABSTRACT (58) Field of Classification Search The present invention relates to RSPO-binding agents, par CPC CO7K 16/30; A61 K 39/395; A61 K 39/39533; ticularly RSPO3-binding agents and methods of uS1ng the A61K 39/39558 agents for treating diseases such as cancer. The present S lication file f 1 h hi invention provides antibodies that specifically bind human ee application file for complete search history. RSPO3 proteins and modulate B-catenin activity. The pres ent invention further provides methods of using agents that (56) References Cited modulate the activity of RSPO3 proteins and inhibit tumor growth. Also described are methods of treating cancer U.S. PATENT DOCUMENTS comprising administering a therapeutically effect amount of an agent or antibody of the present invention to a patient 6,004,528 A 12/1999 Bergstein 6.824,973 B2 11/2004 Tang et al. having a tumor or cancer. 7,193,069 B2 3/2007 Isogai et al. 21 Claims, 20 Drawing Sheets US 9,598.497 B2 Page 2

(56) References Cited WO WO-0212447 A2 2/2002 WO WO-02102972 A2 12/2002 U.S. PATENT DOCUMENTS WO WO-O3O2.9405 A2 4/2003 WO WO-030505O2 A2 6, 2003 2005/0256044 A1 1 1/2005 Boyle et al. WO WO-03054152 A2 7, 2003 2006/00 1925.6 A1 1/2006 Clarke et al. WO WO-2004.005457 A2 1/2004 2006/0057149 A1 3f2006 Johnson et al. WO WO-2004074436 A2 9, 2004 2006, O149049 A1 7/2006 Tang WO WO-2004O98521 A2 11/2004 2006/0263803 Al 11/2006 Tang WO WO-2005040418 A2 5, 2005 2006/0275870 A1 12, 2006 Gu WO WO-2005040828 A2 5, 2005 2007/0042359 A1 2/2007 Throsby et al. WO WO-2006110581 A2 10, 2006 2007/0117751 A1 5/2007 Gurney et al. WO WO-2007.096.149 A1 8/2007 2007/O124581 A1 5, 2007 Khare et al. WO WO-2008020942 A2 2/2008 2007/0237770 A1 10, 2007 Lai et al. WO WO-2008.042236 A2 4, 2008 2007/0244061 A1 10, 2007 Niehrs et al. WO WO-2008046649 A1 4, 2008 2008.0038257 A1 2/2008 Han et al. WO WO-2008075796 A1 6, 2008 2008/0064049 A1 3f2008 Clarke et al. WO WO-2008O88524 A2 7, 2008 2008/0108.565 A1 5/2008 Winston, Jr. et al. WO WO-200900.5809 A2 1/2009 2008/0286261 A1 11/2008 Morgan et al. WO WO-2009045443 A2 4, 2009 2008/0306004 A1 12/2008 Tang WO WO-2010016766 A2 2/2010 2009.0036369 A1 2/2009 Kakitani et al. WO WO-2010121923 A1 10, 2010 2009/0074782 A1 3/2009 Gurney WO WO-2011076932 A1 6, 2011 2009/0118176 A1 5/2009 Emtage et al. WO WO-2012092336 A2 7, 2012 2009/0191205 A1 7/2009 Gurney et al. WO WO-2012140274 A2 10, 2012 2009/0208484 A1 8/2009 Christiano WO WO-2013012747 A1 1/2013 2009, 0220495 A1 9, 2009 Fanidi et al. WO WO-2013149159 A1 10, 2013 2010/0003258 A1 1/2010 Weng et al. WO WO-2014012007 A2 1/2014 2010.00472.57 A1 2/2010 Blanc et al. WO WO-2014165232 A1 10, 2014 2010, 0071078 A1 3, 2010 Niehrs et al. WO WO-2014 192974 A1 12/2014 2010/0278800 Al 11/2010 Boyle et al. WO WO-2015,058132 A2 4, 2015 2010/0292 155 A1 1 1/2010 Tang WO WO-201609.0024 A2 6, 2016 2010/033.0089 A1 12/2010 Damle et al. 2011/O176995 A1 7, 2011 Funahashi 2011/0287444 A1 11/2011 Kanamori et al. Beachy, P.A., et al., “Tissue Repair and Stem Cell Renewal in 2012/00399.12 A1 2/2012 Rawadi et al. Carcinogenesis.” Nature 432(7015):324-331, Nature Publishing 2012,0082659 A1 4, 2012 Land et al. Group, England (2004). 2012fOO88727 A1 4/2012 Niehrs et al. Beerman, H. et al., “Flow Cytometric Analysis of DNA Stemline 2012/0114646 A1 5/2012 Tchessalov et al. Heterogeneity in Primary and Metastatic Breast Cancer,” Cytometry 2012/O16527O A1 6, 2012 Choi et al. 12(2): 147-154, Wiley-Liss, Inc., United States (1991). 2012/0171226 A1 T/2012 Horwitz Bienz, M. and Clevers, H., “Linking Colorectal Cancer to Wnt 2012/01846.16 A9 7, 2012 Rabbani et al. Signaling,” Cell 103(2):311-320, Cell Press, United States (2000). 2012fO1896.39 A1 7/2012 Schebye et al. Bonsing, B.A., et al., “Allelotype Analysis of Flow-Sorted Breast 2012/0263730 A1 10, 2012 Niehrs et al. Cancer Cells Demonstrates Genetically Related Diploid and 2012,032.9994 A1 12, 2012 Chen et al. 2013/00951.16 A1 4/2013 Gurney et al. Aneuploid Subpopulations in Primary Tumors and Lymph Node 2013/0115206 A1 5/2013 Gurney et al. Metastases.” Genes, Chromosomes and Cancer 28(2):173-183. 2013/O121993 A1 5/2013 Gurney Wiley-Liss, Inc., United States (2000). 2013/0209473 A1 8/2013 De Sauvage et al. Bonsing, B.A., et al. “High Levels of DNA Index Heterogeneity in 2013/0336885 Al 12/2013 Hongo et al. Advanced Breast Carcinomas. Evidence for DNA ploidy Differ 2013/0337533 A1 12, 2013 Niehrs et al. ences between Lymphatic and Hematogenous Metastases, Cancer 2014, OO17253 A1 1/2014 Gurney et al. 71(2):382-391, American Cancer Society, United States (1993). 2014/O127223 A1 5/2014 Yamazaki et al. Boyden, L.M., et al., “High Bone Density Due to a Mutation in 39.854 A. 3.3. Sty al LDL-Receptor Related Protein 5” The New England Journal of 2014/0302054 A1 10, 2014 E.eVeS et al.a Miss 2013-1521, Massachussetts Medical Society, 2014/0328859 A1 11/2014 Cong et al. B. ed States ( 3' & 8 2015, 0147333 A1 5, 2015 Storm et al. rennan, K.R., an Brown, A.M.C., “Wnt Proteins in Mammary 2015,0165024 A1 6/2015 Gurney Development and Cancer,” Journal of Mammary Gland Biology and 2016,000.0780 A1 1/2016. An Neoplasia 9(2): 119-131, Kluwer Academic/Plenum Publishers, 2016,0152947 A1 6, 2016 PioSZak United States (2004). Cabrera, C.V., et al., “Phenocopies Induced with Antisense RNA FOREIGN PATENT DOCUMENTS Identify the Wingless Gene.” Cell 50(4):659-663, Cell Press, United States (1987). EP 2157.192 A1 2, 2010 Campbell, A.M.. “Characterisation of monoclonal antibodies,” in EP 1673.475 B1 4/2010 Monoclonal Antibody Technology The Production and Character EP 1427747 B1 4/2012 ization of Rodent and Human Hybridomas, Chapter 10, pp. 186 EP 2997.975 A1 3, 2016 215, Elsevier Science Publishers B.V. The Netherlands (1984). JP 2010.532169 A 10, 2010 Campbell, A.M., “General Properties and Application of Monoclo WO WO-98493O2 A1 11F1998 nal Antibodies,” in Monoclonal Antibody Technology The Produc W W833. A. tion and Characterization of Rodent and Human Hybridomas, WO WO-0021555 A1 4/2000 1, pp. 1-32, , Elsevier Science Publishers B.V., Amsterdam WO WO-0107611 A2 2/2001 (1984). & 8 WO WO-O157190 A2 8, 2001 Carmon, K.S., et al., “R-Spondings Function as Ligands of the WO WO-0177169 A2 10/2001 Orphan Receptors LGR4 and LGR5 to Regulate Wnt/B-Catenin WO WO-01873.38 A1 11 2001 Signaling.” Proceedings of the National Academy of Sciences WO WO-0188092 A2 11/2001 108(28): 11452-11457, National Academy of Sciences, United WO WO-O 1922.97 A2 12/2001 States (2011). US 9,598.497 B2 Page 3

(56) References Cited Gradl, D., et al., “The Wnt/Wg Signal Transducer Beta-Catenin Controls Fibronectin Expression.” Molecular Cell Biology OTHER PUBLICATIONS 19(8):5576-5587, Microbiology, United States (1999). Gura, T., "Systems for Identifying New Drugs are often Faulty.” Carter, P.J., “Potent Antibody Therapeutics by Design.” Nature Science 278(5340): 1041-1042, American Association for the Reviews, Immunology 6(5):343-357, Nature Pub. Group, England Advancement of Science, United States (1997). (2006). Gurney, A., et al., “Inhibition of R-spondin (RSPO) signaling Casset, F., et al., “A Peptide Mimetic of an Anti-CD4 Monoclonal reduces the growth of multiple human tumors.” AACR. Annual Antibody by Rational Design.” Biochemical and Biophysical Meeting 2014, Abstract 1764, Apr. 5-9, 1 pages (2014). Research Communications 307(1): 198-205, Academic Press, Harada, N., et al., “Intestinal Polyposis in Mice with a Dominant United States (2003). Chen, J.Z., et al., “Cloning and Identification of a cDNA that Stable Mutation of the B-catenin Gene.” European Molecular Biol encodes a Novel Human Protein with Thrombospondin Type I ogy Organization Journal 18(21):5931-5942, Wiley-Blackwell, Inc., Repeat Domain, hPWTSR,” Molecular Biology Reports 29:287 England (1999). 292, Kluwer Academic Publishers, Netherlands (2002). Hartmann, C., “Wnt-Signaling and Skeletogenesis,” Journal of Chen, Y., et al., “Selection and Analysis of an Optimized Anti Musculoskelet & Neuronal Interactions 2(3):274-276, International VEGF Antibody: Crystal Structure of an Affinity-matured Fab in Society of Musculoskeletal and Neuronal Interactions, Greece Complex with Antigen.” Journal of Molecular Biology 293(4):865 (2002). 881, Academic Press, England (1999). Hatsell, S., et al., “Beta-Catenin and Tcfs in Mammary Develop Chien, A.J., et al., “Activated Wnt/B-Catenin Signaling in Mela ment and Cancer,” Journal of Mammary Gland Biology and noma is Associated with Decreased Proliferation in Patient Tumors Neoplasia 8(2): 145-158, Kluwer Academic/Plenum Publishers, and a Murine Melanoma Model.” Proceeding of the National United States (2003). Academy of Sciences 106(4): 1193-1198, National Academy of He, T.C., et al., “Identification of c-MYC as a Target of the APC Sciences, United States (2009). Pathway,” Science 281 (5382): 1509-1512, American Association for De Genst, E., et al., “Antibody Repertoire Development in the Advancement of Science, United States (1998). Camelids.” Developmental and Comparative Immunology 30(1- Horesh, Y., et al., “A Rapid Method for Detection of Putative RNAi 2): 187-198, Elsevier Science, United States (2006). Target Genes in Genomic Data.” Bioinformatics 19(Suppl 2):ii73 De Pascalis, R., et al., “Grafting of Abbreviated Complementarity Determining Regions Containing Specificity-Determining Residues i80, Oxford University Press, England (2003). Essential for Ligand Contact to Engineer a Less Immunogenic Hsu, S.Y., et al., “Activation of Orphan Receptors by the Hormone Humanized Monoclonal Antibody,” Journal of Immunology Relaxin,” Science 295:671-674, American Association for the 169(6):3076-3084. The American Association of Immunologists, Advancement of Science, United States (2002). United States (2002). Imbert, A., et al., “Delta N89 Beta-Catenin induces Precocious Dermer, G.B., “Another Anniversary for the War on Cancer.” Development, Differentiation, and Neoplasia in Mammary Gland.” Biotechnology 12:320, Nature Publishing Co., United States The Journal of Cell Biology 153(3):555-568, Rockerfeller Univer (1994). sity Press, United States (2001). Easwaran, V., et al., “Beta-Catenin Regulates Vascular Endothelial International Preliminary Report on Patentability for International Growth Factor Expression in Colon Cancer.” Cancer Research Application No. PCT/US2008/008210, the International Bureau of 63(12):3145-3153, American Association for Cancer Research, WIPO, Geneva, Switzerland, mailed Jan. 5, 2010, 9 pages. United States (2003). International Search Report for International Application No. PCT/ English language Abstract of DE 10339820A1, espacenet database, IS2008/008210, European Patent Office, Netherlands, mailed on Worldwide published Mar 17, 2005. Mar. 2, 2009, 6 pages. English language Abstract of JP2010-532169A, espacenet database, International Search Report for International Application No. PCT/ Worldwide published Jan. 8, 2009. US2012/046746, the International Searching Authority, Alexandria, European Opposition Brief for European Patent No. 2,157,192 filed mailed on Oct. 23, 2012, 3 pages. May 8, 2014, 46 pages. International Search Report for International Application No. PCT/ Extended European Search Report for EP Application No. US2013/050300, from the International Bureau of WO, Geneva 12814264, European Patent Office, Germany, mailed on January 28, Switzerland, mailed Feb. 7, 2014, 7 pages. 2015, 7 pages. Jackson, A.L. and Linsley, P.S., “Noise Amidst the Silence: Off Fischer, L., et al., “Wnt-3A Enhances Bone Morphogenetic Protein Target Effects of siRNAs?,” Trends in Genetics 20011):521-524, 2-Mediated Chondrogenesis of Murine C3H10T1/2 Mesenchymal Elsevier Science Publishers B.V., Netherlands (2004). Cells.” The Journal of Biological Chemistry 277(34):30870-30878, Jain, R.L., “Barriers to Drug Delivery in Solid Tumors,” Scientific JBC Papers in Press, American Society for Biochemistry and American 271(1):58-65, Scientific American, Inc., United States Molecular Biology, United States (2002). (1994). Freshney, R.I., "Culture of Animal Cells.” A Manual of Basic Jemal, A., et al., “Cancer Statistics, 2003, CA-A Cancer Journal Technique 4, Alan R. Liss, Inc., United States (1983). for Clinicians 53(1):05-26, American Cancer Society, United States Fujino, T., et al., “Low-Density Lipoprotein Receptor-Related Pro (2003). tein 5 (LRP5) is Essential for Normal Cholesterol Metabolism and Kamata, T., et al., “R-Spondin, A Novel Gene with Thrombospondin Glucose-Induced Insulin Secretion.” Proceedings of the National Type 1 Domain, was Expressed in the Dorsal Neural Tube and Academy of Sciences 100(1):229-234, National Academy of Sci Affected in Wnts Mutants.” Biochimica et Biophysica Acta ences, United States (2003). 1676(1):51-62, Elsevier Pub. Co., Netherlands (2004). Gazit A. et al., “Human Frizzled 1 Interacts with Transforming Kazanskaya, O., et al., “R-Spondin2 is a Secreted activator of Wnts to Transduce a TCF Dependent Transcriptional Response.” Wnt/.beta.-Catenin Signaling and is required for Xenopus Ocogene 18(44):5959-5966, Nature Publishing Group, England Myogenesis.” Developmental Cell 7:525-534, Cell Press, United (1999). States (2004). Goldblum, S.E., et al., “Thrombospondin-1 Induces Tyrosine Kim, K.A., et al., “Mitogenic Influence of Human R-Spondin1 on Phosphorylation of Adherens Junction Proteins and Regulates an the Intestinal Epithelium.” Science 309(5738): 1256-1259, Ameri Endothelial Paracellular Pathway,” Molecular Biology of the Cell can Association for the Advancement of Science, United States 10(5):1537-1551. The American Society for Cell Biology, United (2005). States (1999). Kim, K.A., et al., “R-Spondin Family Members Regulate the Wnt Gong, Y, et al., “LDL Receptor-Related Protein 5 (LRP5) Affects Pathway by a Common Mechanism.” Molecular Biology of the Cell Bone Accrual and Eye Development,” Cell 107(4):513-523, Cell 19:2588-2596, The American Society for Cell Biology, United Press, United States (2001). States (2008). US 9,598.497 B2 Page 4

(56) References Cited Nam, J.S., et al., “Mouse Cristin/R-Spondin Family Proteins are Novel Ligands for the Fizzled 8 and LRP6 Receptors and Activate OTHER PUBLICATIONS Beta-Catenin-Dependent Gene Expression.” The Journal of Bio logical Chemistry 281 (19): 13247-13257, American Society for Bio Kim, K.A., et al., “R-Spondin Proteins: A Novel Link to Beta chemistry and Molecular Biology, United States (2006). Catenin Activation.” Cell Cycle 5(1):23-26, Landes Bioscience, Non-Final Office Action mailed Jun. 24, 2011 for U.S. Appl. No. United States (2006). 12/167,176, Gurney, A., filed Jul. 2, 2008. Korinek, V., et al., “Constitutive Transcriptional Activation by a Nusse, R. and Varmus, H.E., “Many Tumors Induced by the Mouse Beta-Catenin-Tcf Complex in APC -f- Colon Carccinoma,” Sci Mammary Tumor Virus contain a Provirus Integrated in the Same ence 275(5307):1784-1787, American Association for Advance ment of Science, United States (1997). Region of the Host Genome.” Cell 31(1):99-109, Cell Press, United Kumar, K.K., et al., “Structure and Function of LGR5: An Enig States (1982). matic G-Protein Coupled Receptor Marking Stem Cells.” Protein Non-Final Office Action mailed Feb. 24, 2015 for U.S. Appl. No. Science 23:551-565, Wiley-Blackwell, United States (2014). 13/940,834, Gurney, A.L., et al., filed Jul. 12, 2013. Kuukasjrvi, P. et al., “Overview of Systematic Reviews on Invasive OncoMEd Pharmaceuticals Press Release, “OncoMed Presents Treatment of Stable Coronary Artery Disease.” The International Data on Clinical and Preclinical Anti-Cancer StemCell Programs at Journal of Technology Assessment in Health Care 22(2):219-234, American Association for Cancer Research Annual Meeting.” Apr. Cambridge University Press, England (2006). 21, 2015, 3 pages. Larue, L. and Delmas, V., “The Wnt/Beta-Catenin Pathway in OncoMed Pharmaceuticals, Press Release, "OncoMed Presents Melanoma,” Frontiers in Bioscience 11:733-742, Frontiers in Data on Multiple Anti-Cancer Stem Cell Programs at American Bioscience Publications, United States (2006). Association for Cancer Research Annual Meeting.” Apr. 8, 2014, 4 Li, S.J., et al., "Loss-of-Function Point Mutations and Two-Furin pageS. Domain Derivatives Provide Insights about R-Spondin2 Structure OncoMed Pharmaceuticals Press Release, "OncoMed to Present and Function.” Cellular Signalling 21(6): 916-925, Elsevier Science Data on Multiple Anti-Cancer StemCell Candidates at the Ameri Ltd, England (2009). can Association of Cancer Research Meeting.” Mar. 19, 2015, 2 Lonberg, N., “Human Antibodies from Transgenic Animals.” pageS. Nature Biotechnology 23(9): 1117-1125, Nature Publishing Group, Oshima, H., et al., “Morphological and Molecular Processes of United Kingdom (2005). Polyp Formation in ApcA716 Knockout Mice.” Cancer Research Luo, C-W., et al., “Genomic Analyses of the Evolution of LGR Genes,” Chang Gung Med J 29(1):2-8, Chau-Xiong Zhang, China 57(9): 1644-1649. The American Association for Cancer Research, (2006). United States (1997). Lulu, H.H., et al., “Wnt/beta-Catenin Signaling Pathway as Novel Pandis, N., et al., "Cytogenetic Comparison of Primary Tumors and Cancer Drug Targets.” Current Cancer Drug Targets 4:653-671, Lymph Node Metastases in Breast Cancer Patients.” Genes, Chro Bentham Science Publishers, Netherlands (2004). mosomes & Cancer 22:122-129, Wiley-Liss, Inc., United States MacCallum, R.M., et al., “Antibody-Antigen Interactions: Contact (1998). Analysis and Binding Site Topography,” Journal of Molecular Paul, W.E., "Structure and Function of Immunoglobulins,” in Fun Biology 262(5):732-745, Elsevier, England (1996). damental Immunology, Third Edition, pp. 292-295, Raven Press, Mazerbourg, S., et al., "Leucine-Rich Repeat-Containing, G Pro New York, United States (1993). tein-Coupled Receptor 4 Null Mice Exhibit Intrauterine Growth Perantoni, A.O., “Renal Development: Perspectives on a Wnt Retardation Associated with Embryonic and Perinatal Lethality.” Dependent Process.” Seminars in Cells and Development Biology Molecular Endocrinology 18(9):2241-2254. The Endocrine Society, Abstract, 1 page, Academic Press, United Kingdom (2003). United States (2004). Polakis, P. “Wnt signaling and cancer.” Genes & Development McClanahan, T., et al., “Identification of Overexpression of Orphan 14:1837-1851, Cold Spring Harbor Laboratory Press, United States G Protein-Coupled Receptor GPR49 in Human Colon and Ovarian (2000). Primary Tumors.” Cancer Biology and Therapy 5(4):419-426, Polesskaya, A., et al., “Wnt Signaling Induces the Myogenic Speci Landes Bioscience, United States (2006). fication of Resident CD45+ Adult StemCells during Muscle Regen Meniel, V. and Clarke, A.R., “Wnt-Cadherin Connections in Normal eration.” Cell Abstract, 1 page, MIT Press, United States (2003). and Neoplastic Mammary Epithelium.” Journal of Mammary Gland Reply to European Opposition Brief for European Patent No. Biology and Neoplasia 8(4):435-447, Kluwer Academic/Plenum 2157192 filed Jan. 8, 2015, 42 pages. Publishers, United States (2003). Response to European Opposition Brief Reply for European Patent Michaelson, J.S., and Leder, P., "Beta-Catenin is a Downstream No. 2157192, filed May 14, 2015, 14 pages. Effector of Wnt-Mediated Tumorigenesis in the Mammary Gland.” Reya, T. and Clevers, H., “Wnt Signaling in Stem Cells and Oncogene 20(37):5093-5099, Nature Publishing Group, England Cancer,” Nature 434(7035):843-850, Nature Publishing Group, (2001). England (2005). Miller, J.R., et al., “Mechanism and Function of Signal Transduction Rijsewijk, F., et al., “The Drosophila Homolog of the Mouse by the Wnt/B-catenin and Wnt/Ca2+ Pathways.” Oncogene Mammary Oncogene int-1 is Identical to the Segment Polarity Gene 18(55):7860-7872, Nature Publishing Group, England (1999). wingless,” Cell 50(4):649-657, Cell Press, United States (1987). Milovanovic, T., et al., “Expression of Wnt Genes and Frizzled 1 Rudikoff, S., et al., “Single Amino Acid Substitution Altering and 2 Receptors in Normal Breast Epithelium and Infiltrating Breast Antigen-Binding Specificity.” Proceedings of the National Acad Carcinoma.” International Journal of Oncology 25(5): 1337-1342, emy of Sciences 79(6):1979-1983, The National Academy of Sci D.A. Spandidos, Greece (2004). ences, United States (1982). Morita, H., et al., "Neonatal Lethality of LGR5 Null Mice is Saadi-Kheddouci, S., et al., “Early Development of Polycystic Associated with Ankyloglossia and Gastrointestinal Distension.” Kidney Disease in Transgenic Mice Expressing an Activated Mutant Molecular Cell Biology 24(22):9736-9743, American Society for of the Beta-Catenin Gene.” Oncogene 20(42):5972-5981, Nature Microbiology, United States (2004). Publishing Group, England (2001). Morrison, S.J., et al., “Hematopoietic Stem Cells: Challenges to Shen, C.Y., et al., “Genome-Wide Search for Loss of Heterozygos Expectations.” Current Opinion in Immunology 9(2):216-221. ity. Using Laser Capture Microdissected Tissue of Breast Carci Elsevier Science, United States (1997). noma: An Implication for Mutator Phenotype and Breast Cancer Morrison, S.J.,et al., “Regulatory Mechanisms in Stem Cell Biol Pathogenesis.” Cancer Research 60(14):3884-3892, American ogy,” Cell 88(3):287-298, Elsevier Science, United States (1997). Association of Cancer Research, United States (2000). Morrison, S.J.,et al., “The Biology of Hematopoietic Stem Cells.” Smalley, M.J. and Dale, T.C., “Wnt Signalling in Mammalian Annual Review of Cell and Developmental Biology 11:35-71, Development and Cancer.” Cancer and Metastasis Reviews Annual Reviews, United States (1995). 18(2)215-230, Kluwer Academic Publishers, Netherlands (1999). US 9,598.497 B2 Page 5

(56) References Cited De Lau, W., et al., “Lgr5 Homologues Associate with Wnt Recep tors and Mediate R-spondin Signaling.” Nature 476(7360):293-297. OTHER PUBLICATIONS Macmillan Publishers Limited, England (2011) (D14) as cited in Opposition of EP2173379). Stump, R.J., et al., “A Role for Wnt/Beta-Catenin Signaling in Lens Hsu, S.Y., et al., “Characterization of Two LGR Genes Homologous Epithelial Differentiation.” Developmental Biology Abstract, 1 to Gonadotropin and Thyrotropin Receptors with Extracellular page, Elsevier Inc., United States (2003). Leucine-rich Repeats and a G protein-coupled. Seven Surendran, K., et al., “A Role for Wnt-4 in Renal Fibrosis,' Ameri transmembrane Region.” Molecular Endocrinology 12(12): 1830 can Journal of Physiology. Renal Physciology 282(3): F431-F441. 1845, Endocrine Society, United States (1998) (D29 as cited in American Physiological Society, United Sates (2002). Opposition of EP2173379). Takahashi-Yanaga, F. and Sasaguri, T., “The Wnt/.Beta.-Catenin LifeSpan BioSciences, Inc., Anti-GPR49/LGR5 Antibody (N-Ter Signaling Pathway as a Target in Drug Discovery,” Journal of minus) IHC-plus LS-A1232, Retrieved on May 31, 2016, 2 pages Pharmaceutical Sciences 104:293-302, The Japanese Phamacologi (D38 as cited in Opposition of EP2173379). cal society, Japan (2007). Ohkawara, B., et al., “Rspo3 Binds Syndecan 4 and Induces Tan, B.T., et al., “The Cancer Stem Cell Hypothesis: A Work in Wnt/PCP Signaling via Clathrin-Mediated Endocytosis to Promote Progress.” Laboratory Investigation 86(12): 1203-1207, USCAP, Morphogenesis.” Developmental Cell 20:303-314. Elsevier Inc., United States (2011). United States (2006). Response to attend Oral Proceeding in Opposition to European Tepera, S.B., et al., “A Beta-Catenin Survival Signal is required for Patent 2157192, Opponent OncoMed Pharmaceuticals, Inc., 22 Normal Lobular Development in the Mammary Gland,” Journal of pages, Dec. 18, 2015. Cell Science 116(Pt6): 1137-1149, Company of Biologists, England Sequence Alignment 1: EP2173379 B1 SEQID No. 13 (LGR5 PRT, (2003). top) vs. WO9915660 SEQ ID No. 2 (HG38 PRT, bottom) (D36 as Testsu, O. and McCormick, F. “Beta-Catenin Regulates Expression cited in Opposition of EP2173379), on Jun 2, 2016. of Cyclin D1 in Colon Carcinoma Cells,” Nature 398(6726):422 Sequence Alignment 2: EP2173379 B1 SEQID No. 13 (LGR5 PRT, 426, Nature Publishing Group, England (1999). top) vs. WO2004074436 SEQ ID No. 1 (HG38 PRT, bottom) (D37 Transmittal of third party observations sent on Dec. 17, 2014 in as cited in Opposition of EP2173379), on Jun. 2, 2016. European Application No. 08779934.2, 6 pages. Patent Owner's Response to the Notice of Opposition against UniProt “Thrombospondin-1” identifying No. P07996-TSP1 HU European Patent No. 2173379 B, filed on Jun. 2, 2016, Strawman MAN, accessed at http://www.uniprot.org/uniprot/P07996, Limited vs. OncoMed Pharmaceuticals, Inc., Submitted Nov. 15, accessed on Sep. 25, 2014, 14 pages. 2016, 37 pages. Van Ooyen, A. and Nusse, R., "Structure and Nucelotide Sequence Antagonists of R-spondin 3 for Treatment of Bone Disorders of the Putative Mammary Oncogene Int-1; Proviral Insertions Leave (P-881), dkfz, German Cancer Research Center in the Helmholtz Association, Jul. 12, 2 pages, Jul. 2012. the Protein-Encoding Domain Intact.” Cell 39(1):233-240, Cell Atwood, B.K., et al., “Expression of G Protein-coupled Receptors Press, United States (1984). and Related Proteins in HEK293, AtT20, BV2, and N18 Cell Lines Written Opinion of the International Searching Authority for Inter as Revealed by Microarray Analysis.” BMC Genomics 12:14, national Application No. PCT/US2008/008210, European Patent BioMed Central, England (2011). Office, Netherlands, mailed on Jan. 5, 2010, 8 pages. Chen, P.H., et al., “The Structural Basis of R-Spondin Recognition Written Opinion of the International Searching Authority for Inter by LGR5 and RNF43.” Genes & Development 27(12): 1345-1350 national Application No. PCT/US2012/046746, mailed Oct. 4. and Supplementary Information, Cold Spring Harbor Laboratory 2012, 7 pages. Press, United States (2013). Written Opinion of the International Searching Authority for Inter Cruciat, C.M. and Niehrs, C., “Secreted and Transmembrane wint national Application No. PCT/US2013/050300, mailed Feb. 7, Inhibitors and Activators,” Cold Spring Harbor perspectives in 2014, 11 pages. Biology 5(3):aO15081, Cold Spring Harbor Laboratory Press, Wu, C.-H... and Nusse, R., “Ligand Receptor Interactions in the Wnt United States (2013). Signaling Pathway in Drosophila,” The Journal of Biological De Lau, W.B., et al., “The R-Spondin Protein Family.” Genome Chemistry 277(44):41762-41769, American Society for Biochem Biology 13(3):242, BioMed Central Ltd, England (2012). istry and Molecular Biology, United States (2002). Dkfz, "R-spondins 2 and 3 as inhibitors of angiogenesis: Potential Wu, H., et al., “Humanization of a Murine Monoclonal Antibody by cancer therapeutics (P-732), Technology offer, 2 pages. Simultaneous Optimization of Framework and CDR Residues.” Glinka, A., et al., “LGR4 and LGR5 are R-Spondin Receptors Journal of Molecular Biology 294(1): 151-162, Elsevier, England Mediating Wnt/B-Catenin and Wnt/PCP Signalling.” EMBO (1999). Reports 12(10): 1055-1061, Wiley Blackwell, England (2011). Wu, W., et al., “Mutual Antagonism between Dickkopfl and Dick Hao, H.X., et al., “ZNRF3 Promotes Wnt Receptor Turnover in an kopf2 Regulates Wnt/Beta-Catenin Signalling.” Current Biology R-Spondin-Sensitive Manner,” Nature 485(7397): 195-200 and 10(24): 1611-1614, Elsevier Science Ltd., England (2000). Supplementary Information, Nature Publishing Group, England Zhao, J., et al., “R-spondin1, A Novel Intestinotrophic Mitogen, (2012). Ameliorates Experimental Colitis in Mice,” Gastroenterology Hendrickx, M. and Leyns, L., “Non-Conventional Frizzled Ligands 132(4): 1331-1334, W.B. Saunders, United States (2007). and Wnt Receptors.” Development, Growth & Differentiation Akhmetshina, A., et al., “Activation of Canonical Wnt Signalling is 50(4):229-243, Japanese Society of Developmental Biologists, Required for TGF-B-mediated Fibrosis,” Nature Communications Japan (2008). 3:735:1-12, Nature Publishing Group, England (2012). Jin, Y.R. and Yoon, J.K., “The R-Spondin Family of Proteins: Assignment from Austin Gurney to Oncomed Pharmaceuticals, Inc., Emerging Regulators of WNTSignaling.” The International Journal 2 pages (D12 as cited in Opposition of EP2173379), dated Jan. 2, of Biochemistry & Cell Biology 44(12):2278-2287. Elsevier, Neth 2009. erlands (2012). Blaydon, D.C., et al., “R-spondins in Cutaneous Biology: Nails and Niehrs, C., “The Complex World of WNT Receptor Signalling.” Cancer.” Cell Cycle 6(8):895-897, Taylor & Francis, United States Nature Reviews. Molecular Cell Biology 13(12):767-799, Nature (2007) (D19 as cited in Opposition of EP2173379). Publishing Group, England (2012). Cho, M., et al. “Cardamonin Suppresses Melanogenesis by Inhibi Schuijers, J. and Clevers, H., “Adult Mammalian Stem Cells: the tion of Wnt/B-catenin Signalin.” Biochemical and Biophysical Role of Wnt, Lgr5 and R-Spondins.” The EMBO Journal Research Communication 390:500-505, Elsevier Inc., United States 31(12):2685-2696, Wiley Blackwell, England (2012). (2009). Seshagiri, S., et al., “Recurrent R-spondin Fusions in Colon Can Crystal Image of LGR5 with Binding Sites Overlaid, 1 page (D35 cer,” Nature 488(7413):660-664, Nature Publishing Group, England as cited in Opposition of EP2173379) on Jun. 2, 2016. (2012). US 9,598.497 B2 Page 6

(56) References Cited Reya, T., et al., “A Role for Wnt Signalling in Self-renewal of Haematopoietic Stem Cells.” Nature 423 (6938):409–414, Nature OTHER PUBLICATIONS Publishing Group, England (2003). Sasaki, Y., et al., “Establishment of a novel monoclonal antibody Tanese, K., et al., “G-Protein-Coupled Receptor GPR49 is up against LGR5.” Biochemical and Biophysical Research Communi Regulated in Basal Cell Carcinoma and Promotes Cell Proliferation cations 394(3):498-502, Elsevier Inc., United States (2010). and Tumor Formation.” The American Journal of Pathology Statement of Facts and Arguments in Opposition of EP2081586B1, 173(3):835-843, Elsevier, United States (2008). filed Sep. 23, 2016, 39 pages. Tomaselli, S., et al., “Human RSPO1/R-Spondin1 is Expressed During Early Ovary Development and Augments f3-Catenin Sig Statement of Grounds of Appeal by Appellant, Offensive Opposition naling.” PloS one 6(1): e16366, Public Library of Science, United to European Patent No. 2157192, filed Sep. 1, 2016, 64 pages. States (2011). Storm, E.E., et al., “Targeting PTPRK-RSPO3 Colon Tumours Wang, D., et al., “Structural Basis for R-Spondin Recognition by Promotes Differentiation and Loss of Stem-cell Function.” Nature LGR4/5/6 Receptors.” Genes & Development 27(12): 1339-1344, 529(7584):97-100, Nature Publishing Group, England (2016). Cold Spring Harbor Laboratory Press, United States (2013). Walker, F., et al., “LGR5 Is a Negative Regulator of Tumourigenic Wei. Q., et al., “R-Spondin1 is a High Affinity Ligand for LRP6 and ity, Antagonizes Wnt Signalling and Regulates Cell Adhesion in Induces LRP6 Phosphorylation and Beta-Catenin Signaling.” The Colorectal Cancer Cell Lines.” PLoS One 6(7):e22733, Public Journal of Biological Chemistry 282(21): 15903-15911, American Library of Science, United States, 20 pages (2011). Society for Biochemistry and Molecular Biology, United States Yang, Y, et al., “Wnt5a and Wnt5b Exhibit Distinct Activities in (2007). Coordinating Chondrocyte Proliferation and Differentiation.” Yang, K., et al., “The Evolving Roles of Canonical WNT Signaling Development 130(5):1003-1015, Company of Biologists Limited, in Stem Cells and Tumorigenesis: Implications in Targeted Cancer England (2003). Therapies.” Laboratory Investigation 96(2):116-136, Nature Pub Adams, G.P. and Weiner, L.M., “Monoclonal Antibody Therapy of lishing Group, United States (2016). Cancer,” Nature Biotechnology 23(9): 1147-1157, Nature Publish Alberts, B., et al., Molecular Biology of the Cell, 4th Edition, ing Group, United States (2005) (D23 as cited in Opposition of EP Garland Science, Taylor & Francis Group, 2002, pp. 895-896, 2173379). 1282-1283, 1352 and 1358 (D7 as cited in Opposition of EP Akhmetshina, A., et al., “Activation of Canonical Wnt Signalling is 2081586 B1). Aoki, M., et al., “R-spondin3 is Required for Mouse Placental Required for TGF-B-mediated Fibrosis,” Nature Communications Development.” Developmental Biology 301(1):218-226, Elsevier, 3:735:1-12, Nature Publishing Group, England. United States (2007)(D6 as cited in Opposition of EP2081586 B1). Assignment from Austin Gurney to Oncomed Pharmaceuticals, Inc., Baljinnyam, B., et al., “Recombinant R-spondin2 and Wnt3a Up 2 pages (D12 as cited in Opposition of EP2173379). and Down-regulate Novel Target Genes in C57MG Mouse Mam Assignment of Austin Gurney to Oncomed Pharmaceuticals, mary Epithelial Cells.” PLoS One 7(1):e294.55, Public Library of Screenshot of USPTO Recordal Database, 2 pages, recorded on Jan. Science, United States (2012). 27, 2009 (D13 as cited in Opposition of EP2173379). Fafilek, B., et al., “Troy, a tumor necrosis factor receptor family Barker, N. and Clevers, H., “Mining the Wnt Pathway for Cancer member, interacts with Lgr5 to inhibit Wnt signaling in intestinal Therapeutics.” Nature Reviews/Drug Discovery 5(12):997-1014, stem cells,” Gastroenterology 144(2):381-391, AGA Institute, Nature Publishing Group, United States (2006) (D5 as cited in United States (2013). Opposition of EP2173379). Gong. X., et al., “LGR5-Targeted Antibody-Drug Conjugate Eradi Barker, N., et al., “Identification of Stem Cells in Small Intestine cates Gastrointestinal Tumors and Prevents Recurrence.” Molecular and Colon by Marker Gene Lgr5.” Nature 449(7165): 1003-1007, Cancer Therapeutics 15(7): 1580-1590, American Association for Nature Publishing Group, England (2007) (D33 as cited in Oppo Cancer Research, Inc., United States (2016). sition of EP2173379). Goodwin, A.M., and D'Amore, P.A., “Wnt Signaling in the Bergmann, C., et al., “Mutations in the Gene Encoding the Wnt Vasculature.' Angiogenesis 5(1-2): 1-9, Springer, Germany signaling Component R-spondin 4 (RSPO4) Cause Autosomal (2002)(D10 as cited in Opposition of EP 2081586 B1). Recessive Anonychia, American Journal of Human Genetics Junttila, M.R., et al., “Targeting LGR5" cells with an antibody-drug 79(6): 1105-1109, University of Chicago Press, United States (2006) conjugate for the treatment of colon cancer, Science Translational (D21 as cited in Opposition of EP2173379). Medicine 7(314):314ra186, American Association for the Advance Blaydon, D.C., et al., “R-spondins in Cutaneous Biology: Nails and ment of Science, United States, 12 pages (2015). Cancer.” Cell Cycle 6(8)895-897, Taylor & Francis, United States Junttila, M.R., et al., “Supplementary Materials for Targeting (2007) (D19 as cited in Opposition of EP2173379). LGR5+ cells with an antibody-drug conjugate for the treatment of Chartier, C., et al., “Therapeutic Targeting of Tumor-Derived colon cancer.” Science Translational Medicine 7(314):314ra186, R-Spondin Attenuates f-Catenin Signaling and Tumorigenesis in American Association for the Advancement of Science, United Multiple Cancer Types.” Cancer Research 76(3):713-723, American States, 17 pages (2015). Association for Cancer Research, United States (2016). Kobayashi, S., et al., “LGR5-positive colon cancer stem cells Cho, M., et al. “Cardamonin Suppresses Melanogenesis by Inhibi interconvert with drug-resistant LGR5-negative cells and are tion of Wnt/B-catenin Signaling.” Biochemical and Biophysical capable of tumor reconstitution.” Stem Cells 30(12):2631-2644, Research Communications 390:500-505, Elsevier Inc., United AlphaMed Press, United States (2012). States (2009). Kudryavtseva, E., et al., “Wnt Signaling Genes of Murine Chro Chua, A.W., et al., “The Role of R-spondin2 in Keratinocyte mosome 15 are Involved in Sex-affected Pathways of Inflammatory Proliferation and Epidermal Thickening in Keloid Scarring.” Jour Arthritis.” Arthritis and Rheumatism 64(4): 1057-1068, Wiley nal of Investigative Dermatology 131(3):644-654, Elsevier, United Blackwell, United States (2012). States (2011). Kwon, M.S., et al., "Leucine-rich repeat-containing G-protein Crystal Image of LGR5 with Binding Sites Overlaid, 1 page (D35 coupled receptor 5/GPR49 activates G-Rho GTPase pathway.” as cited in Opposition of EP2173379). Molecules and Cells 36(3):267-272. The Korean Society for De Lau, W., et al., “Lgr5 Homologues Associate with Wnt Recep Molecular and Cellular Biology, South Korea (2013). tors and Mediate R-spondin Signalling.” Nature 476(7360):293 Maeda, K., et al., “Wnt5a-Ror2 Signaling between Osteoblast 297, Macmillan Publishers Limited, England (2011) (D14 as cited lineage Cells and Osteoclast Precursors Enhances in Opposition of EP2173379). Osteoclastogenesis,” Nature Medicine 18(3):405-412, Nature Pub De Lau, W., et al., “The R-spondin/Lgr5/Rnf43 Module: Regulator lishing Company, United States (2012). of Wnt Signal Strength.” Genes and Development 28(4):305-316, Priority Document of the Patent (EP06022070.4) (D8 as cited in Cold Spring Harbor Laboratory Press, United States (2014) (D31 as Opposition of EP 2081586 B1), filed Oct. 20, 2006. cited in Opposition of EP2173379. US 9,598.497 B2 Page 7

(56) References Cited Platanias, L.C., “Soluble protein inhibitors of the TGF-B pathway.” in Cytokines and Cancer, Platanias, L.C., ed., p. 141, Springer OTHER PUBLICATIONS Science+Business Media, Inc., United States (2005) (D30 as cited in Opposition of EP2173379). Doring, E. et al., “Identification and Characterization of the TNFO. Response to attend Oral Proceedings in Opposition to European Antagonist Derived from a Monoclonal Antibody.” Molecular Patent 2157192, Opponent OncoMed Pharmaceuticals, Inc., 22 pages, Dec. 18, 2015. Immunology 31(14): 1059-1067. Elsevier Science Ltd., England Sequence Alignment 1: EP2173379 B1 SEQID NO:13 (LGR5 PRT, (1994). top) vs. WO9915660 SEQ ID NO:2 (HG38 PRT, bottom) (D36 as Guo, Y, et al., “Wnt/B-catenin Signaling: A Promising New Target cited in Opposition of EP2173379). for Fibrosis Diseases.” Physiological Research 61(4):337-346, Sequence Alignment 2: EP2173379 B1 SEQID NO:13 (LGR5 PRT, Praha, Czech Republic (2012). top) vs. WO2004074436 SEQ ID NO: 1 (HG38 PRT, bottom) (D37 Hsu, S.Y., et al., “Characterization of Two LGR Genes Homologous as cited in Opposition of EP2173379). to Gonadotropin and Thyrotropin Receptors with Extracellular Supplementary Information in De Lau, W., et al., “Lgr5 Leucine-rich Repeats and a G protein-Coupled, Seven Homologues Associate with Wnt Receptors and Mediate R-spondin transmembrane Region.” Molecular Endocrinology 12(12): 1830 Signalling.” Nature 476(7360):293-297. Nature Publishing Group, England (2011), 23 pages (D15 as cited in Opposition of EP 1845, Endocrine Society, United States (1998) (D29 as cited in 2173379). Opposition of EP2173379). Theodorou, V., et al., “MMTV Insertional Mutagenesis Identifies International Search Report for Application No. PCT/US2015/ Genes, Gene Families and Pathways Involved in Mammary Can 063480, ISA/US, Alexandria, Virginia, United States, mailed on cer,” Nature Genetics 39(6): 759-769, Nature Publishing Group, May 3, 2016, 5 pages. England (2007). International Search Report for International Application No. PCT/ Unexamined Application for German Patent Application No. 10339 US 15/45210, United States Patent and Trademark Office, United 820.1, filed Aug. 22, 2003 with the Federal Republic of Germany States, mailed on Jan. 6, 2016, 7 pages. German Patent and Trademark Office, 126 pages (D6 and D6A as Kazanskaya, O., et al., “The Wnt Signaling Regulator R-spondin 3 cited in Opposition of EP2173379). Promotes Angioblast and Vascular Development.” Development U.S. Appl. No. 60/947,611, filed Jul. 2, 2007, 98 pages (D11 as cited 135(22):3655-3664, Company of Biologists, England (2008). in Opposition of EP2173379). Klaus, A. and Birchmeier, W. “Wnt Signaling and its Impact on Veeman, M.T., et al., “A Second Canon: Functions and Mechanisms Development and Cancer,” Nature Reviews/Cancer 8(5):387-398. of B-Catenin-Independent Wnt Signaling.” Developmental Cell Nature Publishing Group, United States (2008) (D34 as cited in 5(3):367-377. Cell Press, United States (2003). Opposition of EP2173379). Written Opinion for International Application No. PCT/US2015/ Lam, A.P. and Gottardi, C.J., “B-catenin Signaling: A Novel Media 063480, United States Patent and Trademark Office, United States, tor of Fibrosis and Potential Therapeutic Target,” Current Opinion mailed on May 3, 2016, 9 pages. in Rheumatology 23(6):562-567, Lippincott Williams and Wilkins, Written Opinion for International Application No. PCT/US 15/ United States (2011). 45210, United States Patent and Trademark Office, United States, LifeSpan BioSciences, Inc., Anti-GPR49/LGR5 Antibody (N-Ter mailed on Jan. 6, 2016, 7 pages. minus) IHC-plus LS-A1232, Retrieved on May 31, 2016, 2 pages Written Submission including New Main Request and Auxiliary (D38 as cited in Opposition of EP2173379). Request in preparation for Oral Proceedings in Opposition to Miao, C.G., et al., “Wnt Signaling in Liver Fibrosis: Progress, European Patent 2157192, Opponent OncoMed Pharmaceuticals, Challenges and Potential Directions.” Biochimie 95(12):2326-2335, Inc., 12 pages, Dec. 18, 2015. Elsevier Masson SAS, Paris (2013). Yamamoto, Y, et al., “Overexpression of Orphan G-protein-coupled Ohkawara, B., et al., “Rspo3 Binds Syndecan 4 and Induces Receptor, Gpr49, in Human Hepatocellular Carcinomas with Wnt/PCP Signaling via Clathrin-Mediated Endocytosis to Promote B-catenin Mutations.” Hepatology 37(3):528-533, Wiley, United Morphogenesis.” Developmental Cell 20:303-314. Elseview Inc., States (2003) (D28 as cited in Opposition of EP2173379). United States (2011). Yin, X., et al., “R-spondin2 Activates Hepatic Stellate Cells and Ohkawara, B., et al., “Rspo3 Binds Syndecan 4 and Induces Promotes Liver Fibrosis.” Digestive Diseases and Sciences Wnt/PCP Signaling via Clathrin-Mediated Endocytosis to Promote 59(10):2452-2461, Springer Science + Business Media, United Morphogenesis.' Supplemental Information and Supplemental Fig States (2014). ures, Developmental Cell 20:14 pages (2011). Yoon, J.K., and Lee, J.S., "Cellular Signaling and Biological Peng, W.C., et al., “Structure of StemCell Growth Factor R-spondin Functions of R-spondins,” Cell Signalling 24(2):369-377. Elsevier 1 in Complex with the Ectodomain of its Receptor LGR5,” Cell Science Ltd, England (2012). Reports 3(6):1885-1892, Cell Press, United States (2013) (D16 as cited in Opposition of EP2173379). * cited by examiner U.S. Patent Mar. 21, 2017 Sheet 1 of 20 US 9,598,497 B2

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{}} US 9,598,497 B2 1. 2 RSPO3 BINDING AGENTS AND USES action between RSPO proteins and LGR (leucine-rich repeat THEREOF containing, G protein-coupler receptor) proteins, such as LGR5 (U.S. Patent Publication Nos. 2009/0074782 and CROSS-REFERENCE TO RELATED 2009/0191205), and these data present an alternative path APPLICATIONS way for the activation of B-catenin signaling. The Wnt signaling pathway has been identified as a This application is a Divisional application of U.S. appli potential target for cancer therapy. The Wnt signaling path cation Ser. No. 13/940,834, filed Jul. 12, 2013, which claims way is one of several critical regulators of embryonic pattern priority benefit of U.S. Provisional Application No. 61/671, formation, post-embryonic tissue maintenance, and stem 421, filed Jul. 13, 2012, U.S. Provisional Application No. 10 cell biology. More specifically, Wnt signaling plays an 61/753,184, filed Jan. 16, 2013, U.S. Provisional Applica important role in the generation of cell polarity and cell fate tion No. 61/789,156, filed Mar. 15, 2013, and U.S. Provi specification including self-renewal by stem cell popula sional Application No. 61/826,747, filed May 23, 2013, each tions. Unregulated activation of the Wnt pathway is associ of which is hereby incorporated by reference herein in its ated with numerous human cancers where it is believed the entirety. 15 activation can alter the developmental fate of cells. The activation of the Wnt pathway may maintain tumor cells in REFERENCE TO ASEQUENCE LISTING an undifferentiated state and/or lead to uncontrolled prolif SUBMITTED ELECTRONICALLY VIA eration. Thus carcinogenesis can proceed by overtaking EFS-WEB homeostatic mechanisms which control normal develop ment and tissue repair (reviewed in Reya & Clevers, 2005, The content of the electronically submitted sequence Nature, 434:843-50; Beachy et al., 2004, Nature, 432:324 listing (Name: 2293 0930005 sequencelisting ascii.txt, 31). Size: 166 kilobytes; and Date of Creation: Sep. 29, 2015) is The Wnt signaling pathway was first elucidated in the herein incorporated by reference in its entirety. Drosophila developmental mutant wingless (wg) and from 25 the murine proto-oncogene int-1, now Wnt1 (Nusse & FIELD OF THE INVENTION Varmus, 1982, Cell, 31:99-109; Van Ooyen & Nusse, 1984, Cell, 39:233-40; Cabrera et al., 1987, Cell, 50:659-63; The field of this invention generally relates to antibodies Rijsewijk et al., 1987, Cell, 50:649-57). Wnt genes encode and other agents that bind R-Spondin proteins (RSPO), secreted lipid-modified glycoproteins of which 19 have been particularly human R-Spondin protein RSPO3, as well as to 30 identified in mammals. These secreted ligands activate a methods of using the antibodies or other agents for the receptor complex consisting of a Frizzled (FZD) receptor treatment of diseases such as cancer. family member and low-density lipoprotein (LDL) receptor related protein 5 or 6 (LRP5/6). The FZD receptors are seven BACKGROUND OF THE INVENTION transmembrane domain proteins of the G-protein coupled 35 receptor (GPCR) superfamily and contain a large extracel The R-Spondin (RSPO) family of proteins is conserved lular N-terminal ligand binding domain with 10 conserved among vertebrates and comprises four members, RSPO1, cysteines, known as a cysteine-rich domain (CRD) or Fri RSPO2, RSPO3, and RSPO4. These proteins have been domain. There are ten human FZD receptors, FZD1, FZD2, referred to by a variety of names, including roof plate FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, and specific spondins, hPWTSR (hRSPO3), THS2D (RSPO3), 40 FZD10. Different FZD CRDs have different binding affini Cristin 1-4, and Futrin 1-4. The RSPOs are small secreted ties for specific Wnt proteins (Wu & Nusse, 2002, J. Biol. proteins that overall share approximately 40-60% sequence Chem., 277:41762-9), and FZD receptors have been grouped homology and domain organization. All RSPO proteins into those that activate the canonical B-catenin pathway and contain two furin-like cysteine-rich domains at the N-ter those that activate non-canonical pathways (Miller et al., minus followed by a thrombospondin domain and a basic 45 1999, Oncogene, 18:7860-72). charged C-terminal tail (Kim et al., 2006, Cell Cycle, A role for Wnt signaling in cancer was first uncovered 5:23-26). with the identification of Wnt1 (originally int1) as an onco Studies have shown that RSPO proteins have a role during gene in mammary tumors transformed by the nearby inser vertebrate development (Kamata et al., 2004, Biochim. Bio tion of a murine virus (Nusse & Varmus, 1982, Cell, phys Acta, 1676:51-62) and in Xenopus myogenesis (Kazan 50 31:99-109). Additional evidence for the role of Wnt signal skaya et al., 2004, Dev. Cell, 7:525-534). RSPO1 has also ing in breast cancer has since accumulated. For instance, been shown to function as a potent mitogen for gastrointes transgenic over-expression of B-catenin in the mammary tinal epithelial cells (Kim et al., 2005, Science, 309:1256 glands results in hyperplasias and adenocarcinomas (Imbert 1259). It has been reported that RSPO3 is prominently et al., 2001, J. Cell Biol., 153:555-68; Michaelson & Leder, expressed in or close by endothelial cells and their cellular 55 2001. Oncogene, 20:5093-9) whereas loss of Wnt signaling precursors in Xenopus and mouse. Furthermore, it has been disrupts normal mammary gland development (Tepera et al., Suggested that RSPO3 may act as an angiogenic factor in 2003, J. Cell Sci., 116:1137-49; Hatsell et al., 2003, J. embryogenesis (Kazanskaya et al., 2008, Development, 135: Mammary Gland Biol. Neoplasia, 8: 145-58). In human 3655-3664). RSPO proteins are known to activate B-catenin breast cancer, B-catenin accumulation implicates activated signaling similar to Wnt signaling, however the relationship 60 Wnt signaling in over 50% of carcinomas, and though between RSPO proteins and Wnt signaling is still being specific mutations have not been identified, up-regulation of investigated. It has been reported that RSPO proteins pos Frizzled receptor expression has been observed (Brennan & sess a positive modulatory activity on Wnt ligands (Nam et Brown, 2004, J. Mammary Gland Biol. Neoplasia, 9:119-31; al., 2006, JBC 281:13247-57). This study also reported that Malovanovic et al., 2004, Int. J. Oncol., 25:1337-42). RSPO proteins could function as Frizzled8 and LRP6 recep 65 Activation of the Wnt pathway is also associated with tor ligands and induce B-catenin signaling (Nam et al., 2006, colorectal cancer. Approximately 5-10% of all colorectal JBC 281:13247-57). Recent studies have identified an inter cancers are hereditary with one of the main forms being US 9,598,497 B2 3 4 familial adenomatous polyposis (FAP), an autosomal domi In one aspect, the invention provides a binding agent, nant disease in which about 80% of affected individuals such as an antibody, that specifically binds human RSPO3. contain a germline mutation in the adenomatous polyposis The sequence of human RSPO3 is known in the art and is coli (APC) gene. Mutations have also been identified in included herein as SEQ ID NO:3. In certain embodiments, other Wnt pathway components including Axin and 5 the RSPO3-binding agent binds within amino acids 22-272 B-catenin. Individual adenomas are clonal outgrowths of of human RSPO3. In certain embodiments, the RSPO3 epithelial cells containing a second inactivated allele, and binding agent binds within amino acids 22-207 of human the large number of FAP adenomas inevitably results in the RSPO3. In certain embodiments, the RSPO3-binding agent development of adenocarcinomas through additional muta binds within amino acids 35-135 of human RSPO3. In 10 certain embodiments, the RSPO3-binding agent binds tions in oncogenes and/or tumor Suppressor genes. Further within amino acids 35-86 of human RSPO3. In certain more, activation of the Wnt signaling pathway, including embodiments, the RSPO3-binding agent binds within amino loss-of-function mutations in APC and stabilizing mutations acids 92-135 of human RSPO3. In some embodiments, the in B-catenin, can induce hyperplastic development and RSPO3-binding agent (e.g., an antibody) specifically binds tumor growth in mouse models (Oshima et al., 1997, Cancer 15 at least one other human RSPO selected from the group Res., 57:1644-9; Harada et al., 1999, EMBO.J., 18:5931-42). consisting of RSPO1, RSPO2, and RSPO4. In some embodi Similar to breast cancer and colon cancer, melanoma ments, the RSPO3-binding agent or antibody modulates often has constitutive activation of the Wnt pathway, as B-catenin activity, is an antagonist of 3-catenin signaling, indicated by the nuclear accumulation of B-catenin. Activa inhibits 3-catenin signaling, and/or inhibits activation of tion of the Wnt/B-catenin pathway in Some melanoma B-catenin. In some embodiments, the RSPO3-binding agent tumors and cell lines is due to modifications in pathway inhibits RSPO3 signaling. In some embodiments, the components, such as APC, ICAT, LEF1 and B-catenin (see RSPO3-binding agent inhibits, interferes with, and/or dis e.g., Lame et al. 2006, Frontiers Biosci., 11:733-742). rupts binding of RSPO3 to one or more LGR proteins (e.g., However, there are conflicting reports in the literature as to LGR4, LGR5, and/or LGR6). In some embodiments, the the exact role of Wnt/B-catenin signaling in melanoma. For 25 RSPO3-binding agent inhibits binding of RSPO3 to LGR5. example, one study found that elevated levels of nuclear In certain embodiments, the RSPO3-binding agent is an B-catenin correlated with improved survival from mela antibody which binds human RSPO3. In some embodi noma, and that activated Wnt/B-catenin signaling was asso ments, the antibody binds human RSPO3 and mouse ciated with decreased cell proliferation (Chien et al., 2009, RSPO3. In certain embodiments, the antibody comprises a PNAS, 106:1193-1198). 30 heavy chain CDR1 comprising KASGYTFTDYS (SEQ ID The focus of cancer drug research is shifting toward NO:9), KASGYTFTSYTF (SEQ ID NO:34), or DYSIH targeted therapies aimed at genes, proteins, and pathways (SEQ ID NO:78), a heavy chain CDR2 comprising involved in human cancer. There is a need for new agents IYPSNGDS (SEQID NO:10) or YIYPSNGDSGYNQKFK targeting signaling pathways and new combinations of (SEQ ID NO:79), and a heavy chain CDR3 comprising agents that target multiple pathways that could provide 35 ATYFANYFDY (SEQ ID NO:11), ATYFANNFDY (SEQ therapeutic benefit for cancer patients. Thus, biomolecules ID NO:35), or TYFANNFD (SEQ ID NO:80). In some (e.g., anti-RSPO3 antibodies) that disrupt B-catenin signal embodiments, the antibody further comprises a light chain ing are a potential Source of new therapeutic agents for CDR1 comprising QSVDYDGDSYM (SEQ ID NO:12) or cancer, as well as other B-catenin-associated diseases. KASQSVDYDGDSYMN (SEQ ID NO:81), a light chain 40 CDR2 comprising AAS (SEQ ID NO:13) or AASNLES BRIEF SUMMARY OF THE INVENTION (SEQ ID NO:82), and a light chain CDR3 comprising QQSNEDPLT (SEQID NO:14) or QQSNEDPLTF (SEQ ID The present invention provides binding agents, such as NO:83). In some embodiments, the antibody comprises a antibodies, that bind RSPO3 proteins, as well as composi heavy chain CDR1 comprising KASGYTFTDYS (SEQ ID tions, such as pharmaceutical compositions, comprising the 45 NO:9), a heavy chain CDR2 comprising IYPSNGDS (SEQ binding agents. Binding agents that bind RSPO3 as well as ID NO:10), and a heavy chain CDR3 comprising ATYFAN at least one additional antigen or target, and pharmaceutical NFDY (SEQ ID NO:35), and/or a light chain CDR1 com compositions of Such binding agents, are also provided. In prising QSVDYDGDSYM (SEQ ID NO:12), a light chain certain embodiments, the RSPO3-binding agents are novel CDR2 comprising AAS (SEQ ID NO:13), and a light chain polypeptides. Such as antibodies, antibody fragments, and 50 CDR3 comprising QQSNEDPLT (SEQID NO:14). In some other polypeptides related to such antibodies. The invention embodiments, the antibody comprises a heavy chain CDR1 further provides methods of inhibiting the growth of a tumor comprising DYSIH (SEQ ID NO:78), a heavy chain CDR2 by administering the RSPO3-binding agents to a subject comprising YTYPSNGDSGYNQKFK (SEQID NO:79), and with a tumor. The invention further provides methods of a heavy chain CDR3 comprising TYFANNFD (SEQ ID treating cancer by administering the RSPO3-binding agents 55 NO:80), and/or a light chain CDR1 comprising KASQS to a subject in need thereof. In some embodiments, the VDYDGDSYMN (SEQ ID NO:81), a light chain CDR2 methods of treating cancer or inhibiting tumor growth comprising AASNLES (SEQ ID NO:82), and a light chain comprise targeting cancer stem cells with the RSPO3 CDR3 comprising QQSNEDPLTF (SEQ ID NO:83). In binding agents. In some embodiments, the methods com Some embodiments, the antibody comprises a heavy chain prise disrupting B-catenin signaling. In some embodiments, 60 CDR1 comprising DYSIH (SEQ ID NO:78), a heavy chain the methods comprise modulating (e.g., inhibiting) angio CDR2 comprising YTYPSNGDSGYNQKFK (SEQ ID genesis. In certain embodiments, the methods comprise NO:79), and a heavy chain CDR3 comprising TYFANNFD reducing the frequency of cancer stem cells in a tumor, (SEQ ID NO:80), and/or a light chain CDR1 comprising reducing the number of cancer stem cells in a tumor, KASQSVDYDGDSYMN (SEQ ID NO:81), a light chain reducing the tumorigenicity of a tumor, and/or reducing the 65 CDR2 comprising AASNLES (SEQ ID NO:82), and a light tumorigenicity of a tumor by reducing the number or fre chain CDR3 comprising QQSNEDPLT (SEQID NO:14). In quency of cancer stem cells in the tumor. Some embodiments, the antibody comprises a heavy chain US 9,598,497 B2 5 6 CDR1 comprising KASGYTFTDYS (SEQ ID NO:9) or petes for specific binding to human RSPO3 with an antibody DYSIH (SEQ ID NO:78), a heavy chain CDR2 comprising that comprises a heavy chain variable region comprising IYPSNGDS (SEQ ID NO:10), and a heavy chain CDR3 SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:36, SEQ ID comprising TYFANNFD (SEQ ID NO:80), and/or a light NO:37, SEQID NO:44, SEQID NO:45, or SEQID NO:62, chain CDR1 comprising QSVDYDGDSYM (SEQ ID and a light chain variable region comprising SEQID NO:17, NO:12), a light chain CDR2 comprising AAS (SEQ ID SEQ ID NO:72, or SEQID NO:86. In some embodiments, NO:13), and a light chain CDR3 comprising QQSNEDPLT the antibody with which the RSPO3-binding agent competes (SEQ ID NO:14). is antibody 131R002 or antibody 131R003. In some embodi In certain embodiments, the RSPO3-binding agent is an ments, the antibody with which the RSPO3-binding agent antibody which comprises: (a) a heavy chain CDR1 com 10 competes is a humanized form of antibody 131R002 or prising KASGYTFTDYS (SEQ ID NO:9), KASGYTFTS antibody 131R003. In some embodiments, the antibody with YTF (SEQ ID NO:34), DYSIH (SEQ ID NO:78), or a which the RSPO3-binding agent competes is antibody variant thereof comprising 1, 2, 3, or 4 amino acid Substi h131R006A, h131R006B, h131R005/131R007, h131R008, tutions; (b) a heavy chain CDR2 comprising IYPSNGDS h131R010, or hl31R011. In some embodiments, the binding (SEQ ID NO:10), YTYPSNGDSGYNQKFK (SEQ ID 15 agent competes for specific binding to RSPO3 with an NO:79), or a variant thereof comprising 1, 2, 3, or 4 amino antibody of the invention in an in vitro competitive binding acid Substitutions; (c) a heavy chain CDR3 comprising assay. ATYFANYFDY (SEQ ID NO:11), ATYFANNFDY (SEQ In certain embodiments, the binding agent is an antibody ID NO:35), TYFANNFD (SEQ ID NO:80), or a variant that binds the same epitope, or essentially the same epitope, thereof comprising 1, 2, 3, or 4 amino acid substitutions; (d) on RSPO3 as an antibody of the invention (e.g., 131R002, a light chain CDR1 comprising QSVDYDGDSYM (SEQID 131R003, or humanized forms/variants thereof). In certain NO:12), KASQSVDYDGDSYMN (SEQ ID NO:81), or a embodiments, the binding agent is an antibody that antibody variant thereof comprising 1, 2, 3, or 4 amino acid Substi binds the same epitope, or essentially the same epitope, on tutions; (e) a light chain CDR2 comprising AAS (SEQ ID RSPO3 as antibody h131R005/131R007, h131R008, NO:13), AASNLES (SEQ ID NO:82), or a variant thereof 25 h131R010, or hl31R011. comprising 1, 2, 3, or 4 amino acid Substitutions; and (f) a In still another aspect, the binding agent is an antibody light chain CDR3 comprising QQSNEDPLT (SEQ ID that binds an epitope on RSPO3 that overlaps with the NO:14), QQSNEDPLTF (SEQ ID NO:83), or a variant epitope on RSPO3 bound by an antibody of the invention thereof comprising 1, 2, 3, or 4 amino acid substitutions. In (e.g., 131R002, 131 R003, or humanized forms/variants Some embodiments, the amino acid Substitutions are con 30 thereof). In some embodiments, the binding agent is an servative amino acid Substitutions. In some embodiments, antibody that binds an epitope on RSPO3 that overlaps with the substitutions are made as part of a germline humaniza the epitope on RSPO3 bound by antibody hl31R005/ tion process. 131R007, h131R008, h131R010, or hl31R011. In certain embodiments, the RSPO3-binding agent is an In certain embodiments of each of the aforementioned antibody which comprises: (a) a heavy chain variable region 35 aspects or embodiments, as well as other aspects and/or having at least 80% sequence identity to SEQ ID NO:15, embodiments described elsewhere herein, the binding agent SEQ ID NO:16, SEQ ID NO:36, SEQ ID NO:37, SEQ ID is a bispecific antibody. In some embodiments, the bispecific NO:44, SEQID NO:45, or SEQID NO:62; and/or (b) a light antibody specifically binds human RSPO3 and a second chain variable region having at least 80% sequence identity target. In some embodiments, the bispecific antibody spe to SEQ ID NO:17, SEQ ID NO:72, or SEQ ID NO:86. In 40 cifically binds human RSPO3 and human RSPO1. In some certain embodiments, the RSPO3-binding agent is an anti embodiments, the bispecific antibody specifically binds body that comprises: (a) a heavy chain variable region human RSPO3 and human RSPO2. In some embodiments, having at least 90% sequence identity to SEQ ID NO:15, the bispecific antibody specifically binds human RSPO3 and SEQ ID NO:16, SEQ ID NO:36, SEQ ID NO:37, SEQ ID human RSPO4. In some embodiments, the bispecific anti NO:44, SEQID NO:45, or SEQID NO:62; and/or (b) a light 45 body modulates B-catenin activity. In certain embodiments, chain variable region having at least 90% sequence identity the bispecific antibody inhibits f-catenin activity. In certain to SEQ ID NO:17, SEQID NO:72, or SEQ ID NO:86. embodiments, the bispecific antibody inhibits B-catenin sig In some embodiments, the RSPO3-binding agent is a naling. In certain embodiments, the bispecific antibody monoclonal antibody. In some embodiments, the monoclo inhibits activation of B-catenin. In some embodiments, the nal antibody is an IgG1 antibody. In some embodiments, the 50 bispecific antibody reduces the number of frequency of monoclonal antibody is an IgG2 antibody. In some embodi cancer stem cells. In certain embodiments, the bispecific ments, the RSPO3-binding agent is monoclonal antibody antibody comprises two identical light chains. In certain 131R002 or monoclonal antibody 131R003. In some embodiments, the bispecific antibody is an IgG antibody. In embodiments, the RSPO3-binding agent is an affinity-ma certain embodiments, the bispecific antibody is an IgG1 tured variant of monoclonal antibody 131R002 or monoclo 55 antibody. In certain embodiments, the bispecific antibody is nal antibody 131R003. In some embodiments, the RSPO3 an IgG2 antibody. binding agent is a chimeric antibody comprising the antigen In some embodiments, the bispecific antibody comprises: binding sites from antibody 131R002 or antibody 131R003. a first antigen-binding site that specifically binds human In some embodiments, the RSPO3-binding agent is a RSPO3, wherein the first antigen-binding site comprises a humanized form of antibody 131R002 or antibody 131R003. 60 heavy chain CDR1 comprising KASGYTFTDYS (SEQ ID In some embodiments, the RSPO3-binding agent is antibody NO:9), KASGYTFTSYTF (SEQ ID NO:34), or DYSIH h131R006A, h131R006B, h131R005/131R007, h131R008, (SEQ ID NO:78), a heavy chain CDR2 comprising h131R010, or hl31R011. IYPSNGDS (SEQID NO:10) or YIYPSNGDSGYNQKFK In another aspect, the invention provides a binding agent (SEQ ID NO:79), and a heavy chain CDR3 comprising (e.g., an antibody) that competes for specific binding to 65 ATYFANYFDY (SEQ ID NO:11), ATYFANNFDY (SEQ human RSPO3 with an antibody of the invention. In some ID NO:35), or TYFANNFD (SEQ ID NO:80). In some embodiments, the binding agent (e.g., an antibody) com embodiments, the first antigen-binding site comprises a light US 9,598,497 B2 7 8 chain CDR1 comprising QSVDYDGDSYM (SEQ ID selected from the group consisting of: SEQID NO:15, SEQ NO:12) or KASQSVDYDGDSYMN (SEQ ID NO:81), a ID NO:16, SEQID NO:17, SEQID NO:21, SEQID NO:22, light chain CDR2 comprising AAS (SEQ ID NO:13) or SEQ ID NO:23, SEQ ID NO:27, SEQ ID NO:28, SEQ ID AASNLES (SEQ ID NO:82), and a light chain CDR3 NO:29, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, comprising QQSNEDPLT (SEQ ID NO:14) or QQSNED SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:42, SEQ ID PLTF (SEQ ID NO:83). In some embodiments, the bispe NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, cific antibody further comprises a second antigen-binding SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:62, SEQ ID site that specifically binds human RSPO1. In some embodi NO:63, SEQ ID NO:64, SEQ ID NO:68, SEQ ID NO:69, ments, the bispecific antibody further comprises a second SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID antigen-binding site that specifically binds human RSPO2. 10 NO:86, SEQ ID NO:87, and SEQ ID NO:88. In some Non-limiting examples of antibodies to RSPO1 or antibod embodiments, the polypeptide comprises SEQ ID NO:15 ies to RSPO2 have been described in, for example, Inter and/or SEQ ID NO:17. In some embodiments, the polypep national Patent Application Pub. No. WO 2013/012747. In tide comprises SEQ ID NO:16 and/or SEQ ID NO:17. In Some embodiments, the first and second binding sites com some embodiments, the polypeptide comprises SEQ ID prise a common (e.g., identical) light chain. 15 NO:36 and/or SEQ ID NO:17. In some embodiments, the In some embodiments, the bispecific antibody comprises: polypeptide comprises SEQ ID NO:37 and/or SEQ ID a) a first antigen-binding site that specifically binds human NO:17. In some embodiments, the polypeptide comprises RSPO3, and b) a second antigen-binding site that specifi SEQ ID NO:44 and/or SEQ ID NO:17. In some embodi cally binds human RSPO1, wherein the first antigen-binding ments, the polypeptide comprises SEQ ID NO:45 and/or site comprises a heavy chain CDR1 comprising KASGYT SEQ ID NO:17. In some embodiments, the polypeptide FTDYS (SEQ ID NO:9), KASGYTFTSYTF (SEQ ID comprises SEQ ID NO:62 and/or SEQ ID NO:17. In some NO:34), or DYSIH (SEQ ID NO:78), a heavy chain CDR2 embodiments, the polypeptide comprises SEQ ID NO:44 comprising IYPSNGDS (SEQID NO:10) or YTYPSNGDS and/or SEQ ID NO:72. In some embodiments, the polypep GYNQKFK (SEQ ID NO:79), and a heavy chain CDR3 tide comprises SEQ ID NO:45 and/or SEQ ID NO:72. In comprising ATYFANYFDY (SEQ ID NO:11), ATYFAN 25 some embodiments, the polypeptide comprises SEQ ID NFDY (SEQ ID NO:35), or TYFANNFD (SEQ ID NO:80). NO:62 and/or SEQ ID NO:72. In some embodiments, the In some embodiments, the bispecific antibody comprises: a) polypeptide comprises SEQ ID NO:44 and/or SEQ ID a first antigen-binding site that specifically binds human NO:86. In some embodiments, the polypeptide comprises RSPO3, and b) a second antigen-binding site that specifi SEQ ID NO:45 and/or SEQ ID NO:86. In some embodi cally binds human RSPO2, wherein the first antigen-binding 30 ments, the polypeptide comprises SEQ ID NO:62 and/or site comprises a heavy chain CDR1 comprising KASGYT SEQ ID NO:86. FTDYS (SEQ ID NO:9), KASGYTFTSYTF (SEQ ID In some embodiments, the polypeptide comprises SEQID NO:34), or DYSIH (SEQ ID NO:78), a heavy chain CDR2 NO:21 and/or SEQ ID NO:23. In some embodiments, the comprising IYPSNGDS (SEQID NO:10) or YTYPSNGDS polypeptide comprises SEQ ID NO:22 and/or SEQ ID GYNQKFK (SEQ ID NO:79), and a heavy chain CDR3 35 NO:23. In some embodiments, the polypeptide comprises comprising ATYFANYFDY (SEQ ID NO:11), ATYFAN SEQ ID NO:38 and/or SEQ ID NO:23. In some embodi NFDY (SEQ ID NO:35), or TYFANNFD (SEQ ID NO:80). ments, the polypeptide comprises SEQ ID NO:41 and/or In some embodiments, the first antigen-binding site com SEQ ID NO:23. In some embodiments, the polypeptide prises a light chain CDR1 comprising QSVDYDGDSYM comprises SEQ ID NO:46 and/or SEQ ID NO:23. In some (SEQ ID NO:12) or KASQSVDYDGDSYMN (SEQ ID 40 embodiments, the polypeptide comprises SEQ ID NO:47 NO:81), a light chain CDR2 comprising AAS (SEQ ID and/or SEQ ID NO:23. In some embodiments, the polypep NO:13) or AASNLES (SEQ ID NO:82), and a light chain tide comprises SEQ ID NO:63 and/or SEQ ID NO:23. In CDR3 comprising QQSNEDPLT (SEQ ID NO:14) or some embodiments, the polypeptide comprises SEQ ID QQSNEDPLTF (SEQ ID NO:83). NO:68 and/or SEQ ID NO:23. In some embodiments, the In some embodiments, the bispecific antibody specifically 45 polypeptide comprises SEQ ID NO:46 and/or SEQ ID binds human RSPO3 and comprises: a heavy chain variable NO:73. In some embodiments, the polypeptide comprises region having at least 90% sequence identity to SEQ ID SEQ ID NO.47 and/or SEQ ID NO:73. In some embodi NO:15, SEQ ID NO:16, SEQ ID NO:36, SEQ ID NO:37, ments, the polypeptide comprises SEQ ID NO:63 and/or SEQID NO:44, SEQID NO:45, or SEQID NO:62. In some SEQ ID NO:73. In some embodiments, the polypeptide embodiments, the bispecific antibody specifically binds 50 comprises SEQ ID NO:68 and/or SEQ ID NO:73. In some human RSPO3 and comprises: a heavy chain variable region embodiments, the polypeptide comprises SEQ ID NO:46 having at least 95% sequence identity to SEQ ID NO:15, and/or SEQ ID NO:87. In some embodiments, the polypep SEQ ID NO:16, SEQ ID NO:36, SEQ ID NO:37, SEQ ID tide comprises SEQ ID NO.47 and/or SEQ ID NO:87. In NO:44, SEQID NO:45, or SEQID NO:62. In some embodi some embodiments, the polypeptide comprises SEQ ID ments, the bispecific antibody comprises a first and second 55 NO:63 and/or SEQ ID NO:87. In some embodiments, the binding site, wherein the first and second binding sites polypeptide comprises SEQ ID NO:68 and/or SEQ ID comprise a common (e.g., identical) light chain. In some NO:87. embodiments, the bispecific antibody comprises a light In some embodiments, the polypeptide comprises SEQID chain variable region having at least 95% sequence identity NO:27 and/or SEQ ID NO:29. In some embodiments, the to SEQ ID NO:17, SEQID NO:72, or SEQ ID NO:86. 60 polypeptide comprises SEQ ID NO:28 and/or SEQ ID In certain embodiments of each of the aforementioned NO:29. In some embodiments, the polypeptide comprises aspects, as well as other aspects and/or embodiments SEQ ID NO:39 and/or SEQ ID NO:29. In some embodi described elsewhere herein, the RSPO3-binding agent or ments, the polypeptide comprises SEQ ID NO:42 and/or antibody is isolated. In some embodiments, the RSPO3 SEQ ID NO:29. In some embodiments, the polypeptide binding agent or antibody is Substantially pure. 65 comprises SEQ ID NO:48 and/or SEQ ID NO:29. In some In another aspect, the invention provides polypeptides. In embodiments, the polypeptide comprises SEQ ID NO:49 Some embodiments, the polypeptide comprises a sequence and/or SEQ ID NO:29. In some embodiments, the polypep US 9,598,497 B2 9 10 tide comprises SEQ ID NO:64 and/or SEQ ID NO:29. In In some embodiments, the cell is a tumor cell. In some some embodiments, the polypeptide comprises SEQ ID embodiments, the tumor is a colorectal tumor. In some NO:69 and/or SEQ ID NO:29. In some embodiments, the embodiments, the tumor is an ovarian tumor. In some polypeptide comprises SEQ ID NO:48 and/or SEQ ID NO:74. In some embodiments, the polypeptide comprises embodiments, the tumor is a pancreatic tumor. In some SEQ ID NO:49 and/or SEQ ID NO:74. In some embodi embodiments, the tumor is a lung tumor. In some embodi ments, the polypeptide comprises SEQ ID NO:64 and/or ments, the tumor is a breast tumor. In some embodiments, SEQ ID NO:74. In some embodiments, the polypeptide the tumor expresses elevated levels of at least one RSPO comprises SEQ ID NO:69 and/or SEQ ID NO:74. In some protein. In some embodiments, the tumor expresses elevated embodiments, the polypeptide comprises SEQ ID NO:48 levels of RSPO1. In some embodiments, the tumor and/or SEQID NO:88. In some embodiments, the polypep 10 expresses elevated levels of RSPO2. In some embodiments, tide comprises SEQ ID NO:49 and/or SEQ ID NO:88. In the tumor expresses elevated levels of RSPO3. In some some embodiments, the polypeptide comprises SEQ ID embodiments, the tumor expresses a high level of at least NO:64 and/or SEQ ID NO:88. In some embodiments, the one RSPO protein. In some embodiments, the tumor polypeptide comprises SEQ ID NO:69 and/or SEQ ID NO:88. 15 expresses a high level of RSPO1. In some embodiments, the In some embodiments, the polypeptide is isolated. In tumor expresses a high level of RSPO2. In some embodi certain embodiments, the polypeptide is substantially pure. ments, the tumor expresses a high level of RSPO3. In certain In certain embodiments, the polypeptide is an antibody or embodiments, the RSPO3-binding agent inhibits growth of part of any antibody, such as an antibody fragment. the tumor, for example, by reducing the number and/or In another aspect, the invention provides isolated poly frequency of cancer stem cells in the tumor. In some nucleotide molecules comprising a polynucleotide that embodiments, the tumor contains a RSPO gene fusion. In encodes the antibodies and/or polypeptides of each of the some embodiments, the tumor contains a RSPO2 gene aforementioned aspects, as well as other aspects and/or fusion. In some embodiments, the tumor contains a RSPO3 embodiments described herein. In some embodiments, the gene fusion. polynucleotide comprises a sequence selected from the 25 In another aspect, the invention provides methods of group consisting of SEQID NO:18, SEQID NO:19, SEQID treating cancer in a Subject. In some embodiments, the NO:20, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, method comprises administering to a subject a therapeuti SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID cally effective amount of any of the RSPO3-binding agents NO:40, SEQ ID NO:43, SEQ ID NO:50, SEQ ID NO:51, or antibodies described above, as well as those described SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID 30 elsewhere herein. In some embodiments, the cancer is NO:55, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, pancreatic cancer. In some embodiments, the cancer is SEQ ID NO:70, SEQ ID NO:71, SEQID NO:75, SEQ ID colorectal cancer. In some embodiments, the colorectal NO:76, SEQ ID NO:77, SEQ ID NO:84, SEQ ID NO:85, cancer comprises an inactivating mutation in the adenoma SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID tous polyposis coli (APC) gene. In some embodiments, the NO:92, SEQ ID NO:93, SEQ ID NO:94, and SEQ ID 35 colorectal cancer does not comprise an inactivating mutation NO:95. In some embodiments, the polynucleotide comprises in the APC gene. In some embodiments, the colorectal a polynucleotide that encodes a polypeptide selected from cancer comprises a wild-type APC gene. In some embodi the group consisting of: SEQ ID NO:15, SEQ ID NO:16, ments, the colorectal cancer comprises a RSPO gene fusion. SEQ ID NO:17, SEQ ID NO:21, SEQ ID NO:22, SEQ ID In some embodiments, the colorectal cancer comprises a NO:23, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, 40 RSPO2 gene fusion. In some embodiments, the colorectal SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID cancer comprises a RSPO3 gene fusion. In some embodi NO:39, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:44, ments, the cancer is ovarian cancer. In some embodiments, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID the cancer is lung cancer. In some embodiments, the cancer NO:48, SEQ ID NO:49, SEQ ID NO:62, SEQ ID NO:63, is breast cancer. In some embodiments, the cancer expresses SEQ ID NO:64, SEQ ID NO:68, SEQ ID NO:69, SEQ ID 45 elevated levels of at least one RSPO protein. In some NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:86, embodiments, the cancer is an ovarian cancer that expresses SEQ ID NO:87, and SEQID NO:88. elevated levels of RSPO3. In some embodiments, the cancer The invention further provides expression vectors that is lung cancer that expresses elevated levels of RSPO3. In comprise the polynucleotides, as well as cells that comprise Some embodiments, the cancer is breast cancer that the expression vectors and/or the polynucleotides. In some 50 expresses elevated levels of RSPO3. In some embodiments, embodiments, the cell is a hybridoma cell line. In some the cancer is pancreatic cancer that expresses elevated levels embodiments, the cell is a monoclonal cell line. In some of RSPO3. embodiments, the cell is a prokaryotic cell. In some embodi In another aspect, the invention provides methods of ments, the cell is an eukaryotic cell. treating a disease in a subject wherein the disease is asso In other aspects, the invention provides methods of inhib 55 ciated with activation of B-catenin, increased B-catenin iting growth of a tumor, comprising contacting the tumor signaling, and/or aberrant B-catenin signaling, wherein the with an effective amount of a RSPO3-binding agent or method comprises administering to the Subject a therapeu antibody, including each of those described herein. tically effective amount of a RSPO3-binding agent or anti In another aspect, the invention provides a method of body, including each of those described herein. inhibiting the growth of a tumor in a Subject, comprising 60 In certain embodiments of each of the aforementioned administering to the subject a therapeutically effective aspects, as well as other aspects and/or embodiments amount of a RSPO3-binding agent or antibody, including described elsewhere herein, the treatment methods further each of those described herein. comprise a step of determining the expression level of at In another aspect, the invention provides a method of least one RSPO protein in the tumor or cancer. inhibiting B-catenin signaling in a cell, comprising contact 65 In another aspect, the invention provides a method of ing the cell with an effective amount of a RSPO3-binding identifying a human Subject or selecting a human Subject for agent or antibody, including each of those described herein. treatment with a RSPO3-binding agent or antibody, includ US 9,598,497 B2 11 12 ing but not limited to, each of those described herein. In Shown is a Summary of microarray data from normal, Some embodiments, the method comprises determining if benign, and malignant tissue human samples. Individual tick the Subject has a tumor that has an elevated expression level marks indicate the expression level of RSPO3 mRNA. of a specific RSPO (e.g., RSPO3) as compared to the FIG. 2. Binding studies of RSPO proteins and LGR5. FACS analysis of HEK-293 cells expressing LGR5. HEK expression of the same RSPO protein in normal tissue or to 293 cells were transiently transfected with a cDNA expres a pre-determined level of the same RSPO protein. In some sion vector encoding FLAG-LGR5-CD4TM-GFP and then embodiments, the method comprises identifying a subject subsequently mixed with soluble RSPO1-Fc, RSPO2-Fc, for treatment or selecting a subject for treatment if the tumor RSPO3-Fc, or RSPO4-Fc fusion proteins. An anti-FLAG has an elevated level of RSPO expression. In some embodi antibody was used as a positive control, and soluble FZD8 ments, the method comprises determining if the Subject has 10 Fc was used as a negative control. Specific binding is a tumor that comprises an inactivating mutation in the APC indicated by the presence of signal within the dark lined box gene. In some embodiments, the method comprises identi overlay on each FACS plot. fying a subject for treatment or selecting a Subject for FIG. 3. Anti-RSPO3 antibodies inhibit B-catenin signal treatment if the tumor comprises an inactivating mutation in ing induced by RSPO3 and WNT3A. ATOPflash luciferase 15 reporter assay was used to measure B-catenin signaling in the APC gene. In some embodiments, the method comprises HEK-293 cells after exposure to a combination of WNT3a determining if the Subject has a tumor that comprises a (5 ng/ml) and RSPO3 (10 ng/ml) and in the presence of RSPO gene fusion (e.g., a RSPO3 gene fusion). In some increasing concentrations of anti-RSPO3 antibodies embodiments, the method comprises identifying a subject (131R002 or 131R003). Antibodies were used as 4-fold for treatment or selecting a subject for treatment if the tumor serial dilutions from 20 ug/ml to 0.02 ug/ml. Controls comprises a RSPO gene fusion (e.g., a RSPO3 gene fusion). included exposure to control medium (no WNT3a and no In certain embodiments of each of the aforementioned RSPO), WNT3a alone, or a combination of WNT3a and aspects, as well as other aspects and/or embodiments RSPO3 in the absence of antibody. FIG. 4. Affinity-matured 131R003 antibody variants described elsewhere herein, the treatment methods comprise inhibit B-catenin signaling induced by RSPO3 and WNT3A. administering to the subject the RSPO3-binding agent and at 25 A TOPflash luciferase reporter assay was used to measure least one additional therapeutic agent. B-catenin signaling in HEK-293 cells after exposure to a Pharmaceutical compositions comprising a RSPO3-bind combination of WNT3a and RSPO3 and in the presence of ing agent or antibody described herein and a pharmaceuti increasing concentrations of anti-RSPO3 antibodies cally acceptable carrier are further provided, as are cell lines (131R003 (-A-), 131R003 CDR1 variant (--), or 131R003 30 CDR3 variant (-O-)). Antibodies were used as 5-fold serial that produce the RSPO3-binding agents. Methods of treating dilutions from 20 ug/ml to 0.006 ug/ml. Controls included cancer and/or inhibiting tumor growth in a subject (e.g., a exposure to control medium (no WNT3a and no RSPO)/ human) comprising administering to the Subject an effective cells only (-A-) a control antibody (-V-), WNT3a amount of a pharmaceutical composition comprising the alone (-0-), or a combination of WNT3a and RSPO3 in the RSPO3-binding agents are also provided. absence of antibody (--). 35 FIG. 5. Inhibition of tumor growth with anti-RSPO anti Where aspects or embodiments of the invention are bodies. OV38 ovarian tumor cells were injected subcutane described in terms of a Markush group or other grouping of ously into NOD/SCID mice. Mice were treated with a alternatives, the present invention encompasses not only the combination of anti-RSPO1 antibody 89M5 and anti entire group listed as a whole, but also each member of the RSPO3 antibody 131R003 (-O-), taxol (-A-), a combination 40 of anti-RSPO1 antibody 89M5, anti-RSPO3 antibody group individually and all possible subgroups of the main 131R003, and taxol (-V-), or a control antibody (--). Data group, and also the main group absent one or more of the is shown as tumor volume (mm) over days post-treatment. group members. The present invention also envisages the FIG. 6. Inhibition of tumor growth with anti-RSPO anti explicit exclusion of one or more of any of the group bodies. OV38 ovarian tumor cells were injected subcutane 45 ously into NOD/SCID mice. Mice were treated with a members in the claimed invention. combination of anti-RSPO1 antibody 89M5 and anti RSPO3 antibody 131R002 (-A-), a combination of anti BRIEF DESCRIPTION OF THE FIGURES RSPO1 antibody 89M5 and taxol (-O-), a combination of anti-RSPO3 antibody 131R002 and taxol (--), a combina FIG. 1A. RSPO1 expression in tumors and normal tissues. 50 tion of anti-RSPO1 antibody 89M5, anti-RSPO3 antibody Shown is a Summary of microarray data from normal, 131R002, and taxol (-A-) taxol alone (-V-), or a control benign, and malignant tissue human samples. antibody (--). Data is shown as tumor volume (mm) over FIG. 1B. RSPO1 expression in tumors and normal tissues. days post-treatment. Shown is a Summary of microarray data from normal, FIG. 7A. Inhibition of tumor growth with anti-RSPO3 benign, and malignant tissue human samples. Individual tick 55 antibodies. LU45 lung tumor cells were injected subcutane marks indicate the expression level of RSPO1 mRNA. ously into NOD/SCID mice. Mice were treated with anti FIG. 1C. RSPO2 expression in tumors and normal tissues. RSPO3 antibody 131R002 (-O-) or a control Shown is a Summary of microarray data from normal, antibody (--). benign, and malignant tissue human samples. FIG. 7B. Inhibition of tumor growth with anti-RSPO3 FIG. 1D. RSPO2 expression in tumors and normal tissues. 60 antibodies. LU25 lung tumor cells were injected subcutane Shown is a Summary of microarray data from normal, ously into NOD/SCID mice. Mice were treated with anti benign, and malignant tissue human samples. Individual tick RSPO3 antibody 131R002 (-O-) or a control marks indicate the expression level of RSPO2 mRNA. antibody (--). Data is shown as tumor volume (mm) over FIG. 1E. RSPO3 expression in tumors and normal tissues. days post-treatment. Shown is a Summary of microarray data from normal, 65 FIG. 8. Affinity-matured antibody variants inhibit benign, and malignant tissue human samples. B-catenin signaling induced by RSPO3 and WNT3A. A FIG.1F. RSPO3 expression in tumors and normal tissues. TOPflash luciferase reporter assay was used to measure US 9,598,497 B2 13 14 B-catenin signaling in HEK-293T cells after exposure to a alone (-V-), or a combination of WNT3a and human RSPO3 combination of WNT3a and human RSPO3 and in the in the absence of antibody (-0-). presence of increasing concentrations of anti-RSPO3 anti FIG. 14. Inhibition of tumor growth with anti-RSPO body 131R002 (-A-), 131R006 (-0-), or 131R007 (--). antibodies. LU25 NSCLC lung tumor cells were injected Antibodies were used as 5-fold serial dilutions from 20 subcutaneously into NOD/SCID mice. Mice were treated ug/ml to 0.0064 g/ml. Controls included exposure to con with anti-RSPO3 antibody 131R008 (-A-), paclitaxel trol medium (no WNT3a and no RSPO/cells (-O-)), WNT3a alone (-O-), a combination of anti-RSPO3 antibody alone (-V-), or a combination of WNT3a and human RSPO3 131R008 and paclitaxel (-O-), or a control antibody (--). in the absence of antibody (-0-). Data is shown as tumor volume (mm) over days post FIG. 9. Inhibition of RSPO3 and LGR5 interaction by 10 treatment. anti-RSPO3 antibodies. FACS analysis of HEK-293T cells expressing LGR5. HEK-293T cells were transiently trans DETAILED DESCRIPTION OF THE fected with a cDNA expression vector encoding the extra INVENTION cellular domain of human LGR5 (FLAG-LGR5-CD4TM GFP) and then subsequently mixed with RSPO3-biotin 15 The present invention provides novel agents, including, fusion protein in combination with anti-RSPO3 antibodies but not limited to polypeptides such as antibodies, that bind 131R006 or 131R007. Binding was detected with PE-con RSPO proteins, particularly human RSPO3. The RSPO3 jugated streptavidin. Relative RSPO3-biotin binding is binding agents include, but are not limited to, antagonists of shown on the y-axis and expression of the FLAG-LGR5 B-catenin signaling. The RSPO3-binding agents include, but CD4TM-GFP fusion protein is indicated on the x-axis. are not limited to, inhibitors of RSPO3 and LGR protein Positive binding is indicated by the presence of signal within interactions. Related polypeptides and polynucleotides, the dark lined box overlay on each FACS plot. compositions comprising the RSPO3-binding agents, and FIG. 10. Inhibition of tumor growth with anti-RSPO methods of making the RSPO3-binding agents are also antibodies. NCI-H2030 cells were injected subcutaneously provided. Methods of using the novel RSPO3-binding into NOD/SCID mice. Mice were treated with anti-RSPO3 25 agents, such as methods of inhibiting tumor growth, meth antibody 131 R002 (-O-), carboplatin alone (-A-) a combi ods of treating cancer, methods of modulating angiogenesis, nation of anti-RSPO3 antibody 131R002 and methods of reducing the frequency of cancer stem cells in a carboplatin (-O-), or a control antibody (--). Data is shown tumor, methods of inhibiting B-catenin signaling, and/or as tumor volume (mm) over days post-treatment. methods of identifying and/or selecting Subjects for treat FIG. 11A. Inhibition of tumor growth with anti-RSPO 30 ment, are further provided. antibodies. LU 102 lung tumor cells were injected subcuta Monoclonal antibodies that specifically bind human neously into NOD/SCID mice. Mice were treated with RSPO3 have been identified monoclonal antibodies anti-RSPO3 antibody 131R002 (-O-), carboplatin alone 131R002 and 131R003 (Example 3). Anti-RSPO3 antibod (-A-) a combination of anti-RSPO3 antibody 131R002 and ies 131R002 and 131R003 have binding affinities for human carboplatin (-O-), or a control antibody (--). Data is shown 35 RSPO3 of less than 10 nM (Example 3). Anti-RSPO3 as tumor volume (mm) over days post-treatment. antibodies 131 R002 and 131 R003 inhibit RSPO3-induced FIG. 11B. Gene set enrichment analysis results. B-catenin signaling (Example 4, FIG. 3). Affinity-matured FIG. 12A. Inhibition of tumor growth with anti-RSPO variants of 131R003 inhibit RSPO3-induced B-catenin sig antibodies. PN35 pancreatic tumor cells were injected sub naling and have greater activity than parental 131R003 cutaneously into NOD/SCID mice. Mice were treated with 40 (Example 5, FIG. 4). Anti-RSPO3 antibodies inhibit tumor anti-RSPO3 antibody 131R002 (-O-), a combination of growth as single agents, in combination with anti-RSPO1 gemcitabine and nab-paclitaxel (ABRAXANE) (-A-), a antibodies, and in combination with one or more chemo combination of anti-RSPO3 antibody 131R002 and gemcit therapeutic agents (Examples 6, 7, 11 12 and 14: FIGS. 5-7, abine and nab-paclitaxel (ABRAXANE) (-O-), or a control 10-12 and 14). Humanized anti-RSPO3 antibodies antibody (--). Data is shown as tumor volume (mm) over 45 h131R006 and h131R007 are stronger inhibitors of days post-treatment. All four treatment groups are shown. B-catenin activity than antibody 131R002 (Example 8, FIG. FIG. 12B. Inhibition of tumor growth with anti-RSPO 8). Anti-RSPO3 antibodies h131R006 and h131R007 block antibodies. PN35 pancreatic tumor cells were injected sub binding of RSPO3 to LGR5 (Example 9, FIG. 9). Human cutaneously into NOD/SCID mice. Mice were treated with ized anti-RSPO3 antibody h131R010 isotype IgG1 inhibits anti-RSPO3 antibody 131R002 (-O-), a combination of 50 B-catenin activity similar to the IgG2 isotype antibody gemcitabine and nab-paclitaxel (ABRAXANE) (-A-), a h131R007 (Example 13, FIG. 13). combination of anti-RSPO3 antibody 131R002 and gemcit abine and nab-paclitaxel (ABRAXANE) (-O-), or a control I. DEFINITIONS antibody (--). Data is shown as tumor volume (mm) over days post-treatment. The gemcitabine and nab-paclitaxel 55 To facilitate an understanding of the present invention, a treatment group and the anti-RSPO3 antibody gemcitabine number of terms and phrases are defined below. and nab-paclitaxel treatment are shown on an expanded The terms 'antagonist' and “antagonistic” as used herein scale. refer to any molecule that partially or fully blocks, inhibits, FIG. 13. Inhibition of B-catenin signaling induced by reduces, or neutralizes a biological activity of a target and/or RSPO3 and WNT3A. A TOPflash luciferase reporter assay 60 signaling pathway (e.g., the B-catenin signaling). The term was used to measure B-catenin signaling in HEK-293T cells 'antagonist' is used herein to include any molecule that after exposure to a combination of WNT3a and human partially or fully blocks, inhibits, reduces, or neutralizes the RSPO3 and in the presence of increasing concentrations of activity of a protein (e.g., a RSPO protein). Suitable antago anti-RSPO3 antibody 131R007 (--) or 131R010 (-O-). nist molecules specifically include, but are not limited to, Antibodies were used as 5-fold serial dilutions from 20 65 antagonist antibodies or antibody fragments. ug/ml to 0.0064 g/ml. Controls included exposure to con The terms “modulation' and “modulate” as used herein trol medium (no WNT3a and no RSPO/cells (-A-)), WNT3a refer to a change or an alteration in a biological activity. US 9,598,497 B2 15 16 Modulation includes, but is not limited to, stimulating or The term “monoclonal antibody' encompasses both intact inhibiting an activity. Modulation may be an increase or a and full-length monoclonal antibodies as well as antibody decrease in activity (e.g., a decrease in RSPO signaling; a fragments (e.g., Fab, Fab'. F(ab')2, Fv), single chain (sclv) decrease in B-catenin signaling), a change in binding char antibodies, bispecific antibodies, fusion proteins comprising acteristics, or any other change in the biological, functional, 5 an antibody portion, and any other modified immunoglobu or immunological properties associated with the activity of lin molecule comprising an antigen recognition site (anti a protein, pathway, or other biological point of interest. gen-binding site). Furthermore, “monoclonal antibody' The term “antibody' as used herein refers to an immu refers to Such antibodies made by any number of techniques, noglobulin molecule that recognizes and specifically binds a including but not limited to, hybridoma production, phage target, Such as a protein, polypeptide, peptide, carbohydrate, 10 selection, recombinant expression, and transgenic animals. polynucleotide, lipid, or combinations of the foregoing, The term “humanized antibody” as used herein refers to through at least one antigen-binding site within the variable forms of non-human (e.g., murine) antibodies that are spe region(s) of the immunoglobulin molecule. As used herein, cific immunoglobulin chains, chimeric immunoglobulins, or the term encompasses intact polyclonal antibodies, intact fragments thereof that contain minimal non-human monoclonal antibodies, single chain antibodies, antibody 15 sequences. Typically, humanized antibodies are human fragments (such as Fab, Fab', F(ab')2, and Fv fragments), immunoglobulins in which residues of the CDRs are single chain Fv (scFv) antibodies, multispecific antibodies replaced by residues from the CDRs of a non-human species Such as bispecific antibodies, monospecific antibodies, mon (e.g., mouse, rat, rabbit, or hamster) that have the desired ovalent antibodies, chimeric antibodies, humanized antibod specificity, affinity, and/or binding capability (Jones et al., ies, human antibodies, fusion proteins comprising an anti 1986, Nature, 321:522-525; Riechmann et al., 1988, Nature, gen-binding site of an antibody, and any other modified 332:323-327; Verhoeyen et al., 1988, Science, 239:1534 immunoglobulin molecule comprising an antigen recogni 1536). In some instances, the Fv framework region residues tion site (i.e., antigen-binding site) as long as the antibodies of a human immunoglobulin are replaced with the corre exhibit the desired biological activity. An antibody can be sponding residues in an antibody from a non-human species any of the five major classes of immunoglobulins: IgA, Ig|D. 25 that has the desired specificity, affinity, structural, and/or IgE. IgG, and IgM, or Subclasses (isotypes) thereof (e.g., binding capability. The humanized antibody can be further IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2), based on the modified by the substitution of additional residues either in identity of their heavy chain constant domains referred to as the Fv framework region and/or within the replaced non alpha, delta, epsilon, gamma, and mu, respectively. The human residues to refine and optimize antibody specificity, different classes of immunoglobulins have different and 30 affinity, structural, and/or binding capability. In general, the well-known subunit structures and three-dimensional con humanized antibody will comprise substantially all of at figurations. Antibodies can be naked or conjugated to other least one, and typically two or three of the CDRs that molecules, including but not limited to, toxins and radio correspond to the non-human immunoglobulin whereas all isotopes. or substantially all of the framework regions are those of a The term “antibody fragment” refers to a portion of an 35 human immunoglobulin consensus sequence. The human intact antibody and refers to the antigenic determining ized antibody can also comprise at least a portion of an variable regions of an intact antibody. Examples of antibody immunoglobulin constant region or domain (Fc), typically fragments include, but are not limited to, Fab, Fab'. F(ab')2. that of a human immunoglobulin. Examples of methods and FV fragments, linear antibodies, single chain antibodies, used to generate humanized antibodies are described in, for and multispecific antibodies formed from antibody frag 40 example, U.S. Pat. No. 5.225,539. ments. “Antibody fragment as used herein comprises an The term “human antibody” as used herein refers to an antigen-binding site or epitope-binding site. antibody produced by a human or an antibody having an The term “variable region' of an antibody refers to the amino acid sequence corresponding to an antibody produced variable region of an antibody light chain, or the variable by a human. A human antibody may be made using any of region of an antibody heavy chain, either alone or in 45 the techniques known in the art. This definition of a human combination. The variable regions of the heavy and light antibody specifically excludes a humanized antibody com chains each consist of four framework regions (FR) con prising non-human CDRS. nected by three complementarity determining regions The term "chimeric antibody” as used herein refers to an (CDRs), also known as “hypervariable regions”. The CDRs antibody wherein the amino acid sequence of the immuno in each chain are held together in close proximity by the 50 globulin molecule is derived from two or more species. framework regions and, with the CDRs from the other chain, Typically, the variable region of both light and heavy chains contribute to the formation of the antigen-binding site of the corresponds to the variable region of antibodies derived antibody. There are at least two techniques for determining from one species of mammal (e.g., mouse, rat, rabbit, etc.) CDRS: (1) an approach based on cross-species sequence with the desired specificity, affinity, and/or binding capabil variability (i.e., Kabat et al., 1991, Sequences of Proteins of 55 ity, while the constant regions correspond to sequences in Immunological Interest, 5th Edition, National Institutes of antibodies derived from another species (usually human). Health, Bethesda, Md.), and (2) an approach based on The phrase “affinity-matured antibody” as used herein crystallographic Studies of antigen-antibody complexes (Al refers to an antibody with one or more alterations in one or Lazikani et al., 1997, J. Mol. Biol., 273:927-948). In addi more CDRs thereof that result in an improvement in the tion, combinations of these two approaches are sometimes 60 affinity of the antibody for an antigen, compared to a parent used in the art to determine CDRs. antibody that does not possess those alterations(s). The The term “monoclonal antibody” as used herein refers to definition also includes alterations in non-CDR residues a homogeneous antibody population involved in the highly made in conjunction with alterations to CDR residues. specific recognition and binding of a single antigenic deter Preferred affinity-matured antibodies will have nanomolar minant or epitope. This is in contrast to polyclonal antibod 65 or even picomolar affinities for the target antigen. Affinity ies that typically include a mixture of different antibodies matured antibodies are produced by procedures known in directed against a variety of different antigenic determinants. the art. For example, Marks et al., 1992, Bio/Technology US 9,598,497 B2 17 18 10:779-783, describes affinity maturation by VH and VL non-limiting example, a bispecific antibody may comprise domain shuffling. Random mutagenesis of CDR and/or one antigen-binding site that recognizes an epitope on one framework residues is described by Barbas et al., 1994, protein (e.g., human RSPO3) and further comprise a second, PNAS, 91:38.09-3813; Schier et al., 1995, Gene, 169: 147 different antigen-binding site that recognizes a different 155; Yelton et al., 1995, J. Immunol. 155:1994-2004; Jack epitope on a second protein (e.g., human RSPO2). Gener son et al., 1995, J. Immunol., 154:3310-9; and Hawkins et ally, but not necessarily, reference to binding means specific al., 1992, J. Mol. Biol., 226:889-896. Site-directed muta binding. genesis may also be used to obtain affinity-matured anti The terms “polypeptide' and “peptide' and “protein’ are bodies. used interchangeably herein and refer to polymers of amino The terms "epitope' and “antigenic determinant” are used 10 acids of any length. The polymer may be linear or branched, interchangeably herein and refer to that portion of an antigen it may comprise modified amino acids, and it may be capable of being recognized and specifically bound by a interrupted by non-amino acids. The terms also encompass particular antibody. When the antigen is a polypeptide, an amino acid polymer that has been modified naturally or epitopes can be formed both from contiguous amino acids by intervention; for example, disulfide bond formation, and noncontiguous amino acids juxtaposed by tertiary fold 15 glycosylation, lipidation, acetylation, phosphorylation, or ing of a protein. Epitopes formed from contiguous amino any other manipulation or modification, Such as conjugation acids (also referred to as linear epitopes) are typically with a labeling component. Also included within the defi retained upon protein denaturing, whereas epitopes formed nition are, for example, polypeptides containing one or more by tertiary folding (also referred to as conformational analogs of an amino acid (including, for example, unnatural epitopes) are typically lost upon protein denaturing. An amino acids), as well as other modifications known in the epitope typically includes at least 3, and more usually, at art. It is understood that, because the polypeptides of this least 5 or 8-10 amino acids in a unique spatial conformation. invention may be based upon antibodies, in certain embodi The terms "heteromultimeric molecule' or "heteromulti ments, the polypeptides can occur as single chains or asso mer' or "heteromultimeric complex” or "heteromultimeric ciated chains. polypeptide' are used interchangeably herein to refer to a 25 The terms “polynucleotide' and “nucleic acid are used molecule comprising at least a first polypeptide and a second interchangeably herein and refer to polymers of nucleotides polypeptide, wherein the second polypeptide differs in of any length, and include DNA and RNA. The nucleotides amino acid sequence from the first polypeptide by at least can be deoxyribonucleotides, ribonucleotides, modified one amino acid residue. The heteromultimeric molecule can nucleotides or bases, and/or their analogs, or any substrate comprise a "heterodimer' formed by the first and second 30 that can be incorporated into a polymer by DNA or RNA polypeptide or can form higher order tertiary structures polymerase. where additional polypeptides are present. “Conditions of high stringency” may be identified by The terms “selectively binds' or “specifically binds” those that: (1) employ low ionic strength and high tempera mean that a binding agent or an antibody reacts or associates ture for washing, for example 15 mM NaCl/1.5 mM sodium more frequently, more rapidly, with greater duration, with 35 citrate/0.1% sodium dodecyl sulfate at 50° C.; (2) employ greater affinity, or with some combination of the above to the during hybridization a denaturing agent, Such as formamide, epitope, protein or target molecule than with alternative for example, 50% (v/v) formamide with 0.1% bovine serum Substances, including unrelated proteins. In certain embodi albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM ments “specifically binds' means, for instance, that an sodium phosphate buffer at pH 6.5 in 5xSSC (0.75M NaCl, antibody binds a protein with a K, of about 0.1 mM or less, 40 75 mM sodium citrate) at 42° C.; or (3) employ during but more usually less than about 1 uM. In certain embodi hybridization 50% formamide in 5xSSC, 50 mM sodium ments, “specifically binds' means that an antibody binds a phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5xDen target at times with a K of at least about 0.1 uM or less, at hardt’s solution, sonicated salmon sperm DNA (50 ug/ml), other times at least about 0.01 uM or less, and at other times 0.1% SDS, and 10% dextran sulfate at 42°C., with washes at least about 1 nM or less. Because of the sequence identity 45 at 42° C. in 0.2xSSC and 50% formamide, followed by a between homologous proteins in different species, specific high-stringency wash consisting of 0.1XSSC containing binding can include an antibody that recognizes a protein in EDTA at 55° C. more than one species (e.g., human RSPO3 and mouse The terms “identical' or percent “identity” in the context RSPO3). Likewise, because of homology within certain of two or more nucleic acids or polypeptides, refer to two or regions of polypeptide sequences of different proteins, spe 50 more sequences or Subsequences that are the same or have cific binding can include an antibody (or other polypeptide a specified percentage of nucleotides or amino acid residues or binding agent) that recognizes more than one protein that are the same, when compared and aligned (introducing (e.g., human RSPO3 and human RSPO1). It is understood gaps, if necessary) for maximum correspondence, not con that, in certain embodiments, an antibody or binding moiety sidering any conservative amino acid Substitutions as part of that specifically binds a first target may or may not specifi 55 the sequence identity. The percent identity may be measured cally bind a second target. As such, “specific binding does using sequence comparison Software or algorithms or by not necessarily require (although it can include) exclusive visual inspection. Various algorithms and Software that may binding, i.e. binding to a single target. Thus, an antibody be used to obtain alignments of amino acid or nucleotide may, in certain embodiments, specifically bind more than sequences are well-known in the art. These include, but are one target. In certain embodiments, multiple targets may be 60 not limited to, BLAST, ALIGN, Megalign, BestFit, GCG bound by the same antigen-binding site on the antibody. For Wisconsin Package, and variations thereof. In some embodi example, an antibody may, in certain instances, comprise ments, two nucleic acids or polypeptides of the invention are two identical antigen-binding sites, each of which specifi substantially identical, meaning they have at least 70%, at cally binds the same epitope on two or more proteins (e.g., least 75%, at least 80%, at least 85%, at least 90%, and in RSPO3 and RSPO1). In certain alternative embodiments, an 65 some embodiments at least 95%, 96%, 97%, 98%, 99% antibody may be multispecific and comprise at least two nucleotide or amino acid residue identity, when compared antigen-binding sites with differing specificities. By way of and aligned for maximum correspondence, as measured US 9,598,497 B2 19 20 using a sequence comparison algorithm or by visual inspec similar cancerous lesion at a new location. A "metastatic' or tion. In some embodiments, identity exists over a region of "metastasizing cell is one that loses adhesive contacts with the sequences that is at least about 10, at least about 20, at neighboring cells and migrates via the bloodstream or lymph least about 40-60 residues, at least about 60-80 residues in from the primary site of disease to invade neighboring body length or any integral value therebetween. In some embodi Structures. ments, identity exists over a longer region than 60-80 The terms “cancer stem cell' and “CSC and "tumor stem residues, such as at least about 80-100 residues, and in some cell and “tumor initiating cell are used interchangeably embodiments the sequences are Substantially identical over herein and refer to cells from a cancer or tumor that: (1) have the full length of the sequences being compared, such as the extensive proliferative capacity; 2) are capable of asymmet coding region of a nucleotide sequence. 10 ric cell division to generate one or more types of differen A “conservative amino acid substitution' is one in which tiated cell progeny wherein the differentiated cells have one amino acid residue is replaced with another amino acid reduced proliferative or developmental potential; and (3) are residue having a similar side chain. Families of amino acid capable of symmetric cell divisions for self-renewal or residues having similar side chains have been defined in the self-maintenance. These properties confer on the cancer art, including basic side chains (e.g., lysine, arginine, histi 15 stem cells the ability to form or establish a tumor or cancer dine), acidic side chains (e.g., aspartic acid, glutamic acid), upon serial transplantation into an immunocompromised uncharged polar side chains (e.g., glycine, asparagine, glu host (e.g., a mouse) compared to the majority of tumor cells tamine, serine, threonine, tyrosine, cysteine), nonpolar side that fail to form tumors. Cancer stem cells undergo self chains (e.g., alanine, Valine, leucine, isoleucine, proline, renewal versus differentiation in a chaotic manner to form phenylalanine, methionine, tryptophan), beta-branched side tumors with abnormal cell types that can change over time chains (e.g., threonine, Valine, isoleucine) and aromatic side as mutations occur. chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). The terms “cancer cell' and "tumor cell refer to the total For example, Substitution of a phenylalanine for a tyrosine population of cells derived from a cancer or tumor or is a conservative substitution. Preferably, conservative sub pre-cancerous lesion, including both non-tumorigenic cells, stitutions in the sequences of the polypeptides and antibod 25 which comprise the bulk of the cancer cell population, and ies of the invention do not abrogate the binding of the tumorigenic stem cells (cancer stem cells). As used herein, polypeptide orantibody containing the amino acid sequence, the terms “cancer cell' or “tumor cell” will be modified by to the antigen?s), i.e., the one or more RSPO protein(s) to the term “non-tumorigenic’ when referring solely to those which the polypeptide or antibody binds. Methods of iden cells lacking the capacity to renew and differentiate to tifying nucleotide and amino acid conservative Substitutions 30 distinguish those tumor cells from cancer stem cells. which do not eliminate antigen binding are well-known in The term “tumorigenic' as used herein refers to the the art. functional features of a cancer stem cell including the The term “vector as used herein means a construct, properties of self-renewal (giving rise to additional tumori which is capable of delivering, and usually expressing, one genic cancer stem cells) and proliferation to generate all or more gene(s) or sequence(s) of interest in a host cell. 35 other tumor cells (giving rise to differentiated and thus Examples of vectors include, but are not limited to, viral non-tumorigenic tumor cells). vectors, naked DNA or RNA expression vectors, plasmid, The term “tumorigenicity” as used herein refers to the cosmid, or phage vectors, DNA or RNA expression vectors ability of a random sample of cells from the tumor to form associated with cationic condensing agents, and DNA or palpable tumors upon serial transplantation into immuno RNA expression vectors encapsulated in liposomes. 40 compromised hosts (e.g., mice). This definition also includes A polypeptide, antibody, polynucleotide, vector, cell, or enriched and/or isolated populations of cancer stem cells composition which is "isolated' is a polypeptide, antibody, that form palpable tumors upon serial transplantation into polynucleotide, vector, cell, or composition which is in a immunocompromised hosts (e.g., mice). form not found in nature. Isolated polypeptides, antibodies, The term 'subject” refers to any animal (e.g., a mammal), polynucleotides, vectors, cells, or compositions include 45 including, but not limited to, humans, non-human primates, those which have been purified to a degree that they are no canines, felines, rodents, and the like, which is to be the longer in a form in which they are found in nature. In some recipient of a particular treatment. Typically, the terms embodiments, a polypeptide, antibody, polynucleotide, vec “subject' and “patient” are used interchangeably herein in tor, cell, or composition which is isolated is substantially reference to a human Subject. pure. 50 The term “pharmaceutically acceptable” refers to a prod The term “substantially pure' as used herein refers to uct or compound approved (or approvable) by a regulatory material which is at least 50% pure (i.e., free from contami agency of the Federal government or a state government or nants), at least 90% pure, at least 95% pure, at least 98% listed in the U.S. Pharmacopeia or other generally recog pure, or at least 99% pure. nized pharmacopeia for use in animals, including humans. The terms "cancer and "cancerous” as used herein refer 55 The terms “pharmaceutically acceptable excipient, carrier to or describe the physiological condition in mammals in or adjuvant” or “acceptable pharmaceutical carrier” refer to which a population of cells are characterized by unregulated an excipient, carrier or adjuvant that can be administered to cell growth. Examples of cancer include, but are not limited a Subject, together with at least one binding agent (e.g., an to, carcinoma, blastoma, sarcoma, and hematologic cancers antibody) of the present disclosure, and which does not Such as lymphoma and leukemia. 60 destroy the activity of the binding agent. The excipient, The terms “tumor and “neoplasm' as used herein refer to carrier, or adjuvant should be non-toxic when administered any mass of tissue that results from excessive cell growth or with a binding agent in doses sufficient to deliver a thera proliferation, either benign (noncancerous) or malignant peutic effect. (cancerous) including pre-cancerous lesions. The terms “effective amount’ or “therapeutically effective The term “metastasis” as used herein refers to the process 65 amount’ or “therapeutic effect” refer to an amount of a by which a cancer spreads or transfers from the site of origin binding agent, an antibody, polypeptide, polynucleotide, to other regions of the body with the development of a small organic molecule, or other drug effective to “treat' a US 9,598,497 B2 21 22 disease or disorder in a Subject or mammal. In the case of (“RSPO3-binding agents'). In certain embodiments, the cancer, the therapeutically effective amount of a drug (e.g., RSPO3-binding agent specifically binds at least one other an antibody) has a therapeutic effect and as such can reduce human RSPO. In some embodiments, the at least one other the number of cancer cells; decrease tumorigenicity, tum human RSPO bound by a RSPO3-binding agent is selected origenic frequency or tumorigenic capacity; reduce the from the group consisting of RSPO1, RSPO2, and RSPO4. number or frequency of cancer stem cells; reduce the tumor In some embodiments, the RSPO3-binding agent is an size; reduce the cancer cell population; inhibit and/or stop antibody that binds a common epitope on RSPO1, RSPO2, cancer cell infiltration into peripheral organs including, for and/or RSPO4. In some embodiments, the RSPO3-binding example, the spread of cancer into Soft tissue and bone; agent is a bispecific antibody that binds a first epitope on inhibit and/or stop tumor or cancer cell metastasis; inhibit 10 RSPO3 and binds a second, different epitope on RSPO1, and/or stop tumor or cancer cell growth; relieve to some RSPO2, and/or RSPO4. The full-length amino acid (aa) extent one or more of the symptoms associated with the sequences for human RSPO1, RSPO2, RSPO3, and RSPO4 cancer, reduce morbidity and mortality; improve quality of are known in the art and are provided herein as SEQ ID life; or a combination of such effects. To the extent the agent, NO:1 (RSPO1), SEQ ID NO:2 (RSPO2), SEQ ID NO:3 for example an antibody, prevents growth and/or kills exist 15 (RSPO3), and SEQ ID NO:4 (RSPO4). ing cancer cells, it can be referred to as cytostatic and/or In certain embodiments, the antigen-binding site of a cytotoxic. RSPO-binding agent (e.g., an antibody or a bispecific anti The terms “treating” or “treatment” or “to treat” or body) described herein is capable of binding (or binds) one, “alleviating or “to alleviate' refer to both 1) therapeutic two, three, or four RSPOs. In certain embodiments, the measures that cure, slow down, lessen symptoms of, and/or antigen-binding site of a RSPO-binding agent (e.g., an halt progression of a diagnosed pathologic condition or antibody or a bispecific antibody) described herein is disorder and 2) prophylactic or preventative measures that capable of binding (or binds) RSPO3 as well as one, two, or prevent or slow the development of a targeted pathologic three other RSPOs. For example, in certain embodiments, condition or disorder. Thus those in need of treatment the antigen-binding site of a RSPO3-binding agent is include those already with the disorder; those prone to have 25 capable of specifically binding RSPO3 as well as at least one the disorder; and those in whom the disorder is to be other RSPO selected from the group consisting of RSPO1, prevented. In some embodiments, a Subject is successfully RSPO2, and RSPO4. In certain embodiments, the RSPO3 “treated according to the methods of the present invention binding agent specifically binds RSPO3 and RSPO1. In if the patient shows one or more of the following: a certain embodiments, the RSPO3-binding agent specifically reduction in the number of or complete absence of cancer 30 binds RSPO3 and RSPO2. In certain embodiments, the cells; a reduction in the tumor size; inhibition of or an RSPO3-binding agent specifically binds RSPO3 and absence of cancer cell infiltration into peripheral organs RSPO4. In certain embodiments, the RSPO3-binding agent including the spread of cancer cells into soft tissue and bone; specifically binds RSPO3, RSPO1, and RSPO2. In certain inhibition of or an absence of tumor or cancer cell metas embodiments, the RSPO3-binding agent specifically binds tasis; inhibition oran absence of cancer growth; relief of one 35 RSPO3, RSPO1, and RSPO4. In certain embodiments, the or more symptoms associated with the specific cancer, RSPO3-binding agent specifically binds RSPO3, RSPO2, reduced morbidity and mortality; improvement in quality of and RSPO4. In some embodiments, the RSPO3-binding life; reduction in tumorigenicity; reduction in the number or agent specifically binds human RSPO3. In some embodi frequency of cancer stem cells; or some combination of ments, the RSPO3-binding agent (e.g., antibody) specifi effects. 40 cally binds both human RSPO3 and mouse RSPO3. As used in the present disclosure and claims, the singular In certain embodiments, the agent-binding agent is an forms “a”, “an and “the include plural forms unless the antibody that specifically binds within amino acids 22-272 context clearly dictates otherwise. of human RSPO3. In certain embodiments, the agent-bind It is understood that wherever embodiments are described ing agent is an antibody that specifically binds within amino herein with the language "comprising otherwise analogous 45 acids 22-207 of human RSPO3. In certain embodiments, the embodiments described in terms of “consisting of and/or antigen-binding agent is an antibody that specifically binds “consisting essentially of are also provided. It is also within amino acids 35-135 of human RSPO3. In certain understood that wherever embodiments are described herein embodiments, the antigen-binding agent is an antibody that with the language "consisting essentially of otherwise specifically binds within amino acids 35-86 of human analogous embodiments described in terms of "consisting 50 RSPO3. In certain embodiments, the antigen-binding agent of are also provided. is an antibody that specifically binds within amino acids The term “and/or as used in a phrase such as “A and/or 92-135 of human RSPO3. In certain embodiments, the B” herein is intended to include both A and B: A or B: A RSPO3-binding agent binds within SEQID NO:5. In certain (alone); and B (alone). Likewise, the term “and/or as used embodiments, the RSPO3-binding agent or antibody binds a in a phrase such as "A, B, and/or C is intended to encom 55 furin-like cysteine-rich domain of RSPO3. In some embodi pass each of the following embodiments: A, B, and C: A, B, ments, the agent or antibody binds at least one amino acid or C. A or C. A or B: B or C.; A and C.; A and B; B and C: within a furin-like cysteine-rich domain of RSPO3. In A (alone); B (alone); and C (alone). certain embodiments, the RSPO3-binding agent or antibody binds within sequence SEQ ID NO:6 or SEQ ID NO:7. In II. RSPO-BINDING AGENTS 60 certain embodiments, the RSPO3-binding agent or antibody binds within sequence SEQID NO:6 and SEQID NO:7. In The present invention provides agents that specifically some embodiments, the RSPO3-binding agent binds the bind human RSPO proteins. These agents are referred to thrombospondin domain of RSPO3. In some embodiments, herein as “RSPO-binding agents'. In some embodiments, the RSPO3-binding agent or antibody binds at least one the RSPO-binding agent is an antibody. In some embodi 65 amino acid within the thrombospondin domain of RSPO3. ments, the RSPO-binding agent is a polypeptide. In certain In some embodiments, the RSPO3-binding agent or anti embodiments, the RSPO-binding agent binds RSPO3 body binds within SEQ ID NO:8. US 9,598,497 B2 23 24 In certain embodiments, the RSPO-binding agent or anti 20-fold or more, about 30-fold or more, about 50-fold or body binds at least one RSPO protein with a dissociation more, or about 100-fold or more. Modulation of the affinities constant (K) of about 1 uM or less, about 100 nM or less, of the two antigen-binding sites may affect the biological about 40 nM or less, about 20 nM or less, about 10 nM or activity of the bispecific antibody. For example, decreasing less, about 1 nM or less, or about 0.1 nM or less. In certain the affinity of the antigen-binding site for RSPO3 or the embodiments, a RSPO3-binding agent or antibody binds second target, may have a desirable effect, for example RSPO3 with a dissociation constant (K) of about 1 uM or decreased toxicity of the binding agent and/or increased less, about 100 nM or less, about 40 nM or less, about 20 nM therapeutic index. or less, about 10 nM or less, about 1 nM or less, or about 0.1 By way of non-limiting example, the bispecific antibody nM or less. In some embodiments, a RSPO3-binding agent 10 may comprise (a) a first antigen-binding site that binds or antibody binds RSPO3 with a K, of about 20 nM or less. human RSPO3 with a K, between about 0.1 nM and about In some embodiments, a RSPO3-binding agent or antibody 10 nM, and (b) a second antigen-binding site that specifi binds RSPO3 with a K, of about 10 nM or less. In some cally binds a second target (e.g., human RSPO2) with a K. embodiments, a RSPO3-binding agent or antibody binds between about 0.1 nM and about 20 nM, between about 0.5 RSPO3 with a K, of about 1 nM or less. In some embodi 15 nM and about 20 nM, or between about 1.0 nM and 10 nM. ments, a RSPO3-binding agent or antibody binds RSPO3 In certain embodiments, the RSPO-binding agent (e.g., an with a K, of about 0.5 nM or less. In some embodiments, a antibody) binds to at least one human RSPO protein with a RSPO3-binding agent or antibody binds RSPO3 with a K, half maximal effective concentration (ECs) of about 1 uM of about 0.1 nM or less. In certain embodiments, a RSPO3 or less, about 100 nM or less, about 40 nM or less, about 20 binding agent or antibody described herein binds at least one nM or less, about 10 nM or less, about 1 nM or less, or about other RSPO. In certain embodiments, a RSPO3-binding 0.1 nM or less. In certain embodiments, a RSPO3-binding agent or antibody described herein that binds at least one agent (e.g., an antibody) binds to human RSPO3 with a half other RSPO, binds at least one other RSPO with a K of maximal effective concentration (ECs) of about 1 uM or about 100 nM or less, about 20 nM or less, about 10 nM or less, about 100 nM or less, about 40 nM or less, about 20 nM less, about 1 nM or less or about 0.1 nM or less. For 25 or less, about 10 nM or less, about 1 nM or less, or about 0.1 example, in some embodiments, a RSPO3-binding agent or nM or less. In certain embodiments, a RSPO3-binding agent antibody also binds RSPO1, RSPO2, and/or RSPO4 with a (e.g., an antibody) also binds to human RSPO1, RSPO2, K of about 10 nM or less. In some embodiments, the and/or RSPO4 with an ECs of about 40 nM or less, about RSPO-binding agent binds both human RSPO and mouse 20 nM or less, about 10 nM or less, about 1 nM or less or RSPO with a K, of about 10 nM or less. In some embodi 30 about 0.1 nM or less. ments, a RSPO3-binding agent binds both human RSPO3 In certain embodiments, the RSPO3-binding agent is an and mouse RSPO3 with a K of about 1 nM or less. In some antibody. In some embodiments, the antibody is a recombi embodiments, a RSPO3-binding agent binds both human nant antibody. In some embodiments, the antibody is a RSPO3 and mouse RSPO3 with a K of about 0.1 nM or monoclonal antibody. In some embodiments, the antibody is less. In some embodiments, the dissociation constant of the 35 a chimeric antibody. In some embodiments, the antibody is binding agent (e.g., an antibody) to a RSPO3 protein is the a humanized antibody. In some embodiments, the antibody dissociation constant determined using a RSPO3 fusion is a human antibody. In some embodiments, the antibody is protein comprising at least a portion of the RSPO3 protein an IgA, Ig|D, IgE, IgG, or IgM antibody. In certain embodi immobilized on a Biacore chip. In some embodiments, the ments, the antibody is an IgG1 antibody. In certain embodi dissociation constant of the binding agent (e.g., an antibody) 40 ments, the antibody is an IgG2 antibody. In certain embodi to a RSPO3 protein is the dissociation constant determined ments, the antibody is an antibody fragment comprising an using the binding agent captured by an anti-human IgG antigen-binding site. In some embodiments, the antibody is antibody on a Biacore chip and a RSPO3 protein. a bispecific antibody or a multispecific antibody. In some In some embodiments, the RSPO3-binding agent is a embodiments, the antibody is a monovalent antibody. In bispecific antibody which comprises a first antigen-binding 45 Some embodiments, the antibody is a monospecific antibody. site that specifically binds RSPO3 and a second antigen In some embodiments, the antibody is a bivalent antibody. In binding site that specifically binds a second target. In some Some embodiments, the antibody is conjugated to a cyto embodiments, a RSPO3-binding agent or antibody binds toxic moiety. In some embodiments, the antibody is isolated. both RSPO3 and the second target with a K of about 100 In some embodiments, the antibody is Substantially pure. nM or less. In some embodiments, a RSPO3-binding agent 50 The RSPO3-binding agents (e.g., antibodies) of the pres or antibody binds both RSPO3 and the second target with a ent invention can be assayed for specific binding by any K of about 50 nM or less. In some embodiments, a method known in the art. The immunoassays which can be RSPO3-binding agent or antibody binds both RSPO3 and used include, but are not limited to, competitive and non the second target with a K of about 20 nM or less. In some competitive assay systems using techniques such as Biacore embodiments, a RSPO3-binding agent or antibody binds 55 analysis, FACS analysis, immunofluorescence, immunocy both RSPO3 and the second target with a K of about 10 nM tochemistry, Western blot analysis, radioimmunoassays, or less. In some embodiments, a RSPO3-binding agent or ELISA, 'sandwich’ immunoassays, immunoprecipitation antibody binds both RSPO3 and the second target with a K, assays, precipitation reactions, gel diffusion precipitin reac of about 1 nM or less. In some embodiments, the affinity of tions, immunodiffusion assays, agglutination assays, one of the antigen-binding sites may be weaker than the 60 complement-fixation assays, immunoradiometric assays, affinity of the other antigen-binding site. For example, the fluorescent immunoassays, and protein A immunoassays. K of one antigen binding site may be about 1 nM and the Such assays are routine and well-known in the art (see, e.g., K, of the second antigen-binding site may be about 10 nM. Ausubel et al., Editors, 1994-present, Current Protocols in In some embodiments, the difference in affinity between the Molecular Biology, John Wiley & Sons, Inc., New York, two antigen-binding sites may be about 2-fold or more, 65 N.Y.). about 3-fold or more, about 5-fold or more, about 8-fold or For example, the specific binding of an antibody to human more, about 10-fold or more, about 15-fold or more, about RSPO3 may be determined using ELISA. An ELISA assay US 9,598,497 B2 25 26 comprises preparing antigen, coating wells of a 96 well h131R008, h131R010, or h131R011; two or more of the microtiter plate with antigen, adding the RSPO3-binding CDRs of 131R002, 131R003, or the humanized variants antibody or other RSPO3-binding agent conjugated to a thereof, including hl31R005/131R007, h131R006A, detectable compound Such as an enzymatic Substrate (e.g. h131R006B, h131R008, h131R010, or hl31R011; three or horseradish peroxidase or alkaline phosphatase) to the well, 5 more of the CDRs of 131R002, 131R003, or the humanized incubating for a period of time and detecting the presence of variants thereof, including h131R005/131R007, the antibody bound to the antigen. In some embodiments, h131R006A, h131R006B, h131R008, h131R010, or the RSPO3-binding antibody or agent is not conjugated to a h131R011; four or more of the CDRs of 131R002, 131 R003, detectable compound, but instead a second conjugated anti or the humanized variants thereof, including h131R005/ body that recognizes the RSPO3-binding agent or antibody 10 131R007, h131R006A, h131R006B, h131R008, h131R010, (e.g., an anti-Fc antibody) and is conjugated to a detectable or h131R011; five or more of the CDRs of 131R002, compound is added to the well. In some embodiments, 131R003, or the humanized variants thereof, including instead of coating the well with the antigen, the RSPO3 131R005/131R007, h131R006A, h131R006B, or binding agent or antibody can be coated to the well and a h131R008, h131R010, or hl31R011; or all six of the CDRs second antibody conjugated to a detectable compound can 15 of 131R002, 131R003, or the humanized variants thereof, be added following the addition of the antigen to the coated including hl31R005/131R007, h131R006A, h131R006B, well. One of skill in the art would be knowledgeable as to h131R008, h131R010, or hl31R011. the parameters that can be modified to increase the signal detected as well as other variations of ELISAs known in the TABLE 1. art. In another example, the specific binding of an antibody to 131ROO2/131ROO3 and Humanized Variants human RSPO3 may be determined using FACS. A FACS HC CDR1 KASGYTFTDYS (SEQ ID NO: 9) or screening assay may comprise generating a cDNA construct KASGYTFTSYTE (SEQ ID NO: 34) o that expresses an antigen as a fusion protein (e.g., RSPO3-Fc DYSIH (SEO ID NO : 78) 25 or RSPO3-CD4TM), transfecting the construct into cells, HC CDR2 IYPSNGDS (SEO ID NO: 1.O) or expressing the antigen on the Surface of the cells, mixing the YIYPSNGDSGYNOKFK (SEO ID NO: 79) RSPO3-binding agent with the transfected cells, and incu bating for a period of time. The cells bound by the RSPO3 HC CDR3 ATYFANYFDY (SEQ ID NO: 11) o ATYFANNEDY (SEQ ID NO: 35) o binding agent may be identified using a secondary antibody TYFANNED (SEQ ID NO: 8O conjugated to a detectable compound (e.g., PE-conjugated 30 anti-Fc antibody) and a flow cytometer. One of skill in the C CDR1 OSVDYDGDSYM (SEO ID NO:12) o art would be knowledgeable as to the parameters that can be KASOSVDYDGDSYMN (SEO ID NO : 81) modified to optimize the signal detected as well as other C CDR2 AAS (SEQ ID NO: 13) o variations of FACS that may enhance screening (e.g., AASNLES (SEQ ID NO : 82) screening for blocking antibodies). 35 The binding affinity of an antibody or other binding-agent C CDR3 QOSNEDPLT (SEQ ID NO: 14) o to an antigen (e.g., RSPO3) and the off-rate of an antibody OOSNEDPLTF (SEQ ID NO: 83) antigen interaction can be determined by competitive bind ing assays. One example of a competitive binding assay is In certain embodiments, the invention provides a RSPO3 a radioimmunoassay comprising the incubation of labeled 40 binding agent (e.g., an antibody) that specifically binds antigen (e.g., Hor'I), or fragment or variant thereof, with human RSPO3, wherein the RSPO3-binding agent com the antibody of interest in the presence of increasing prises a heavy chain CDR1 comprising KASGYTFTDYS amounts of unlabeled antigen followed by the detection of (SEQ ID NO:9), KASGYTFTSYTF (SEQ ID NO:34), or the antibody bound to the labeled antigen. The affinity of the DYSIH (SEQ ID NO:78), a heavy chain CDR2 comprising antibody for the antigen and the binding off-rates can be 45 IYPSNGDS (SEQID NO:10) or YIYPSNGDSGYNQKFK determined from the data by Scatchard plot analysis. In (SEQ ID NO:79), and a heavy chain CDR3 comprising Some embodiments, Biacore kinetic analysis is used to ATYFANYFDY (SEQ ID NO:11), ATYFANNFDY (SEQ determine the binding on and off rates of antibodies or ID NO:35), or TYFANNFD (SEQ ID NO:80). In some agents that bind an antigen (e.g., RSPO3). In some embodi embodiments, the RSPO3-binding agent further comprises a ments, Biacore kinetic analysis comprises analyzing the 50 light chain CDR1 comprising QSVDYDGDSYM (SEQ ID binding and dissociation of antibodies from chips with NO:12) or KASQSVDYDGDSYMN (SEQ ID NO:81), a immobilized antigen (e.g., RSPO3) on their surface. In some light chain CDR2 comprising AAS (SEQ ID NO:13) or embodiments, Biacore kinetic analysis comprises analyzing AASNLES (SEQ ID NO:82), and a light chain CDR3 the binding and dissociation of antigen (e.g., RSPO3) from comprising QQSNEDPLT (SEQ ID NO:14) or QQSNED chips with immobilized antibody (e.g., anti-RSPO3 anti- 55 PLTF (SEQID NO:83). In some embodiments, the RSPO3 body) on their surface. binding agent comprises a light chain CDR1 comprising In certain embodiments, the invention provides a RSPO3 QSVDYDGDSYM (SEQID NO:12) or KASQSVDYDGD binding agent (e.g., an antibody) that specifically binds SYMN (SEQ ID NO:81), a light chain CDR2 comprising human RSPO3, wherein the RSPO3-binding agent (e.g., an AAS (SEQ ID NO:13) or AASNLES (SEQID NO:82), and antibody) comprises one, two, three, four, five, and/or six of 60 a light chain CDR3 comprising QQSNEDPLT (SEQ ID the CDRs of antibody 131R002, antibody 131R003, or the NO:14) or QQSNEDPLTF (SEQ ID NO:83). In certain humanized variants thereof, including hl31R005/131R007, embodiments, the RSPO3-binding agent comprises: (a) a h131R006A, h131R006B, h131R008, h131R010, or heavy chain CDR1 comprising KASGYTFTDYS (SEQ ID h131R011 (see Table 1). In some embodiments, the RSPO3 NO:9), a heavy chain CDR2 comprising IYPSNGDS (SEQ binding agent comprises one or more of the CDRs of 65 ID NO:10), and a heavy chain CDR3 comprising ATY 131R002, 131R003, or the humanized variants thereof, FANYFDY (SEQ ID NO:11), and (b) a light chain CDR1 including h131R005/131R007, h131R006A, h131R006B, comprising QSVDYDGDSYM (SEQ ID NO:12), a light US 9,598,497 B2 27 28 chain CDR2 comprising AAS (SEQ ID NO:13), and a light chain CDR1 comprising QSVDYDGDSYM (SEQ ID chain CDR3 comprising QQSNEDPLT (SEQID NO:14). In NO:12), KASQSVDYDGDSYMN (SEQ ID NO:81), or a certain embodiments, the RSPO3-binding agent comprises: variant thereof comprising 1, 2, 3, or 4 amino acid Substi (a) a heavy chain CDR1 comprising KASGYTFTDYS (SEQ tutions; (e) a light chain CDR2 comprising AAS (SEQ ID ID NO:9), a heavy chain CDR2 comprising IYPSNGDS NO:13), AASNLES (SEQ ID NO:82), or a variant thereof (SEQ ID NO:10), and a heavy chain CDR3 comprising comprising 1, 2, 3, or 4 amino acid substitutions; and (f) a ATYFANNFDY (SEQ ID NO:35), and (b) a light chain light chain CDR3 comprising QQSNEDPLT (SEQ ID CDR1 comprising QSVDYDGDSYM (SEQ ID NO:12), a NO:14), QQSNEDPLTF (SEQ ID NO:83), or a variant light chain CDR2 comprising AAS (SEQ ID NO:13), and a thereof comprising 1, 2, 3, or 4 amino acid substitutions. In light chain CDR3 comprising QQSNEDPLT (SEQ ID 10 certain embodiments, the amino acid Substitutions are con NO:14). In certain embodiments, the RSPO3-binding agent servative substitutions. In some embodiments, the substitu comprises: (a) a heavy chain CDR1 comprising KASGYT tions are made as part of a germline humanization process. FTSYTF (SEQID NO:34), a heavy chain CDR2 comprising In certain embodiments, the invention provides a RSPO3 IYPSNGDS (SEQ ID NO:10), and a heavy chain CDR3 binding agent (e.g., an antibody) that specifically binds comprising ATYFANYFDY (SEQ ID NO:11), and (b) a 15 RSPO3, wherein the RSPO3-binding agent comprises a light chain CDR1 comprising QSVDYDGDSYM (SEQ ID heavy chain variable region having at least about 80% NO:12), a light chain CDR2 comprising AAS (SEQ ID sequence identity to SEQ ID NO:15, SEQ ID NO:16, SEQ NO:13), and a light chain CDR3 comprising QQSNEDPLT ID NO:36, SEQID NO:37, SEQID NO:44, SEQID NO:45, (SEQ ID NO:14). In certain embodiments, the RSPO3 or SEQID NO:62 and/or a light chain variable region having binding agent comprises: (a) a heavy chain CDR1 compris at least 80% sequence identity to SEQ ID NO:17, SEQ ID ing KASGYTFTSYTF (SEQ ID NO:34), a heavy chain NO:72, or SEQ ID NO:86. In certain embodiments, the CDR2 comprising IYPSNGDS (SEQ ID NO:10), and a RSPO3-binding agent comprises a heavy chain variable heavy chain CDR3 comprising ATYFANNFDY (SEQ ID region having at least about 85%, at least about 90%, at least NO:35), and (b) a light chain CDR1 comprising QSVDY about 95%, at least about 97%, or at least about 99% DGDSYM (SEQ ID NO:12), a light chain CDR2 compris 25 sequence identity to SEQ ID NO:15. In certain embodi ing AAS (SEQ ID NO:13), and a light chain CDR3 com ments, the RSPO3-binding agent comprises a heavy chain prising QQSNEDPLT (SEQ ID NO:14). In certain variable region having at least about 85%, at least about embodiments, the RSPO3-binding agent comprises: (a) a 90%, at least about 95%, at least about 97%, or at least about heavy chain CDR1 comprising DYSIH (SEQ ID NO:78), a 99% sequence identity to SEQID NO:16. In certain embodi heavy chain CDR2 comprising YTYPSNGDSGYNQKFK 30 ments, the RSPO3-binding agent comprises a heavy chain (SEQ ID NO:79), and a heavy chain CDR3 comprising variable region having at least about 85%, at least about TYFANNFD (SEQ ID NO:80), and (b) a light chain CDR1 90%, at least about 95%, at least about 97%, or at least about comprising KASQSVDYDGDSYMN (SEQ ID NO:81), a 99% sequence identity to SEQID NO:36. In certain embodi light chain CDR2 comprising AASNLES (SEQ ID NO:82), ments, the RSPO3-binding agent comprises a heavy chain and a light chain CDR3 comprising QQSNEDPLTF (SEQ 35 variable region having at least about 85%, at least about ID NO:83). In certain embodiments, the RSPO3-binding 90%, at least about 95%, at least about 97%, or at least about agent comprises: (a) a heavy chain CDR1 comprising 99% sequence identity to SEQID NO:37. In certain embodi DYSIH (SEQ ID NO:78), a heavy chain CDR2 comprising ments, the RSPO3-binding agent comprises a heavy chain YIYPSNGDSGYNQKFK (SEQ ID NO:79), and a heavy variable region having at least about 85%, at least about chain CDR3 comprising TYFANNFD (SEQID NO:80), and 40 90%, at least about 95%, at least about 97%, or at least about (b) a light chain CDR1 comprising KASQSVDYDGD 99% sequence identity to SEQID NO:44. In certain embodi SYMN (SEQ ID NO:81), a light chain CDR2 comprising ments, the RSPO3-binding agent comprises a heavy chain AASNLES (SEQ ID NO:82), and a light chain CDR3 variable region having at least about 85%, at least about comprising QQSNEDPLT (SEQ ID NO:14). In certain 90%, at least about 95%, at least about 97%, or at least about embodiments, the RSPO3-binding agent comprises: (a) a 45 99% sequence identity to SEQID NO:45. In certain embodi heavy chain CDR1 comprising KASGYTFTDYS (SEQ ID ments, the RSPO3-binding agent comprises a heavy chain NO:9) or DYSIH (SEQ ID NO:78), a heavy chain CDR2 variable region having at least about 85%, at least about comprising IYPSNGDS (SEQ ID NO:10), and a heavy 90%, at least about 95%, at least about 97%, or at least about chain CDR3 comprising TYFANNFD (SEQID NO:80), and 99% sequence identity to SEQID NO:62. In certain embodi (b) a light chain CDR1 comprising QSVDYDGDSYM 50 ments, the RSPO3-binding agent comprises a light chain (SEQ ID NO:12), a light chain CDR2 comprising AAS variable region having at least about 85%, at least about (SEQ ID NO:13), and a light chain CDR3 comprising 90%, at least about 95%, at least about 97%, or at least about QQSNEDPLT (SEQ ID NO:14). 99% sequence identity to SEQID NO:17. In certain embodi In certain embodiments, the invention provides a RSPO3 ments, the RSPO3-binding agent comprises a light chain binding agent (e.g., an antibody or bispecific antibody) that 55 variable region having at least about 85%, at least about specifically binds human RSPO3, wherein the RSPO3 90%, at least about 95%, at least about 97%, or at least about binding agent comprises: (a) a heavy chain CDR1 compris 99% sequence identity to SEQID NO:72. In certain embodi ing KASGYTFTDYS (SEQ ID NO:9), KASGYTFTSYTF ments, the RSPO3-binding agent comprises a light chain (SEQ ID NO:34), DYSIH (SEQ ID NO:78), or a variant variable region having at least about 85%, at least about thereof comprising 1, 2, 3, or 4 amino acid substitutions; (b) 60 90%, at least about 95%, at least about 97%, or at least about a heavy chain CDR2 comprising IYPSNGDS (SEQ ID 99% sequence identity to SEQID NO:86. In certain embodi NO:10), YIYPSNGDSGYNQKFK (SEQ ID NO:79), or a ments, the RSPO3-binding agent comprises a heavy chain variant thereof comprising 1, 2, 3, or 4 amino acid Substi variable region having at least about 95% sequence identity tutions; (c) a heavy chain CDR3 comprising ATY to SEQID NO:15, SEQID NO:16, SEQID NO:36, SEQID FANYFDY (SEQ ID NO:11), ATYFANNFDY (SEQ ID 65 NO:37, SEQ ID NO:44, SEQ ID NO:45, or SEQ ID NO:62 NO:35), TYFANNFD (SEQID NO:80), or a variant thereof and/or a light chain variable region having at least about comprising 1, 2, 3, or 4 amino acid substitutions; (d) a light 95% sequence identity to SEQ ID NO:17, SEQ ID NO:72, US 9,598,497 B2 29 30 or SEQ ID NO:86. In certain embodiments, the RSPO3 ments, the RSPO3-binding agent comprises a heavy chain binding agent comprises a heavy chain variable region variable region consisting essentially of SEQID NO:45 and comprising SEQ ID NO:15, SEQ ID NO:16, SEQ ID a light chain variable region consisting essentially of SEQ NO:36, SEQID NO:37, SEQID NO:44, SEQID NO:45, or ID NO:72. In certain embodiments, the RSPO3-binding SEQ ID NO:62, and/or a light chain variable region com agent comprises a heavy chain variable region consisting prising SEQID NO:17, SEQ ID NO:72, or SEQID NO:86. essentially of SEQ ID NO:62 and a light chain variable In certain embodiments, the RSPO3-binding agent com region consisting essentially of SEQ ID NO:72. In certain prises a heavy chain variable region comprising SEQ ID embodiments, the RSPO3-binding agent comprises a heavy NO:15, SEQ ID NO:16, SEQ ID NO:36, SEQ ID NO:37, chain variable region consisting of SEQ ID NO:44 and a SEQ ID NO:44, SEQ ID NO:45, or SEQ ID NO:62 and a 10 light chain variable region consisting of SEQID NO:72. In light chain variable region comprising SEQID NO:17, SEQ certain embodiments, the RSPO3-binding agent comprises a ID NO:72, or SEQ ID NO:86. In certain embodiments, the heavy chain variable region consisting of SEQ ID NO:45 RSPO3-binding agent comprises a heavy chain variable and a light chain variable region consisting of SEQ ID region consisting essentially of SEQ ID NO:15, SEQ ID NO:72. In certain embodiments, the RSPO3-binding agent NO:16, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:44, 15 comprises a heavy chain variable region consisting of SEQ SEQID NO:45, or SEQID NO:62, and a light chain variable ID NO:62 and a light chain variable region consisting of region consisting essentially of SEQ ID NO:17, SEQ ID SEQ ID NO:72. NO:72, or SEQ ID NO:86. In certain embodiments, the In certain embodiments, the RSPO3-binding agent com RSPO3-binding agent comprises a heavy chain variable prises a heavy chain variable region comprising SEQ ID region consisting of SEQ ID NO:15, SEQ ID NO:16, SEQ NO:44 and a light chain variable region comprising SEQID ID NO:36, SEQID NO:37, SEQID NO:44, SEQID NO:45, NO:86. In certain embodiments, the RSPO3-binding agent or SEQ ID NO:62, and a light chain variable region con comprises a heavy chain variable region comprising SEQID sisting of SEQ ID NO:17, SEQ ID NO:72, or SEQ ID NO:45 and a light chain variable region comprising SEQID NO:86. NO:86. In certain embodiments, the RSPO3-binding agent In certain embodiments, the RSPO3-binding agent com 25 comprises a heavy chain variable region comprising SEQID prises a heavy chain variable region comprising SEQ ID NO:62 and a light chain variable region comprising SEQID NO:44 and a light chain variable region comprising SEQID NO:86. In certain embodiments, the RSPO3-binding agent NO:17. In certain embodiments, the RSPO3-binding agent comprises a heavy chain variable region consisting essen comprises a heavy chain variable region comprising SEQID tially of SEQ ID NO:44 and a light chain variable region NO:45 and a light chain variable region comprising SEQID 30 consisting essentially of SEQID NO:86. In certain embodi NO:17. In certain embodiments, the RSPO3-binding agent ments, the RSPO3-binding agent comprises a heavy chain comprises a heavy chain variable region comprising SEQID variable region consisting essentially of SEQID NO:45 and NO:62 and a light chain variable region comprising SEQID a light chain variable region consisting essentially of SEQ NO:17. In certain embodiments, the RSPO3-binding agent ID NO:86. In certain embodiments, the RSPO3-binding comprises a heavy chain variable region consisting essen 35 agent comprises a heavy chain variable region consisting tially of SEQ ID NO:44 and a light chain variable region essentially of SEQ ID NO:62 and a light chain variable consisting essentially of SEQID NO:17. In certain embodi region consisting essentially of SEQ ID NO:86. In certain ments, the RSPO3-binding agent comprises a heavy chain embodiments, the RSPO3-binding agent comprises a heavy variable region consisting essentially of SEQID NO:45 and chain variable region consisting of SEQ ID NO:44 and a a light chain variable region consisting essentially of SEQ 40 light chain variable region consisting of SEQID NO:86. In ID NO:17. In certain embodiments, the RSPO3-binding certain embodiments, the RSPO3-binding agent comprises a agent comprises a heavy chain variable region consisting heavy chain variable region consisting of SEQ ID NO:45 essentially of SEQ ID NO:62 and a light chain variable and a light chain variable region consisting of SEQ ID region consisting essentially of SEQ ID NO:17. In certain NO:86. In certain embodiments, the RSPO3-binding agent embodiments, the RSPO3-binding agent comprises a heavy 45 comprises a heavy chain variable region consisting of SEQ chain variable region consisting of SEQ ID NO:44 and a ID NO:62 and a light chain variable region consisting of light chain variable region consisting of SEQID NO:17. In SEQ ID NO:86. certain embodiments, the RSPO3-binding agent comprises a In certain embodiments, the invention provides a RSPO3 heavy chain variable region consisting of SEQ ID NO:45 binding agent (e.g., an antibody) that specifically binds and a light chain variable region consisting of SEQ ID 50 RSPO3, wherein the RSPO3-binding agent comprises: (a) a NO:17. In certain embodiments, the RSPO3-binding agent heavy chain having at least 90% sequence identity to SEQ comprises a heavy chain variable region consisting of SEQ ID NO:27, SEQID NO:28, SEQID NO:39, SEQID NO:42, ID NO:62 and a light chain variable region consisting of SEQID NO:48, SEQID NO:49, SEQID NO:64, or SEQID SEQ ID NO:17. NO:69; and/or (b) a light chain having at least 90% sequence In certain embodiments, the RSPO3-binding agent com 55 identity to SEQ ID NO:29, SEQ ID NO:74, or SEQ ID prises a heavy chain variable region comprising SEQ ID NO:88. In some embodiments, the RSPO3-binding agent NO:44 and a light chain variable region comprising SEQID comprises: (a) a heavy chain having at least 95% sequence NO:72. In certain embodiments, the RSPO3-binding agent identity to SEQID NO:27, SEQID NO:28, SEQID NO:39, comprises a heavy chain variable region comprising SEQID SEQ ID NO:42, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:45 and a light chain variable region comprising SEQID 60 NO:64, or SEQID NO:69; and/or (b) a light chain having at NO:72. In certain embodiments, the RSPO3-binding agent least 95% sequence identity to SEQ ID NO:29, SEQ ID comprises a heavy chain variable region comprising SEQID NO:74, or SEQ ID NO:88. In some embodiments, the NO:62 and a light chain variable region comprising SEQID RSPO3-binding agent comprises a heavy chain comprising NO:72. In certain embodiments, the RSPO3-binding agent SEQ ID NO:27 and/or a light chain comprising SEQ ID comprises a heavy chain variable region consisting essen 65 NO:29. In some embodiments, the RSPO3-binding agent tially of SEQ ID NO:44 and a light chain variable region comprises a heavy chain comprising SEQID NO:28 and/or consisting essentially of SEQID NO:72. In certain embodi a light chain comprising SEQ ID NO:29. In some embodi US 9,598,497 B2 31 32 ments, the RSPO3-binding agent comprises a heavy chain comprises a heavy chain comprising SEQID NO:64 and/or comprising SEQ ID NO:39 and/or a light chain comprising a light chain comprising SEQ ID NO:88. In some embodi SEQ ID NO:29. In some embodiments, the RSPO3-binding ments, the RSPO3-binding agent comprises a heavy chain agent comprises a heavy chain comprising SEQ ID NO:42 comprising SEQ ID NO:69 and/or a light chain comprising and/or a light chain comprising SEQ ID NO:29. In some SEQ ID NO:88. In some embodiments, the RSPO3-binding embodiments, the RSPO3-binding agent comprises a heavy agent comprises a heavy chain consisting essentially of SEQ chain comprising SEQ ID NO:48 and/or a light chain ID NO:27, SEQID NO:28, SEQID NO:39, SEQID NO:42, comprising SEQ ID NO:29. In some embodiments, the SEQID NO:48, SEQID NO:49, SEQID NO:64, or SEQID RSPO3-binding agent comprises a heavy chain comprising NO:69; and a light chain consisting essentially of SEQ ID SEQ ID NO:49 and/or a light chain comprising SEQ ID 10 NO:74. In some embodiments, the RSPO3-binding agent NO:29. In some embodiments, the RSPO3-binding agent comprises a heavy chain consisting of SEQID NO:27, SEQ comprises a heavy chain comprising SEQID NO:64 and/or ID NO:28, SEQID NO:39, SEQID NO:42, SEQID NO:48, a light chain comprising SEQ ID NO:29. In some embodi SEQ ID NO:49, SEQ ID NO:64, or SEQ ID NO:69 and a ments, the RSPO3-binding agent comprises a heavy chain light chain consisting of SEQ ID NO:74. In some embodi comprising SEQ ID NO:69 and/or a light chain comprising 15 ments, the RSPO3-binding agent comprises a heavy chain SEQ ID NO:29. In some embodiments, the RSPO3-binding consisting essentially of SEQ ID NO:48, SEQ ID NO:49, agent comprises a heavy chain comprising SEQ ID NO:48 SEQ ID NO:64, or SEQ ID NO:69; and a light chain and/or a light chain comprising SEQ ID NO:88. In some consisting essentially of SEQ ID NO:88. In some embodi embodiments, the RSPO3-binding agent comprises a heavy ments, the RSPO3-binding agent comprises a heavy chain chain comprising SEQ ID NO:49 and/or a light chain consisting of SEQ ID NO:48, SEQ ID NO:49, SEQ ID comprising SEQ ID NO:88. In some embodiments, the NO:64, or SEQ ID NO:69 and a light chain consisting of RSPO3-binding agent comprises a heavy chain comprising SEQ ID NO:88. SEQ ID NO:64 and/or a light chain comprising SEQ ID In certain embodiments, the RSPO3-binding agent com NO:88. In some embodiments, the RSPO3-binding agent prises a heavy chain comprising SEQID NO:48 and a light comprises a heavy chain comprising SEQID NO:69 and/or 25 chain comprising SEQ ID NO:29. In certain embodiments, a light chain comprising SEQ ID NO:88. In some embodi the RSPO3-binding agent comprises a heavy chain compris ments, the RSPO3-binding agent comprises a heavy chain ing SEQ ID NO:49 and a light chain variable region com consisting essentially of SEQ ID NO:27, SEQ ID NO:28, prising SEQ ID NO:29. In certain embodiments, the SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:48, SEQ ID RSPO3-binding agent comprises a heavy chain comprising NO:49, SEQID NO:64, or SEQID NO:69; and a light chain 30 SEQID NO:64 and a light chain variable region comprising consisting essentially of SEQ ID NO:29. In some embodi SEQ ID NO:29. In certain embodiments, the RSPO3-bind ments, the RSPO3-binding agent comprises a heavy chain ing agent comprises a heavy chain comprising SEQ ID consisting of SEQ ID NO:28, SEQ ID NO:28, SEQ ID NO:69 and a light chain variable region comprising SEQID NO:39, SEQ ID NO:42, SEQ ID NO:48, SEQ ID NO:49, NO:29. In certain embodiments, the RSPO3-binding agent SEQ ID NO:64, or SEQ ID NO:69 and a light chain 35 comprises a heavy chain consisting essentially of SEQ ID consisting of SEQ ID NO:29. In some embodiments, the NO:48 and a light chain consisting essentially of SEQ ID RSPO3-binding agent comprises a heavy chain consisting of NO:29. In certain embodiments, the RSPO3-binding agent SEQ ID NO:28, SEQ ID NO:28, SEQ ID NO:39, SEQ ID comprises a heavy chain consisting essentially of SEQ ID NO:42, SEQID NO:48, SEQID NO:49, SEQID NO:64, or NO:49 and a light chain consisting essentially of SEQ ID SEQ ID NO:69 and a light chain consisting of SEQ ID 40 NO:29. In certain embodiments, the RSPO3-binding agent NO:88. In some embodiments, the RSPO3-binding agent comprises a heavy chain consisting essentially of SEQ ID comprises a heavy chain comprising SEQID NO:27 and/or NO:64 and a light chain consisting essentially of SEQ ID a light chain comprising SEQ ID NO:74. In some embodi NO:29. In certain embodiments, the RSPO3-binding agent ments, the RSPO3-binding agent comprises a heavy chain comprises a heavy chain consisting essentially of SEQ ID comprising SEQ ID NO:28 and/or a light chain comprising 45 NO:69 and a light chain consisting essentially of SEQ ID SEQ ID NO:74. In some embodiments, the RSPO3-binding NO:29. In certain embodiments, the RSPO3-binding agent agent comprises a heavy chain comprising SEQ ID NO:39 comprises a heavy chain consisting of SEQ ID NO:48 and and/or a light chain comprising SEQ ID NO:74. In some a light chain consisting of SEQ ID NO:29. In certain embodiments, the RSPO3-binding agent comprises a heavy embodiments, the RSPO3-binding agent comprises a heavy chain comprising SEQ ID NO:42 and/or a light chain 50 chain consisting of SEQ ID NO:49 and a light chain con comprising SEQ ID NO:74. In some embodiments, the sisting of SEQ ID NO:29. In certain embodiments, the RSPO3-binding agent comprises a heavy chain comprising RSPO3-binding agent comprises a heavy chain consisting of SEQ ID NO:48 and/or a light chain comprising SEQ ID SEQ ID NO:64 and a light chain consisting of SEQ ID NO:74. In some embodiments, the RSPO3-binding agent NO:29. In certain embodiments, the RSPO3-binding agent comprises a heavy chain comprising SEQID NO:49 and/or 55 comprises a heavy chain consisting of SEQ ID NO:69 and a light chain comprising SEQ ID NO:74. In some embodi a light chain consisting of SEQ ID NO:29. ments, the RSPO3-binding agent comprises a heavy chain In certain embodiments, the RSPO3-binding agent com comprising SEQ ID NO:64 and/or a light chain comprising prises a heavy chain comprising SEQID NO:48 and a light SEQ ID NO:74. In some embodiments, the RSPO3-binding chain comprising SEQ ID NO:74. In certain embodiments, agent comprises a heavy chain comprising SEQ ID NO:69 60 the RSPO3-binding agent comprises a heavy chain compris and/or a light chain comprising SEQ ID NO:74. In some ing SEQ ID NO:49 and a light chain variable region com embodiments, the RSPO3-binding agent comprises a heavy prising SEQ ID NO:74. In certain embodiments, the chain comprising SEQ ID NO:48 and/or a light chain RSPO3-binding agent comprises a heavy chain comprising comprising SEQ ID NO:88. In some embodiments, the SEQID NO:64 and a light chain variable region comprising RSPO3-binding agent comprises a heavy chain comprising 65 SEQ ID NO:74. In certain embodiments, the RSPO3-bind SEQ ID NO:49 and/or a light chain comprising SEQ ID ing agent comprises a heavy chain comprising SEQ ID NO:88. In some embodiments, the RSPO3-binding agent NO:69 and a light chain variable region comprising SEQID US 9,598,497 B2 33 34 NO:74. In certain embodiments, the RSPO3-binding agent embodiments, a RSPO3-binding agent comprises the heavy comprises a heavy chain consisting essentially of SEQ ID chain variable region and/or light chain variable region of NO:48 and a light chain consisting essentially of SEQ ID the 131R002 antibody in a humanized form of the antibody. NO:74. In certain embodiments, the RSPO3-binding agent In certain embodiments, a RSPO3-binding agent comprises comprises a heavy chain consisting essentially of SEQ ID the heavy chain CDRs and/or light chain CDRs of the NO:49 and a light chain consisting essentially of SEQ ID 131R002 antibody in a humanized form of the antibody. In NO:74. In certain embodiments, the RSPO3-binding agent some embodiments, the humanized version of 131R002 is comprises a heavy chain consisting essentially of SEQ ID an IgG1 antibody. In some embodiments, the humanized NO:64 and a light chain consisting essentially of SEQ ID version of 131R002 is an IgG2 antibody. NO:74. In certain embodiments, the RSPO3-binding agent 10 In certain embodiments, a RSPO3-binding agent com comprises a heavy chain consisting essentially of SEQ ID prises, consists essentially of or consists of the antibody NO:69 and a light chain consisting essentially of SEQ ID 131 ROO2. NO:74. In certain embodiments, the RSPO3-binding agent In certain embodiments, a RSPO3-binding agent com comprises a heavy chain consisting of SEQ ID NO:48 and prises the heavy chain variable region and light chain a light chain consisting of SEQ ID NO:74. In certain 15 variable region of the 131 R003 antibody. In some embodi embodiments, the RSPO3-binding agent comprises a heavy ments, the RSPO3-binding agent comprises the heavy chain chain consisting of SEQ ID NO:49 and a light chain con variable region of the 131R003 antibody wherein the heavy sisting of SEQ ID NO:74. In certain embodiments, the chain variable region from 131R003 has been affinity RSPO3-binding agent comprises a heavy chain consisting of matured. In some embodiments, the RSPO3-binding agent SEQ ID NO:64 and a light chain consisting of SEQ ID comprises the heavy chain variable region of the 131R003 NO:74. In certain embodiments, the RSPO3-binding agent antibody wherein the heavy chain variable region comprises comprises a heavy chain consisting of SEQ ID NO:69 and at least one modified or altered CDR as compared to the a light chain consisting of SEQ ID NO:74. parent 131R003 antibody. In some embodiments, the RSPO In certain embodiments, the RSPO3-binding agent com binding agent comprises the heavy chain variable region of prises a heavy chain comprising SEQID NO:48 and a light 25 the 131 R003 antibody wherein the heavy chain variable chain comprising SEQ ID NO:88. In certain embodiments, region comprises a modified CDR1 as compared to the the RSPO3-binding agent comprises a heavy chain compris parent 131R003 antibody. In some embodiments, the RSPO ing SEQ ID NO:49 and a light chain variable region com binding agent comprises the heavy chain variable region of prising SEQ ID NO:88. In certain embodiments, the the 131 R003 antibody wherein the heavy chain variable RSPO3-binding agent comprises a heavy chain comprising 30 region comprises a modified CDR2 as compared to the SEQID NO:64 and a light chain variable region comprising parent 131R003 antibody. In some embodiments, the RSPO SEQ ID NO:88. In certain embodiments, the RSPO3-bind binding agent comprises the heavy chain variable region of ing agent comprises a heavy chain comprising SEQ ID the 131 R003 antibody wherein the heavy chain variable NO:69 and a light chain variable region comprising SEQID region comprises a modified CDR3 as compared to the NO:88. In certain embodiments, the RSPO3-binding agent 35 parent 131R003 antibody. In some embodiments, the RSPO comprises a heavy chain consisting essentially of SEQ ID binding agent comprises the heavy chain variable region of NO:48 and a light chain consisting essentially of SEQ ID the 131 R003 antibody wherein the heavy chain variable NO:88. In certain embodiments, the RSPO3-binding agent region comprises a modified CDR1 and CDR3 as compared comprises a heavy chain consisting essentially of SEQ ID to the parent 131R003 antibody. In certain embodiments, a NO:49 and a light chain consisting essentially of SEQ ID 40 RSPO3-binding agent comprises the heavy chain and light NO:88. In certain embodiments, the RSPO3-binding agent chain of the 131R003 antibody (with or without the leader comprises a heavy chain consisting essentially of SEQ ID sequence). In certain embodiments, a RSPO3-binding agent NO:64 and a light chain consisting essentially of SEQ ID is the 131R003 antibody. In certain embodiments, a RSPO3 NO:88. In certain embodiments, the RSPO3-binding agent binding agent is a variant of the 131R003 antibody that comprises a heavy chain consisting essentially of SEQ ID 45 comprises a different heavy chain CDR1 as compared to the NO:69 and a light chain consisting essentially of SEQ ID parent 131R003 antibody. In certain embodiments, a NO:88. In certain embodiments, the RSPO3-binding agent RSPO3-binding agent is a variant of the 131 R003 antibody comprises a heavy chain consisting of SEQ ID NO:48 and that comprises a different heavy chain CDR3 as compared to a light chain consisting of SEQ ID NO:88. In certain the parent 131R003 antibody. In certain embodiments, a embodiments, the RSPO3-binding agent comprises a heavy 50 RSPO3-binding agent is a variant of the 131 R003 antibody chain consisting of SEQ ID NO:49 and a light chain con that comprises a different heavy chain CDR1 and a different sisting of SEQ ID NO:88. In certain embodiments, the heavy chain CDR3 as compared to the parent 131R003 RSPO3-binding agent comprises a heavy chain consisting of antibody. In certain embodiments, a RSPO3-binding agent SEQ ID NO:64 and a light chain consisting of SEQ ID comprises the heavy chain variable region and/or light chain NO:88. In certain embodiments, the RSPO3-binding agent 55 variable region of the 131R003 antibody or of any of the comprises a heavy chain consisting of SEQ ID NO:69 and variants of 131R003 in a chimeric form of the antibody. In a light chain consisting of SEQ ID NO:88. certain embodiments, a RSPO3-binding agent comprises the In certain embodiments, a RSPO3-binding agent com heavy chain variable region and/or light chain variable prises the heavy chain variable region and light chain region of the 131R003 antibody or of any of the variants of variable region of the 131R002 antibody. In certain embodi 60 131R003 in a humanized form of the antibody. In certain ments, a RSPO3-binding agent comprises the heavy chain embodiments, a RSPO3-binding agent comprises the heavy and light chain of the 131R002 antibody (with or without the chain CDRs and/or light chain CDRs of the 131R003 leader sequence). In certain embodiments, a RSPO3-binding antibody or of any of the variants of 131R003 in a human agent is the 131R002 antibody. In certain embodiments, a ized form of the antibody. In some embodiments, the human RSPO3-binding agent comprises the heavy chain variable 65 ized version of 131R003 or of 131R003 variants is an IgG1 region and/or light chain variable region of the 131R002 antibody. In some embodiments, the humanized version of antibody in a chimeric form of the antibody. In certain 131R003 or of 131R003 variants is an IgG2 antibody. US 9,598,497 B2 35 36 In certain embodiments, a RSPO3-binding agent com embodiments, a RSPO3-binding agent is the h131R010 prises, consists essentially of, or consists of the antibody antibody. In certain embodiments, a RSPO3-binding agent is 131R003. In certain embodiments, a RSPO3-binding agent the h131R011 antibody. In certain embodiments, a RSPO3 comprises, consists essentially of, or consists of a variant of binding agent comprises the heavy chain variable region the antibody 131R003. 5 and/or light chain variable region of the h131R010 or In certain embodiments, a RSPO3-binding agent com h131R011 antibody in a chimeric form of the antibody. In prises the heavy chain variable region and light chain certain embodiments, a RSPO3-binding agent comprises the variable region of the 131R006B antibody. In certain heavy chain CDRs and/or light chain CDRs of the embodiments, a RSPO3-binding agent comprises the heavy h131R010 or hl31R011 antibody. In some embodiments, chain and light chain of the 131R006B antibody (with or 10 the anti-RSPO3 antibody is h131R010. In some embodi without the leader sequence). In certain embodiments, a ments, the anti-RSPO3 antibody is h131R011. RSPO3-binding agent is the 131R006B antibody. In certain In some embodiments, the RSPO3-binding agent com embodiments, a RSPO3-binding agent comprises the heavy prises a heavy chain variable region encoded by the plasmid chain variable region and/or light chain variable region of deposited with American Type Culture Collection (ATCC), the 131R006B antibody in a chimeric form of the antibody. 15 and designated PTA-120420. In some embodiments, the In certain embodiments, a RSPO3-binding agent comprises RSPO3-binding agent comprises a light chain variable the heavy chain variable region and/or light chain variable region encoded by the plasmid deposited with ATCC and region of the 131R006B antibody in a humanized form of designated PTA-120421. In some embodiments, the RSPO3 the antibody. In certain embodiments, a RSPO3-binding binding agent comprises a heavy chain variable region agent comprises the heavy chain CDRS and/or light chain encoded by the plasmid deposited with ATCC and desig CDRs of the 131R006B antibody in a humanized form of the nated PTA-120420, and a light chain variable region antibody. In some embodiments, the humanized version of encoded by the plasmid deposited with ATCC and desig 131R006B is an IgG1 antibody. In some embodiments, the nated PTA-120421. In some embodiments, the RSPO3 humanized version of 131R006B is an IgG2 antibody. binding agent comprises a heavy chain encoded by the In certain embodiments, a RSPO3-binding agent com 25 plasmid deposited with ATCC and designated PTA-120420. prises, consists essentially of, or consists of the antibody In some embodiments, the RSPO3-binding agent comprises 131R006B. In certain embodiments, a RSPO3-binding agent a light chain encoded by the plasmid deposited with ATCC comprises, consists essentially of, or consists of a variant of and designated PTA-120421. In some embodiments, the the antibody 131R006B. RSPO3-binding agent comprises a heavy chain encoded by In certain embodiments, a RSPO3-binding agent com 30 the plasmid deposited with ATCC and designated PTA prises the heavy chain variable region and light chain 120420, and a light chain encoded by the plasmid deposited variable region of the 131R005/131R007 antibody. In cer with ATCC and designated PTA-120421. tain embodiments, a RSPO3-binding agent comprises the In certain embodiments, a RSPO3-binding agent com heavy chain and light chain of the 131R005/131R007 anti prises, consists essentially of or consists of the antibody body (with or without the leader sequence). In certain 35 h131R010. In certain embodiments, a RSPO3-binding agent embodiments, a RSPO3-binding agent is the 131R005/ comprises, consists essentially of, or consists of a variant of 131R007 antibody. In certain embodiments, a RSPO3-bind the antibody h131R010. ing agent comprises the heavy chain variable region and/or In certain embodiments, a RSPO3-binding agent com light chain variable region of the 131R005/131R007 anti prises, consists essentially of or consists of the antibody body in a chimeric form of the antibody. In certain embodi 40 h131R011. In certain embodiments, a RSPO3-binding agent ments, a RSPO3-binding agent comprises the heavy chain comprises, consists essentially of, or consists of a variant of variable region and/or light chain variable region of the the antibody h131R011. 131R005/131R007 antibody in a humanized form of the In certain embodiments, the invention provides a RSPO3 antibody. In certain embodiments, a RSPO3-binding agent binding agent that is a bispecific antibody. In some embodi comprises the heavy chain CDRs and/or light chain CDRs of 45 ments, the RSPO3-binding agent is a bispecific antibody the 131R005/131R007 antibody in a humanized form of the comprising a first antigen-binding site that specifically binds antibody. In some embodiments, the humanized version of human RSPO3. In some embodiments, the RSPO3-binding 131R005/131R007 is an IgG1 antibody. In some embodi agent is a bispecific antibody comprising a first antigen ments, the humanized version of 131R005/131R007 is an binding site that specifically binds human RSPO3 and a IgG2 antibody. In some embodiments, the anti-RSPO3 anti 50 second antigen-binding site that binds a second target. In body is 131R008. some embodiments, the RSPO3-binding agent is a bispecific In certain embodiments, a RSPO3-binding agent com antibody comprising: a first antigen-binding site that spe prises, consists essentially of, or consists of the antibody cifically binds human RSPO3, wherein the first antigen 131R005/131R007. In certain embodiments, a RSPO3-bind binding site comprises a heavy chain CDR1 comprising ing agent comprises, consists essentially of, or consists of a 55 KASGYTFTDYS (SEQ ID NO:9), KASGYTFTSYTF variant of the antibody 131R005/131R007. (SEQ ID NO:34), or DYSIH (SEQ ID NO:78), a heavy In certain embodiments, a RSPO3-binding agent com chain CDR2 comprising IYPSNGDS (SEQ ID NO:10) or prises, consists essentially of, or consists of the antibody YTYPSNGDSGYNQKFK (SEQ ID NO:79), and a heavy 131R008. In certain embodiments, a RSPO3-binding agent chain CDR3 comprising ATYFANYFDY (SEQID NO:11), comprises, consists essentially of, or consists of a variant of 60 ATYFANNFDY (SEQ ID NO:35), ORTYFANNFD (SEQ the antibody 131R008. ID NO:80). In some embodiments, the RSPO3-binding In certain embodiments, a RSPO3-binding agent com agent is a bispecific antibody comprising: a first antigen prises the heavy chain variable region and light chain binding site that specifically binds human RSPO3, wherein variable region of the h131R010 or hl31R011 antibody. In the first antigen-binding site comprises a heavy chain CDR1 certain embodiments, a RSPO3-binding agent comprises the 65 comprising KASGYTFTDYS (SEQ ID NO:9), a heavy heavy chain and light chain of the hil31R010 or 131R011 chain CDR2 comprising IYPSNGDS (SEQ ID NO:10), and antibody (with or without the leader sequence). In certain a heavy chain CDR3 comprising ATYFANNFDY (SEQ ID US 9,598,497 B2 37 38 NO:35). In some embodiments, the RSPO3-binding agent is In some embodiments, the RSPO3-binding agent is a bispe a bispecific antibody comprising: a first antigen-binding site cific antibody comprising a first heavy chain variable region that specifically binds human RSPO3, wherein the first comprising SEQ ID NO:45. In some embodiments, the antigen-binding site comprises a heavy chain CDR1 com RSPO3-binding agent is a bispecific antibody comprising a prising KASGYTFTSYTF (SEQ ID NO:34), a heavy chain first heavy chain variable region comprising SEQID NO:62. CDR2 comprising IYPSNGDS (SEQ ID NO:10), and a In some embodiments, the RSPO3-binding agent is a bispe heavy chain CDR3 comprising ATYFANNFDY (SEQ ID cific antibody comprising a first light chain variable region NO:35). In some embodiments, the RSPO3-binding agent is comprising SEQ ID NO:17. In some embodiments, the a bispecific antibody comprising: a first antigen-binding site RSPO3-binding agent is a bispecific antibody comprising a that specifically binds human RSPO3, wherein the first 10 first light chain variable region comprising SEQID NO:72. antigen-binding site comprises a heavy chain CDR1 com In some embodiments, the RSPO3-binding agent is a bispe prising KASGYTFTSYTF (SEQ ID NO:34), a heavy chain cific antibody comprising a first light chain variable region CDR2 comprising IYPSNGDS (SEQ ID NO:10), and a comprising SEQ ID NO:86. heavy chain CDR3 comprising ATYFANYFDY (SEQ ID In some embodiments, the RSPO3-binding agent is a NO:11). In some embodiments, the RSPO3-binding agent is 15 bispecific antibody comprising a first heavy chain variable a bispecific antibody comprising: a first antigen-binding site region comprising SEQID NO:15, SEQID NO:16, SEQID that specifically binds human RSPO3, wherein the first NO:36, SEQID NO:37, SEQID NO:44, SEQID NO:45, or antigen-binding site comprises a heavy chain CDR1 com SEQ ID NO:62 and a first heavy chain constant region prising DYSIH (SEQ ID NO:80), a heavy chain CDR2 comprising SEQ ID NO:60 or SEQ ID NO:61. In some comprising YTYPSNGDSGYNQKFK (SEQID NO:79), and embodiments, the RSPO3-binding agent is a bispecific anti a heavy chain CDR3 comprising TYFANNFD (SEQ ID body comprising a first heavy chain variable region com NO:80). In some embodiments, the RSPO3-binding agent is prising SEQ ID NO:44 and a first heavy chain constant a bispecific antibody comprising: a first antigen-binding site region comprising SEQ ID NO:60 or SEQ ID NO:61. In that specifically binds human RSPO3, wherein the first some embodiments, the RSPO3-binding agent is a bispecific antigen-binding site comprises a heavy chain CDR1 com 25 antibody comprising a first heavy chain variable region prising DYSIH (SEQID NO:80) or KASGYTFTDYS (SEQ comprising SEQ ID NO:45 and a first heavy chain constant ID NO:9), a heavy chain CDR2 comprising IYPSNGDS region comprising SEQ ID NO:60 or SEQ ID NO:61. In (SEQ ID NO:10), and a heavy chain CDR3 comprising some embodiments, the RSPO3-binding agent is a bispecific TYFANNFD (SEQ ID NO:80). In some embodiments, the antibody comprising a first heavy chain variable region RSPO3-binding agent is a bispecific antibody comprising: a 30 comprising SEQ ID NO:62 and a first heavy chain constant first antigen-binding site that specifically binds human region comprising SEQ ID NO:60 or SEQ ID NO:61. RSPO3, wherein the first antigen-binding site comprises (a) In certain embodiments, the RSPO3-binding agent is a a heavy chain CDR1 comprising KASGYTFTDYS (SEQID bispecific antibody that specifically binds human RSPO3 NO:9), KASGYTFTSYTF (SEQ ID NO:34), or DYSIH and a second target. In some embodiments, the RSPO3 (SEQ ID NO:78), a heavy chain CDR2 comprising 35 binding agent is a bispecific antibody that specifically binds IYPSNGDS (SEQID NO:10) or YIYPSNGDSGYNQKFK human RSPO3 and a second human RSPO. In some embodi (SEQ ID NO:79), and a heavy chain CDR3 comprising ments, the RSPO3-binding agent is a bispecific antibody that ATYFANYFDY (SEQ ID NO:11), ATYFANNFDY (SEQ specifically binds human RSPO3 and a second human RSPO ID NO:35) or TYFANNFD (SEQ ID NO:80), and a second selected from the group consisting of RSPO1, RSPO2, and antigen-binding site, wherein the first antigen-binding site 40 RSPO4. Non-limiting examples of antibodies to human and the second antigen-binding site comprise a common RSPO have been described in, for example, International (i.e., identical) light chain. In some embodiments, the bispe Patent Pub. No. WO 2013/O12747. cific antibody comprises a first antigen-binding site com In some embodiments, the RSPO3-binding agent is a prising a light chain CDR1 comprising QSVDYDGDSYM bispecific antibody that specifically binds human RSPO3 (SEQ ID NO:12) or KASQSVDYDGDSYMN (SEQ ID 45 and human RSPO1. In some embodiments, the bispecific NO:81), a light chain CDR2 comprising AAS (SEQ ID antibody comprises: a) a first antigen-binding site that NO:13) or AASNLES (SEQ ID NO:82), and a light chain specifically binds human RSPO3, and b) a second antigen CDR3 comprising QQSNEDPLT (SEQ ID NO:14) or binding site that specifically binds human RSPO1, wherein QQSNEDPLTF (SEQ ID NO:83). the first antigen-binding site comprises a heavy chain CDR1 In some embodiments, the RSPO3-binding agent is a 50 comprising KASGYTFTDYS (SEQ ID NO:9), KASGYT bispecific antibody comprising a first heavy chain variable FTSYTF (SEQ ID NO:34), or DYSIH (SEQ ID NO:78), a region having at least about 80% sequence identity to SEQ heavy chain CDR2 comprising IYPSNGDS (SEQ ID ID NO:15, SEQID NO:16, SEQID NO:36, SEQID NO:37, NO:10) or YIYPSNGDSGYNQKFK (SEQ ID NO:79), and SEQ ID NO:44, SEQ ID NO:45, or SEQ ID NO:62. In a heavy chain CDR3 comprising ATYFANYFDY (SEQ ID certain embodiments, the RSPO3-binding agent is a bispe 55 NO:11), ATYFANNFDY (SEQ ID NO:35) or TYFANNFD cific antibody comprising a first heavy chain variable region (SEQ ID NO:80); and wherein both the first and second having at least about 85%, at least about 90%, at least about antigen-binding sites comprise a common light chain. 95%, at least about 97%, or at least about 99% sequence In some embodiments, the RSPO3-binding agent is a identity to SEQID NO:15, SEQID NO:16, SEQID NO:36, bispecific antibody that specifically binds human RSPO3 SEQID NO:37, SEQID NO:44, SEQID NO:45, or SEQID 60 and human RSPO2. In some embodiments, the bispecific NO:62. In some embodiments, the bispecific antibody com antibody comprises: a) a first antigen-binding site that prises a light chain variable region at least about 85%, at specifically binds human RSPO3, and b) a second antigen least about 90%, at least about 95%, at least about 97%, or binding site that specifically binds human RSPO2, wherein at least about 99% sequence identity to SEQID NO:17, SEQ the first antigen-binding site comprises a heavy chain CDR1 ID NO:72, or SEQ ID NO:86. In some embodiments, the 65 comprising KASGYTFTDYS (SEQ ID NO:9), KASGYT RSPO3-binding agent is a bispecific antibody comprising a FTSYTF (SEQ ID NO:34), or DYSIH (SEQ ID NO:78), a first heavy chain variable region comprising SEQID NO:44. heavy chain CDR2 comprising IYPSNGDS (SEQ ID US 9,598,497 B2 39 40 NO:10) or YTYPSNGDSGYNQKFK (SEQ ID NO:79), and sponding to positions 240 and 282 of IgG1 (SEQID NO:56) a heavy chain CDR3 comprising ATYFANYFDY (SEQ ID are replaced with lysine. In some embodiments, the RSPO3 NO:11), ATYFANNFDY (SEQ ID NO:35) or TYFANNFD binding agent is a bispecific antibody which comprises a first (SEQ ID NO:80); and wherein both the first and second human IgG2 constant region with amino acid substitutions at antigen-binding sites comprise a common light chain. positions corresponding to positions 249 and 288 of IgG2 In some embodiments, the RSPO3-binding agent is a (SEQ ID NO:57), wherein the amino acids at positions bispecific antibody that comprises a heavy chain variable corresponding to positions 249 and 288 of IgG2 (SEQ ID region from the anti-RSPO3 antibody 131R003. In some NO:57) are replaced with glutamate or aspartate, and a embodiments, the RSPO3-binding agent is a bispecific anti second human IgG2 constant region with amino acid Sub body which comprises a heavy chain variable region from a 10 stitutions at positions corresponding to positions 236 and variant of the anti-RSPO3 antibody 131R003. In some 278 of IgG2 (SEQ ID NO:57), wherein the amino acids at embodiments, the RSPO3-binding agent is a bispecific anti positions corresponding to positions 236 and 278 of IgG2 body that comprises a heavy chain variable region from the (SEQ ID NO:57) are replaced with lysine. In some embodi anti-RSPO3 antibody 131R006B. In some embodiments, the ments, the RSPO-binding agent is a bispecific antibody RSPO3-binding agent is a bispecific antibody that comprises 15 which comprises a first human IgG3 constant region with a heavy chain variable region from the anti-RSPO3 antibody amino acid Substitutions at positions corresponding to posi h131R005/131R007. In some embodiments, the RSPO3 tions 300 and 339 of IgG3 (SEQ ID NO:58), wherein the binding agent is a bispecific antibody that comprises a heavy amino acids at positions corresponding to positions 300 and chain variable region from the anti-RSPO3 antibody 339 of IgG3 (SEQ ID NO:58) are replaced with glutamate h131 RO10 or h131 RO11. or aspartate, and a second human IgG3 constant region with In some embodiments, the RSPO3-binding agent is a amino acid Substitutions at positions corresponding to posi bispecific antibody that comprises a first CH3 domain and a tions 287 and 329 of IgG3 (SEQ ID NO:58), wherein the second CH3 domain, each of which is modified to promote amino acids at positions corresponding to positions 287 and formation of heteromultimers. In some embodiments, the 329 of IgG3 (SEQ ID NO:58) are replaced with lysine. In first and second CH3 domains are modified using a knobs 25 some embodiments, the RSPO-binding agent is a bispecific into-holes technique. In some embodiments, the first and antibody which comprises a first human IgG4 constant second CH3 domains comprise changes in amino acids that region with amino acid Substitutions at positions corre result in altered electrostatic interactions. In some embodi sponding to positions 250 and 289 of IgG4 (SEQID NO:59), ments, the first and second CH3 domains comprise changes wherein the amino acids at positions corresponding to in amino acids that result in altered hydrophobic/hydrophilic 30 positions 250 and 289 of IgG4 (SEQID NO:59) are replaced interactions. with glutamate or aspartate, and a second human IgG4 In some embodiments, the RSPO3-binding agent is a constant region with amino acid substitutions at positions bispecific antibody that comprises heavy chain constant corresponding to positions 237 and 279 of IgG4 (SEQ ID regions selected from the group consisting of: (a) a first NO:59), wherein the amino acids at positions corresponding human IgG1 constant region, wherein the amino acids 35 to positions 237 and 279 of IgG4 (SEQ ID NO:59) are corresponding to positions 253 and 292 of IgG1 (SEQ ID replaced with lysine. NO:56) are replaced with glutamate or aspartate, and a In some embodiments, the RSPO3-binding agent is a second human IgG1 constant region, wherein the amino bispecific antibody which comprises a first human IgG1 acids corresponding to positions 240 and 282 of IgG1 (SEQ constant region with amino acid substitutions at positions ID NO:56) are replaced with lysine; (b) a first human IgG2 40 corresponding to positions 253 and 292 of IgG1 (SEQ ID constant region, wherein the amino acids corresponding to NO:56), wherein the amino acids are replaced with gluta positions 249 and 288 of IgG2 (SEQID NO:57) are replaced mate, and a second human IgG1 constant region with amino with glutamate or aspartate, and a second human IgG2 acid substitutions at positions corresponding to positions constant region wherein the amino acids corresponding to 240 and 282 of IgG1 (SEQ ID NO:56), wherein the amino positions 236 and 278 of IgG2 (SEQID NO:57) are replaced 45 acids are replaced with lysine. In some embodiments, the with lysine; (c) a first human IgG3 constant region, wherein RSPO3-binding agent is a bispecific antibody which com the amino acids corresponding to positions 300 and 339 of prises a first human IgG1 constant region with amino acid IgG3 (SEQ ID NO:58) are replaced with glutamate or Substitutions at positions corresponding to positions 253 and aspartate, and a second human IgG3 constant region wherein 292 of IgG1 (SEQ ID NO:56), wherein the amino acids are the amino acids corresponding to positions 287 and 329 of 50 replaced with aspartate, and a second human IgG1 constant IgG3 (SEQ ID NO:58) are replaced with lysine; and (d) a region with amino acid Substitutions at positions corre first human IgG4 constant region, wherein the amino acids sponding to positions 240 and 282 of IgG1 (SEQID NO:56), corresponding to positions 250 and 289 of IgG4 (SEQ ID wherein the amino acids are replaced with lysine. NO:59) are replaced with glutamate or aspartate, and a In some embodiments, the RSPO3-binding agent is a second IgG4 constant region wherein the amino acids cor 55 bispecific antibody which comprises a first human IgG2 responding to positions 237 and 279 of IgG4 (SEQ ID constant region with amino acid substitutions at positions NO:59) are replaced with lysine. corresponding to positions 249 and 288 of IgG2 (SEQ ID In some embodiments, the RSPO3-binding agent is a NO:57), wherein the amino acids are replaced with gluta bispecific antibody which comprises a first human IgG1 mate, and a second human IgG2 constant region with amino constant region with amino acid substitutions at positions 60 acid substitutions at positions corresponding to positions corresponding to positions 253 and 292 of IgG1 (SEQ ID 236 and 278 of IgG2 (SEQ ID NO:57), wherein the amino NO:56), wherein the amino acids at positions corresponding acids are replaced with lysine. In some embodiments, the to positions 253 and 292 of IgG1 (SEQ ID NO:56) are RSPO3-binding agent is a bispecific antibody which com replaced with glutamate or aspartate, and a second human prises a first human IgG2 constant region with amino acid IgG1 constant region with amino acid Substitutions at posi 65 Substitutions at positions corresponding to positions 249 and tions corresponding to positions 240 and 282 of IgG1 (SEQ 288 of IgG2 (SEQ ID NO:57), wherein the amino acids are ID NO:56), wherein the amino acids at positions corre replaced with aspartate, and a second human IgG2 constant US 9,598,497 B2 41 42 region with amino acid Substitutions at positions corre ments, the affinity of the second antigen-binding site is sponding to positions 236 and 278 of IgG2 (SEQID NO:57), increased. In some embodiments, the affinity of the second wherein the amino acids are replaced with lysine. antigen-binding site is decreased. In some embodiments, the In some embodiments, the RSPO3-binding agent is a affinities of both the RSPO3 and the second antigen-binding bispecific antibody which comprises a heavy chain constant sites are altered. region of SEQID NO:60. In some embodiments, the RSPO The invention provides polypeptides, including, but not binding agent is a bispecific antibody which comprises a limited to, antibodies that specifically bind human RSPO heavy chain constant region of SEQ ID NO:61. In some proteins. In some embodiments, the polypeptides bind embodiments, the RSPO3-binding agent is a bispecific anti human RSPO3. In some embodiments, the polypeptides body which comprises a first heavy chain constant region of 10 bind human RSPO3 and at least one additional human RSPO SEQID NO:60 and a second heavy chain constant region of selected from the group consisting of RSPO1, RSPO2, and SEQ ID NO:61. RSPO4. In some embodiments, the RSPO3-binding agent is a In certain embodiments, the polypeptide comprises one, bispecific antibody which binds RSPO3 with a K of about two, three, four, five, and/or six of the CDRs of antibody 50 nM or less, about 25 nM or less, about 10 nM or less, 15 131R002, 131R003, or variants of 131R003 including about 1 nM or less, or about 0.1 nM or less. In some h131R005/131R007, h131R006A, h131R006B, h131R010, embodiments, the RSPO3-binding agent is a bispecific anti and h131R011 (see Table 1 herein). In some embodiments, body which binds a second target (e.g., RSPO2) with a K, the polypeptide comprises CDRs with up to four (i.e., 0, 1, of about 50 nM or less, about 25 nM or less, about 10 nM 2, 3, or 4) amino acid substitutions per CDR. In certain or less, about 1 nM or less, or about 0.1 nM or less. In some embodiments, the heavy chain CDR(s) are contained within embodiments, the RSPO3-binding agent is a bispecific anti a heavy chain variable region. In certain embodiments, the body which binds RSPO3 with a K of about 50 nM or less light chain CDR(s) are contained within a light chain and binds a second target (e.g., RSPO2) with a K of about variable region. 50 nM or less. In some embodiments, the RSPO3-binding In some embodiments, the invention provides a polypep agent is a bispecific antibody which binds RSPO3 with a K, 25 tide that specifically binds human RSPO3, wherein the of about 25 nM or less and binds a second target (e.g., polypeptide comprises an amino acid sequence having at RSPO2) with a K, of about 25 nM or less. In some least about 80% sequence identity to SEQ ID NO:15, SEQ embodiments, the RSPO3-binding agent is a bispecific anti ID NO:16, SEQID NO:36, SEQID NO:37, SEQID NO:44, body which binds RSPO3 with a K of about 10 nM or less SEQ ID NO:45, or SEQ ID NO:62, and/or an amino acid and binds a second target (e.g., RSPO2) with a K of about 30 sequence having at least about 80% sequence identity to 10 nM or less. In some embodiments, the RSPO3-binding SEQ ID NO:17, SEQ ID NO:72, or SEQ ID NO:86. In agent is a bispecificantibody which binds RSPO3 with a K. certain embodiments, the polypeptide comprises an amino of about 1 nM or less and binds a second target (e.g., acid sequence having at least about 85%, at least about 90%, RSPO2) with a K of about 1 nM or less. at least about 95%, at least about 97%, or at least about 99% In some embodiments, the RSPO3-binding agent is a 35 sequence identity to SEQ ID NO:15, SEQ ID NO:16, SEQ bispecific antibody which comprises one antigen-binding ID NO:36, SEQID NO:37, SEQID NO:44, SEQID NO:45, site with a binding affinity that is weaker than the binding or SEQID NO:62. In certain embodiments, the polypeptide affinity of the second antigen-binding site. For example, in comprises an amino acid sequence having at least about some embodiments, the bispecific antibody may bind 85%, at least about 90%, at least about 95%, at least about RSPO3 with a K, ranging from about 0.1 nM to 1 nM and 40 97%, or at least about 99% sequence identity to SEQ ID may bind a second target (e.g., RSPO2) with a K, ranging NO:17, SEQ ID NO:72, or SEQ ID NO:86. In certain from about 1 nM to 10 nM. Or the bispecific antibody may embodiments, the polypeptide comprises an amino acid bind RSPO3 with a K, ranging from about 1 nM to 10 nM sequence having at least about 95% sequence identity to and may bind a second target (e.g., RSPO2) with a K. SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:36, SEQ ID ranging from about 0.1 nM to 1 nM. In some embodiments, 45 NO:37, SEQID NO:44, SEQID NO:45, or SEQID NO:62, the bispecific antibody may bind RSPO3 with a K, ranging and/or an amino acid sequence having at least about 95% from about 0.1 nM to 1 nM and may bind a second target sequence identity to SEQ ID NO:17, SEQ ID NO:72, or (e.g., RSPO2) with a K, ranging from about 1 nM to 10 nM. SEQ ID NO:86. In certain embodiments, the polypeptide Or the bispecific antibody may bind RSPO3 with a K. comprises an amino acid sequence comprising SEQ ID ranging from about 1 nM to 10 nM and may bind a second 50 NO:15 and/or an amino acid sequence comprising SEQ ID target (e.g., RSPO2) with a K, ranging from about 0.1 nM NO:17. In certain embodiments, the polypeptide comprises to 1 nM. In some embodiments, the difference in affinity an amino acid sequence comprising SEQ ID NO:16 and/or between the two antigen-binding sites may be about 2-fold an amino acid sequence comprising SEQ ID NO:17. In or more, about 3-fold or more, about 5-fold or more, about certain embodiments, the polypeptide comprises an amino 8-fold or more, about 10-fold or more, about 15-fold or 55 acid sequence comprising SEQ ID NO:36 and/or an amino more, about 30-fold or more, about 50-fold or more, or about acid sequence comprising SEQ ID NO:17. In certain 100-fold or more. In some embodiments, at least one amino embodiments, the polypeptide comprises an amino acid acid residue in at least one CDR of the antigen-binding site sequence comprising SEQID NO:37 and/or an amino acid for RSPO3 is substituted with a different amino acid so that sequence comprising SEQ ID NO:17. In certain embodi the affinity of the RSPO3-binding site is altered. In some 60 ments, the polypeptide comprises an amino acid sequence embodiments, the affinity of the RSPO3-binding site is comprising SEQID NO:44 and/or an amino acid sequence increased. In some embodiments, the affinity of the RSPO3 comprising SEQ ID NO:17. In certain embodiments, the binding site is decreased. In some embodiments, at least one polypeptide comprises an amino acid sequence comprising amino acid residue in at least one CDR of the antigen SEQ ID NO:45 and/or an amino acid sequence comprising binding site for the second target (e.g., RSPO2) is substi 65 SEQ ID NO:17. In certain embodiments, the polypeptide tuted with a different amino acid so that the affinity of the comprises an amino acid sequence comprising SEQ ID second antigen-binding site is altered. In some embodi NO:62 and/or an amino acid sequence comprising SEQ ID US 9,598,497 B2 43 44 NO:17. In certain embodiments, the polypeptide comprises SEQID NO:49, SEQ ID NO:64, or SEQID NO:69, and/or an amino acid sequence comprising SEQ ID NO:15 and/or an amino acid sequence comprising SEQID NO:29, SEQID an amino acid sequence comprising SEQ ID NO:72. In NO:74, or SEQ ID NO:88. In certain embodiments, the certain embodiments, the polypeptide comprises an amino polypeptide comprises an amino acid sequence comprising acid sequence comprising SEQ ID NO:16 and/or an amino 5 SEQ ID NO:48 and/or an amino acid sequence comprising acid sequence comprising SEQ ID NO:72. In certain SEQ ID NO:29. In certain embodiments, the polypeptide embodiments, the polypeptide comprises an amino acid comprises an amino acid sequence comprising SEQ ID sequence comprising SEQID NO:36 and/or an amino acid NO:49 and/or an amino acid sequence comprising SEQ ID sequence comprising SEQ ID NO:72. In certain embodi NO:29. In certain embodiments, the polypeptide comprises ments, the polypeptide comprises an amino acid sequence 10 an amino acid sequence comprising SEQ ID NO:64 and/or comprising SEQ ID NO:37 and/or an amino acid sequence an amino acid sequence comprising SEQ ID NO:29. In comprising SEQ ID NO:72. In certain embodiments, the certain embodiments, the polypeptide comprises an amino polypeptide comprises an amino acid sequence comprising acid sequence comprising SEQ ID NO:69 and/or an amino SEQ ID NO:44 and/or an amino acid sequence comprising acid sequence comprising SEQ ID NO:29. In certain SEQ ID NO:72. In certain embodiments, the polypeptide 15 embodiments, the polypeptide comprises an amino acid comprises an amino acid sequence comprising SEQ ID sequence comprising SEQID NO:48 and/or an amino acid NO:45 and/or an amino acid sequence comprising SEQ ID sequence comprising SEQ ID NO:74. In certain embodi NO:72. In certain embodiments, the polypeptide comprises ments, the polypeptide comprises an amino acid sequence an amino acid sequence comprising SEQ ID NO:62 and/or comprising SEQ ID NO:49 and/or an amino acid sequence an amino acid sequence comprising SEQ ID NO:72. In comprising SEQ ID NO:74. In certain embodiments, the certain embodiments, the polypeptide comprises an amino polypeptide comprises an amino acid sequence comprising acid sequence comprising SEQ ID NO:44 and/or an amino SEQ ID NO:64 and/or an amino acid sequence comprising acid sequence comprising SEQ ID NO:86. In certain SEQ ID NO:74. In certain embodiments, the polypeptide embodiments, the polypeptide comprises an amino acid comprises an amino acid sequence comprising SEQ ID sequence comprising SEQID NO:45 and/or an amino acid 25 NO:69 and/or an amino acid sequence comprising SEQ ID sequence comprising SEQ ID NO:86. In certain embodi NO:74. In certain embodiments, the polypeptide comprises ments, the polypeptide comprises an amino acid sequence an amino acid sequence comprising SEQ ID NO:48 and/or comprising SEQ ID NO:62 and/or an amino acid sequence an amino acid sequence comprising SEQ ID NO:88. In comprising SEQ ID NO:86. certain embodiments, the polypeptide comprises an amino In some embodiments, the invention provides a polypep 30 acid sequence comprising SEQ ID NO:49 and/or an amino tide that specifically binds human RSPO3, wherein the acid sequence comprising SEQ ID NO:88. In certain polypeptide comprises an amino acid sequence having at embodiments, the polypeptide comprises an amino acid least about 80% sequence identity to SEQ ID NO:21, SEQ sequence comprising SEQID NO:64 and/or an amino acid ID NO:22, SEQID NO:38, SEQID NO:41, SEQID NO:46, sequence comprising SEQ ID NO:88. In certain embodi SEQID NO:47, SEQ ID NO:63, or SEQID NO:68, and/or 35 ments, the polypeptide comprises an amino acid sequence an amino acid sequence having at least about 80% sequence comprising SEQ ID NO:69 and/or an amino acid sequence identity to SEQ ID NO:23, SEQ ID NO:73, or SEQ ID comprising SEQ ID NO:88. In certain embodiments, the NO:87. In certain embodiments, the polypeptide comprises polypeptide comprises an amino acid sequence consisting an amino acid sequence having at least about 85%, at least essentially of SEQ ID NO:27, SEQ ID NO:28, SEQ ID about 90%, at least about 95%, at least about 97%, or at least 40 NO:39, SEQ ID NO:42, SEQ ID NO:48, SEQ ID NO:49, about 99% sequence identity to SEQ ID NO:21, SEQ ID SEQ ID NO:64, or SEQ ID NO:69 and/or an amino acid NO:22, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:46, sequence consisting essentially of SEQ ID NO:29, SEQ ID SEQ ID NO:47, SEQ ID NO:63, or SEQ ID NO:68. In NO:74, SEQ ID NO:88. In certain embodiments, the poly certain embodiments, the polypeptide comprises an amino peptide comprises an amino acid sequence consisting essen acid sequence having at least about 85%, at least about 90%, 45 tially of SEQ ID NO:48 and/or an amino acid sequence at least about 95%, at least about 97%, or at least about 99% consisting essentially of SEQID NO:29. In certain embodi sequence identity to SEQ ID NO:23, SEQ ID NO:73, or ments, the polypeptide comprises an amino acid sequence SEQ ID NO:87. In certain embodiments, the polypeptide consisting essentially of SEQ ID NO:49 and/or an amino comprises an amino acid sequence having at least about 95% acid sequence consisting essentially of SEQ ID NO:29. In sequence identity to SEQ ID NO:21, SEQ ID NO:22, SEQ 50 certain embodiments, the polypeptide comprises an amino ID NO:38, SEQID NO:41, SEQID NO:46, SEQID NO:47, acid sequence consisting essentially of SEQ ID NO:64 SEQ ID NO:63, or SEQ ID NO:68, and/or an amino acid and/or an amino acid sequence consisting essentially of SEQ sequence having at least about 95% sequence identity to ID NO:29. In certain embodiments, the polypeptide com SEQ ID NO:23, SEQ ID NO:73, or SEQ ID NO:87. In prises an amino acid sequence consisting essentially of SEQ certain embodiments, the polypeptide comprises an amino 55 ID NO:69 and/or an amino acid sequence consisting essen acid sequence comprising SEQ ID NO:21, SEQID NO:22, tially of SEQ ID NO:29. In certain embodiments, the SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:46, SEQ ID polypeptide comprises an amino acid sequence consisting NO:47, SEQID NO:63, or SEQID NO:68, and/or an amino essentially of SEQID NO:48 and/or an amino acid sequence acid sequence comprising SEQ ID NO:23, SEQ ID NO:73, consisting essentially of SEQID NO:74. In certain embodi or SEQID NO:87. In certain embodiments, the polypeptide 60 ments, the polypeptide comprises an amino acid sequence consists essentially of SEQID NO:21, SEQID NO:22, SEQ consisting essentially of SEQ ID NO:49 and/or an amino ID NO:38, SEQID NO:41, SEQID NO:46, SEQID NO:47, acid sequence consisting essentially of SEQ ID NO:74. In SEQID NO:63, or SEQID NO:68, and/or SEQ ID NO:23, certain embodiments, the polypeptide comprises an amino SEQ ID NO:73, or SEQ ID NO:87. acid sequence consisting essentially of SEQ ID NO:64 In certain embodiments, the polypeptide comprises an 65 and/or an amino acid sequence consisting essentially of SEQ amino acid sequence comprising SEQ ID NO:27, SEQ ID ID NO:74. In certain embodiments, the polypeptide com NO:28, SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:48, prises an amino acid sequence consisting essentially of SEQ US 9,598,497 B2 45 46 ID NO:69 and/or an amino acid sequence consisting essen NO:69 and/or SEQ ID NO:29. In certain embodiments, the tially of SEQ ID NO:74. In certain embodiments, the polypeptide comprises an amino acid sequence comprising polypeptide comprises an amino acid sequence consisting SEQ ID NO:48 and/or an amino acid sequence comprising essentially of SEQID NO:48 and/or an amino acid sequence SEQ ID NO:74. In certain embodiments, the polypeptide consisting essentially of SEQID NO:88. In certain embodi 5 comprises an amino acid sequence comprising SEQ ID ments, the polypeptide comprises an amino acid sequence NO:49 and/or an amino acid sequence comprising SEQ ID consisting essentially of SEQ ID NO:49 and/or an amino NO:74. In certain embodiments, the polypeptide comprises acid sequence consisting essentially of SEQ ID NO:88. In an amino acid sequence comprising SEQ ID NO:64 and/or certain embodiments, the polypeptide comprises an amino an amino acid sequence comprising SEQ ID NO:74. In acid sequence consisting essentially of SEQ ID NO:64 10 certain embodiments, the polypeptide comprises an amino and/or an amino acid sequence consisting essentially of SEQ acid sequence comprising SEQ ID NO:69 and/or an amino ID NO:88. In certain embodiments, the polypeptide com acid sequence comprising SEQ ID NO:74. In certain prises an amino acid sequence consisting essentially of SEQ embodiments, the polypeptide consists essentially of SEQ ID NO:69 and/or an amino acid sequence consisting essen ID NO:27 and/or SEQ ID NO:74. In certain embodiments, tially of SEQ ID NO:88. 15 the polypeptide consists essentially of SEQID NO:28 and/or In some embodiments, the invention provides a polypep SEQ ID NO:74. In certain embodiments, the polypeptide tide that specifically binds human RSPO3, wherein the consists essentially of SEQ ID NO:48 and/or SEQ ID polypeptide comprises an amino acid sequence having at NO:74. In certain embodiments, the polypeptide consists least about 80% sequence identity to SEQ ID NO:27, SEQ essentially of SEQ ID NO:49 and/or SEQ ID NO:74. In ID NO:28, SEQID NO:39, SEQID NO:42, SEQID NO:48, certain embodiments, the polypeptide consists essentially of SEQID NO:49, SEQ ID NO:64, or SEQID NO:69, and/or SEQ ID NO:64 and/or SEQ ID NO:74. In certain embodi an amino acid sequence having at least about 80% sequence ments, the polypeptide consists essentially of SEQ ID identity to SEQ ID NO:29, SEQ ID NO:74, or SEQ ID NO:69 and/or SEQ ID NO:74. In certain embodiments, the NO:88. In certain embodiments, the polypeptide comprises polypeptide comprises an amino acid sequence comprising an amino acid sequence having at least about 85%, at least 25 SEQ ID NO:48 and/or an amino acid sequence comprising about 90%, at least about 95%, at least about 97%, or at least SEQ ID NO:88. In certain embodiments, the polypeptide about 99% sequence identity to SEQ ID NO:27, SEQ ID comprises an amino acid sequence comprising SEQ ID NO:28, SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:48, NO:49 and/or an amino acid sequence comprising SEQ ID SEQ ID NO:49, SEQ ID NO:64, or SEQ ID NO:69. In NO:88. In certain embodiments, the polypeptide comprises certain embodiments, the polypeptide comprises an amino 30 an amino acid sequence comprising SEQ ID NO:64 and/or acid sequence having at least about 85%, at least about 90%, an amino acid sequence comprising SEQ ID NO:88. In at least about 95%, at least about 97%, or at least about 99% certain embodiments, the polypeptide comprises an amino sequence identity to SEQ ID NO:29, SEQ ID NO:74, or acid sequence comprising SEQ ID NO:69 and/or an amino SEQ ID NO:88. In certain embodiments, the polypeptide acid sequence comprising SEQ ID NO:88. In certain comprises an amino acid sequence having at least about 95% 35 embodiments, the polypeptide consists essentially of SEQ sequence identity to SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:48 and/or SEQ ID NO:88. In certain embodiments, ID NO:39, SEQID NO:42, SEQID NO:48, SEQID NO:49, the polypeptide consists essentially of SEQID NO:49 and/or SEQ ID NO:64, or SEQ ID NO:69, and/or an amino acid SEQ ID NO:88. In certain embodiments, the polypeptide sequence having at least about 95% sequence identity to consists essentially of SEQ ID NO:64 and/or SEQ ID SEQID NO:29, SEQID NO:74, SEQID NO:88. In certain 40 NO:88. In certain embodiments, the polypeptide consists embodiments, the polypeptide comprises an amino acid essentially of SEQ ID NO:69 and/or SEQ ID NO:88. sequence comprising SEQID NO:27, SEQID NO:28, SEQ In some embodiments, a RSPO3-binding agent comprises ID NO:39, SEQID NO:42, SEQID NO:48, SEQID NO:49, a polypeptide comprising a sequence selected from the SEQ ID NO:64, or SEQ ID NO:69, and/or an amino acid group consisting of: SEQ ID NO:15, SEQ ID NO:16, SEQ sequence comprising SEQ ID NO:29, SEQ ID NO:74, or 45 ID NO:17, SEQID NO:21, SEQID NO:22, SEQID NO:23, SEQ ID NO:88. In certain embodiments, the polypeptide SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID comprises an amino acid sequence comprising SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, NO:48 and/or an amino acid sequence comprising SEQ ID SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:29. In certain embodiments, the polypeptide comprises NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, an amino acid sequence comprising SEQ ID NO:49 and/or 50 SEQ ID NO:49, SEQ ID NO:62, SEQ ID NO:63, SEQ ID an amino acid sequence comprising SEQ ID NO:29. In NO:64, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:72, certain embodiments, the polypeptide comprises an amino SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:86, SEQ ID acid sequence comprising SEQ ID NO:64 and/or an amino NO:87, and SEQ ID NO:88. acid sequence comprising SEQ ID NO:29. In certain Many proteins, including antibodies, contain a signal embodiments, the polypeptide comprises an amino acid 55 sequence that directs the transport of the proteins to various sequence comprising SEQID NO:69 and/or an amino acid locations. Signal sequences (also referred to as signal pep sequence comprising SEQ ID NO:29. In certain embodi tides or leader sequences) are located at the N-terminus of ments, the polypeptide consists essentially of SEQ ID nascent polypeptides. They target the polypeptide to the NO:27 and/or SEQ ID NO:29. In certain embodiments, the endoplasmic reticulum and the proteins are sorted to their polypeptide consists essentially of SEQ ID NO:28 and/or 60 destinations, for example, to the inner space of an organelle, SEQ ID NO:29. In certain embodiments, the polypeptide to an interior membrane, to the cells outer membrane, or to consists essentially of SEQ ID NO:48 and/or SEQ ID the cell exterior via secretion. Most signal sequences are NO:29. In certain embodiments, the polypeptide consists cleaved from the protein by a signal peptidase after the essentially of SEQ ID NO:49 and/or SEQ ID NO:29. In proteins are transported to the endoplasmic reticulum. The certain embodiments, the polypeptide consists essentially of 65 cleavage of the signal sequence from the polypeptide usually SEQ ID NO:64 and/or SEQ ID NO:29. In certain embodi occurs at a specific site in the amino acid sequence and is ments, the polypeptide consists essentially of SEQ ID dependent upon amino acid residues within the signal US 9,598,497 B2 47 48 sequence. Although there is usually one specific cleavage (e.g., antibody) competes for specific binding to RSPO3 site, more than one cleavage site may be recognized and/or with an antibody that comprises a heavy chain variable may be used by a signal peptidase resulting in a non region comprising SEQID NO:62 and a light chain variable homogenous N-terminus of the polypeptide. For example, region comprising SEQ ID NO:86. the use of different cleavage sites within a signal sequence In certain embodiments, a RSPO3-binding agent (e.g., can result in a polypeptide expressed with different N-ter antibody) competes for specific binding to RSPO3 with an minal amino acids. Accordingly, in Some embodiments, the antibody that comprises a heavy chain comprising SEQ ID polypeptides as described herein may comprise a mixture of NO:27, SEQ ID NO:28, SEQ ID NO:39, SEQ ID NO:42, polypeptides with different N-termini. In some embodi SEQID NO:48, SEQID NO:49, SEQID NO:64, or SEQID ments, the N-termini differ in length by 1, 2, 3, 4, or 5 amino 10 NO:69 and a light chain comprising SEQID NO:29, SEQID acids. In some embodiments, the polypeptide is Substantially NO:74, or SEQ ID NO:88. In certain embodiments, a homogeneous, i.e., the polypeptides have the same N-ter RSPO3-binding agent (e.g., antibody) competes for specific minus. In some embodiments, the signal sequence of the binding to RSPO3 with an antibody that comprises a heavy polypeptide comprises one or more (e.g., one, two, three, chain comprising SEQ ID NO:48 and a light chain com four, five, six, seven, eight, nine, ten, etc.) amino acid 15 prising SEQID NO:29. In certain embodiments, a RSPO3 substitutions and/or deletions as compared to a “native' or binding agent (e.g., antibody) competes for specific binding "parental signal sequence. In some embodiments, the signal to RSPO3 with an antibody that comprises a heavy chain sequence of the polypeptide comprises amino acid Substi comprising SEQ ID NO:49 and a light chain comprising tutions and/or deletions that allow one cleavage site to be SEQ ID NO:29. In certain embodiments, a RSPO3-binding dominant, thereby resulting in a Substantially homogeneous agent (e.g., antibody) competes for specific binding to polypeptide with one N-terminus. In some embodiments, a RSPO3 with an antibody that comprises a heavy chain signal sequence of the polypeptide affects the expression comprising SEQ ID NO:64 and a light chain comprising level of the polypeptide, e.g., increased expression or SEQ ID NO:29. In certain embodiments, a RSPO3-binding decreased expression. agent (e.g., antibody) competes for specific binding to In certain embodiments, a RSPO3-binding agent (e.g., 25 RSPO3 with an antibody that comprises a heavy chain antibody) competes for specific binding to RSPO3 with an comprising SEQ ID NO:69 and a light chain comprising antibody that comprises a heavy chain variable region SEQ ID NO:29. In certain embodiments, a RSPO3-binding comprising SEQ ID NO:15, SEQ ID NO:16, SEQ ID agent (e.g., antibody) competes for specific binding to NO:36, SEQID NO:37, SEQID NO:44, SEQID NO:45, or RSPO3 with an antibody that comprises a heavy chain SEQID NO:62, and a light chain variable region comprising 30 comprising SEQ ID NO:48 and a light chain comprising SEQ ID NO:17, SEQ ID NO:72, or SEQ ID NO:86. In SEQ ID NO:74. In certain embodiments, a RSPO3-binding certain embodiments, a RSPO3-binding agent (e.g., anti agent (e.g., antibody) competes for specific binding to body) competes for specific binding to RSPO3 with an RSPO3 with an antibody that comprises a heavy chain antibody that comprises a heavy chain variable region comprising SEQ ID NO:49 and a light chain comprising comprising SEQID NO:44 and a light chain variable region 35 SEQ ID NO:74. In certain embodiments, a RSPO3-binding comprising SEQ ID NO:17. In certain embodiments, a agent (e.g., antibody) competes for specific binding to RSPO3-binding agent (e.g., antibody) competes for specific RSPO3 with an antibody that comprises a heavy chain binding to RSPO3 with an antibody that comprises a heavy comprising SEQ ID NO:64 and a light chain comprising chain variable region comprising SEQID NO:45 and a light SEQ ID NO:74. In certain embodiments, a RSPO3-binding chain variable region comprising SEQID NO:17. In certain 40 agent (e.g., antibody) competes for specific binding to embodiments, a RSPO3-binding agent (e.g., antibody) com RSPO3 with an antibody that comprises a heavy chain petes for specific binding to RSPO3 with an antibody that comprising SEQ ID NO:69 and a light chain comprising comprises a heavy chain variable region comprising SEQID SEQ ID NO:74. In certain embodiments, a RSPO3-binding NO:62 and a light chain variable region comprising SEQID agent (e.g., antibody) competes for specific binding to NO:17. In certain embodiments, a RSPO3-binding agent 45 RSPO3 with an antibody that comprises a heavy chain (e.g., antibody) competes for specific binding to RSPO3 comprising SEQ ID NO:48 and a light chain comprising with an antibody that comprises a heavy chain variable SEQ ID NO:88. In certain embodiments, a RSPO3-binding region comprising SEQID NO:44 and a light chain variable agent (e.g., antibody) competes for specific binding to region comprising SEQID NO:72. In certain embodiments, RSPO3 with an antibody that comprises a heavy chain a RSPO3-binding agent (e.g., antibody) competes for spe 50 comprising SEQ ID NO:49 and a light chain comprising cific binding to RSPO3 with an antibody that comprises a SEQ ID NO:88. In certain embodiments, a RSPO3-binding heavy chain variable region comprising SEQID NO:45 and agent (e.g., antibody) competes for specific binding to a light chain variable region comprising SEQID NO:72. In RSPO3 with an antibody that comprises a heavy chain certain embodiments, a RSPO3-binding agent (e.g., anti comprising SEQ ID NO:64 and a light chain comprising body) competes for specific binding to RSPO3 with an 55 SEQ ID NO:88. In certain embodiments, a RSPO3-binding antibody that comprises a heavy chain variable region agent (e.g., antibody) competes for specific binding to comprising SEQID NO:62 and a light chain variable region RSPO3 with an antibody that comprises a heavy chain comprising SEQ ID NO:72. In certain embodiments, a comprising SEQ ID NO:69 and a light chain comprising RSPO3-binding agent (e.g., antibody) competes for specific SEQ ID NO:88. binding to RSPO3 with an antibody that comprises a heavy 60 In certain embodiments, a RSPO3-binding agent com chain variable region comprising SEQID NO:44 and a light petes with antibody 131R002 or antibody 131R003 for chain variable region comprising SEQID NO:86. In certain specific binding to human RSPO3. In certain embodiments, embodiments, a RSPO3-binding agent (e.g., antibody) com a RSPO3-binding agent competes with a variant of antibody petes for specific binding to RSPO3 with an antibody that 131R003 for specific binding to human RSPO3. In certain comprises a heavy chain variable region comprising SEQID 65 embodiments, a RSPO3-binding agent competes with a NO:45 and a light chain variable region comprising SEQID humanized version of antibody 131 R003 for specific bind NO:86. In certain embodiments, a RSPO3-binding agent ing to human RSPO3. In certain embodiments, a RSPO3 US 9,598,497 B2 49 50 binding agent competes with antibody h131R005/131R007 embodiments, the RSPO3-binding agent is an antibody that for specific binding to human RSPO3. In certain embodi binds an epitope on RSPO3 that overlaps with the epitope on ments, a RSPO3-binding agent competes with antibody RSPO3 bound by antibody h131R011. h131R008 for specific binding to human RSPO3. In certain In certain embodiments, the RSPO-binding agent (e.g., an embodiments, a RSPO3-binding agent competes with anti 5 antibody) described herein binds at least one human RSPO body h131R006A or antibody h131R006B for specific bind protein and modulates RSPO activity. In some embodi ing to human RSPO3. In certain embodiments, a RSPO3 ments, the RSPO-binding agent is a RSPO antagonist and binding agent competes with antibody h131R010 for decreases RSPO activity. In some embodiments, the RSPO specific binding to human RSPO3. In certain embodiments, binding agent is a RSPO antagonist and decreases B-catenin a RSPO3-binding agent competes with antibody h131R011 10 activity. for specific binding to human RSPO3. In some embodi In certain embodiments, a RSPO3-binding agent (e.g., an ments, a RSPO3-binding agent or antibody competes for antibody) described herein binds human RSPO3 and modu specific binding to RSPO3 in an in vitro competitive binding lates RSPO3 activity. In some embodiments, a RSPO3 assay. In some embodiments, the RSPO3 is human RSPO3. binding agent is a RSPO3 antagonist and decreases RSPO3 In some embodiments, the RSPO3 is mouse RSPO3. 15 activity. In some embodiments, a RSPO3-binding agent is a In certain embodiments, a RSPO3-binding agent (e.g., an RSPO3 antagonist and decreases B-catenin activity. antibody) binds the same epitope, or essentially the same In certain embodiments, the RSPO-binding agent (e.g., an epitope, on RSPO3 as an antibody of the invention. In antibody) is an antagonist of at least one human RSPO certain embodiments, a RSPO3-binding agent (e.g., an anti protein. In some embodiments, the RSPO-binding agent is body) binds the same epitope, or essentially the same an antagonist of at least one RSPO and inhibits RSPO epitope, on RSPO3 as antibody 131R002 or antibody activity. In certain embodiments, the RSPO-binding agent 131R003. In certain embodiments, a RSPO3-binding agent inhibits RSPO activity by at least about 10%, at least about (e.g., an antibody) binds the same epitope, or essentially the 20%, at least about 30%, at least about 50%, at least about same epitope, on RSPO3 as a variant of antibody 131R003. 75%, at least about 90%, or about 100%. In some embodi In certain embodiments, a RSPO3-binding agent (e.g., an 25 ments, the RSPO-binding agent inhibits activity of one, two, antibody) binds the same epitope, or essentially the same three, or four RSPO proteins. In some embodiments, the epitope, on RSPO3 as a humanized version of antibody RSPO-binding agent inhibits activity of human RSPO1, 131R003. In certain embodiments, a RSPO3-binding agent RSPO2, RSPO3, and/or RSPO4. In some embodiments, the (e.g., an antibody) binds the same epitope, or essentially the RSPO-binding agent is a RSPO3-binding agent. In some same epitope, on RSPO3 as antibody h131R006A or anti 30 embodiments, the RSPO3-binding agent inhibits RSPO3 body h131R006B. In certain embodiments, a RSPO3-bind activity. In certain embodiments, a RSPO3-binding agent ing agent (e.g., an antibody) binds the same epitope, or that inhibits human RSPO3 activity is antibody 131R002, essentially the same epitope, on RSPO3 as antibody antibody 131R003, or a variant of 131R003. In certain h131R005/131R007. In certain embodiments, a RSPO3 embodiments, a RSPO3-binding agent that inhibits human binding agent (e.g., an antibody) binds the same epitope, or 35 RSPO3 activity is a humanized version of antibody essentially the same epitope, on RSPO3 as antibody 131R002, antibody 131R003, or a variant of 131R003. In h131R008. In certain embodiments, a RSPO3-binding agent certain embodiments, a RSPO3-binding agent that inhibits (e.g., an antibody) binds the same epitope, or essentially the human RSPO3 activity is antibody h131R006A or antibody same epitope, on RSPO3 as antibody h131R010. In certain h131R006B. In certain embodiments, a RSPO3-binding embodiments, a RSPO3-binding agent (e.g., an antibody) 40 agent that inhibits human RSPO3 activity is antibody binds the same epitope, or essentially the same epitope, on h131R005/131R007. In certain embodiments, a RSPO3 RSPO3 as antibody h131R011. binding agent that inhibits human RSPO3 activity is anti In another embodiment, a RSPO3-binding agent is an body h131R008. In certain embodiments, a RSPO3-binding antibody that binds an epitope on RSPO3 that overlaps with agent that inhibits human RSPO3 activity is antibody the epitope on RSPO3 bound by an antibody of the inven 45 h131R010. In certain embodiments, a RSPO3-binding agent tion. In some embodiments, the RSPO3-binding agent is an that inhibits human RSPO3 activity is antibody h131R011. antibody that binds an epitope on RSPO3 that overlaps with In certain embodiments, the RSPO-binding agent (e.g., the epitope on RSPO3 bound by antibody 131R002 or antibody) is an antagonist of at least one human RSPO antibody 131R003. In another embodiment, the RSPO3 protein. In certain embodiments, the RSPO-binding agent binding agent is an antibody that binds an epitope on RSPO3 50 inhibits RSPO signaling by at least about 10%, at least about that overlaps with the epitope on RSPO3 bound by a variant 20%, at least about 30%, at least about 50%, at least about of antibody 131R003. In some embodiments, the RSPO3 75%, at least about 90%, or about 100%. In some embodi binding agent is an antibody that binds an epitope on RSPO3 ments, the RSPO-binding agent inhibits signaling by one, that overlaps with the epitope on RSPO3 bound by a two, three, or four RSPO proteins. In some embodiments, humanized version of antibody 131 R003. In certain embodi 55 the RSPO-binding agent inhibits signaling of human ments, the RSPO3-binding agent is an antibody that binds an RSPO1, RSPO2, RSPO3, and/or RSPO4. In some embodi epitope on RSPO3 that overlaps with the epitope on RSPO3 ments, the RSPO-binding agent is a RSPO3-binding agent. bound by antibody h131R006A or antibody h131R006B. In In some embodiments, the RSPO3-binding agent inhibits certain embodiments, the RSPO3-binding agent is an anti human RSPO3 signaling. In certain embodiments, a body that binds an epitope on RSPO3 that overlaps with the 60 RSPO3-binding agent that inhibits RSPO3 signaling is anti epitope on RSPO3 bound by antibody h131R005/131R007. body 131R002, antibody 131R003, or a variant of 131R003. In certain embodiments, the RSPO3-binding agent is an In certain embodiments, a RSPO3-binding agent that inhib antibody that binds an epitope on RSPO3 that overlaps with its RSPO3 signaling is a humanized version of antibody the epitope on RSPO3 bound by antibody h131R008. In 131R002, antibody 131R003, or a variant of 131R003. In certain embodiments, the RSPO3-binding agent is an anti 65 certain embodiments, a RSPO3-binding agent that inhibits body that binds an epitope on RSPO3 that overlaps with the human RSPO3 signaling is antibody h131R006A or anti epitope on RSPO3 bound by antibody h131R010. In certain body h131R006B. In certain embodiments, a RSPO3-bind US 9,598,497 B2 51 52 ing agent that inhibits human RSPO3 signaling is antibody of human RSPO3 to at least one LGR protein is antibody h131R005/131R007. In certain embodiments, a RSPO3 h131R010. In certain embodiments, a RSPO3-binding agent binding agent that inhibits human RSPO3 signaling is anti that inhibits binding of human RSPO3 to at least one LGR body h131R008. In certain embodiments, a RSPO3-binding protein is antibody h131R011. agent that inhibits human RSPO3 signaling is antibody In certain embodiments, the RSPO-binding agent (e.g., h131R010. In certain embodiments, a RSPO3-binding agent antibody) blocks binding of at least one RSPO to a receptor. that inhibits human RSPO3 signaling is antibody h131R011. In certain embodiments, the RSPO-binding agent blocks In certain embodiments, the RSPO-binding agent (e.g., binding of a human RSPO protein to one or more of its antibody) is an antagonist of B-catenin signaling. In certain receptors. In some embodiments, the RSPO-binding agent embodiments, the RSPO-binding agent inhibits B-catenin 10 blocks binding of a RSPO to at least one LGR protein. In signaling by at least about 10%, at least about 20%, at least some embodiments, the RSPO-binding agent blocks binding about 30%, at least about 50%, at least about 75%, at least of at least one RSPO protein to LGR4, LGR5, and/or LGR6. about 90%, or about 100%. In some embodiments, the In some embodiments, a RSPO3-binding agent blocks bind RSPO-binding agent that inhibits f-catenin signaling is a ing of RSPO3 to LGR4. In some embodiments, a RSPO3 RSPO3-binding agent. In some embodiments, the RSPO3 15 binding agent blocks binding of RSPO3 to LGR5. In some binding agent inhibits B-catenin signaling. In certain embodiments, a RSPO3-binding agent blocks binding of embodiments, a RSPO3-binding agent that inhibits RSPO3 to LGR6. In certain embodiments, the blocking of B-catenin signaling is antibody 131R002, antibody binding of a RSPO-binding agent to at least one LGR protein 131R003, or a variant of 131R003. In certain embodiments, is at least about 10%, at least about 25%, at least about 50%, a RSPO3-binding agent that inhibits 3-catenin signaling is a at least about 75%, at least about 90%, or at least about 95%. humanized version of antibody 131 R002, antibody In certain embodiments, a RSPO-binding agent that blocks 131R003, or a variant of 131R003. In certain embodiments, binding of at least one RSPO protein to at least one LGR a RSPO3-binding agent that inhibits B-catenin signaling is protein further inhibits f-catenin signaling. In certain antibody h131R006A or antibody h131R006B. In certain embodiments, a RSPO3-binding agent that blocks binding embodiments, a RSPO3-binding agent that inhibits 25 of human RSPO3 to at least one LGR protein is antibody B-catenin signaling is antibody h131R005/131R007. In cer 131R002, antibody 131R003, or a variant of 131R003. In tain embodiments, a RSPO3-binding agent that inhibits certain embodiments, a RSPO3-binding agent that blocks B-catenin signaling is antibody h131R008. In certain binding of human RSPO3 to at least one LGR protein is a embodiments, a RSPO3-binding agent that inhibits humanized version of antibody 131 R002, antibody B-catenin signaling is antibody h131R010. In certain 30 131R003, or a variant of 131R003. In certain embodiments, embodiments, a RSPO3-binding agent that inhibits a RSPO3-binding agent that blocks binding of human B-catenin signaling is antibody h131R011. RSPO3 to at least one LGR protein is antibody h131R006A In certain embodiments, the RSPO-binding agent (e.g., or antibody h131R006B. In certain embodiments, a RSPO3 antibody) inhibits binding of at least one RSPO protein to a binding agent that blocks binding of human RSPO3 to at receptor. In certain embodiments, the RSPO-binding agent 35 least one LGR protein is antibody h131R005/131R007. In inhibits binding of a human RSPO protein to one or more of certain embodiments, a RSPO3-binding agent that blocks its receptors. In some embodiments, the RSPO-binding binding of human RSPO3 to at least one LGR protein is agent inhibits binding of a RSPO protein to at least one LGR antibody h131R008. In certain embodiments, a RSPO3 protein. In some embodiments, the RSPO-binding agent binding agent that blocks binding of human RSPO3 to at inhibits binding of a RSPO protein to LGR4, LGR5, and/or 40 least one LGR protein is antibody h131R010. In certain LGR6. In some embodiments, a RSPO3-binding agent embodiments, a RSPO3-binding agent that blocks binding inhibits binding of RSPO3 to LGR4. In some embodiments, of human RSPO3 to at least one LGR protein is antibody a RSPO3-binding agent inhibits binding of RSPO3 to h131 RO11. LGR5. In some embodiments, a RSPO3-binding agent In certain embodiments, the RSPO-binding agent (e.g., an inhibits binding of RSPO3 to LGR6. In certain embodi 45 antibody) inhibits 3-catenin signaling. It is understood that ments, the inhibition of binding of a RSPO-binding agent to a RSPO-binding agent that inhibits f-catenin signaling may, at least one LGR protein is at least about 10%, at least about in certain embodiments, inhibit signaling by one or more 25%, at least about 50%, at least about 75%, at least about receptors in the B-catenin signaling pathway but not neces 90%, or at least about 95%. In certain embodiments, a sarily inhibit signaling by all receptors. In certain alternative RSPO-binding agent that inhibits binding of at least one 50 embodiments, B-catenin signaling by all human receptors RSPO to at least one LGR protein further inhibits B-catenin may be inhibited. In certain embodiments, B-catenin signal signaling. In certain embodiments, a RSPO3-binding agent ing by one or more receptors selected from the group that inhibits binding of human RSPO3 to at least one LGR consisting of LGR4, LGR5, and LGR6 is inhibited. In protein is antibody 131R002, antibody 131R003, or a variant certain embodiments, the inhibition of B-catenin signaling of 131R003. In certain embodiments, a RSPO3-binding 55 by a RSPO-binding agent is a reduction in the level of agent that inhibits binding of human RSPO3 to at least one B-catenin signaling of at least about 10%, at least about 25%, LGR protein is a humanized version of antibody 131R002, at least about 50%, at least about 75%, at least about 90%, antibody 131R003, or a variant of 131R003. In certain or at least about 95%. In some embodiments, a RSPO3 embodiments, a RSPO3-binding agent that inhibits binding binding agent that inhibits f-catenin signaling is antibody of human RSPO3 to at least one LGR protein is antibody 60 131R002, antibody 131R003, or a variant of 131R003. In h131R006A or antibody h131R006B. In certain embodi some embodiments, a RSPO3-binding agent that inhibits ments, a RSPO3-binding agent that inhibits binding of B-catenin signaling is a humanized version of antibody human RSPO3 to at least one LGR protein is antibody 131R002, antibody 131R003, or a variant of 131R003. In h131R005/131R007. In certain embodiments, a RSPO3 some embodiments, a RSPO3-binding agent that inhibits binding agent that inhibits binding of human RSPO3 to at 65 B-catenin signaling is antibody h131R006A or antibody least one LGR protein is antibody h131R008. In certain h131R006B. In some embodiments, a RSPO3-binding agent embodiments, a RSPO3-binding agent that inhibits binding that inhibits B-catenin signaling is antibody h131R005/ US 9,598,497 B2 53 54 131R007. In some embodiments, a RSPO3-binding agent the frequency of cancer stem cells in the tumor, inhibit tumor that inhibits f-catenin signaling is antibody h131R008. In growth, trigger cell death of tumor cells, induce cells in a some embodiments, a RSPO3-binding agent that inhibits tumor to differentiate, differentiate tumorigenic cells to a B-catenin signaling is antibody h131R010. In some embodi non-tumorigenic State, induce expression of differentiation ments, a RSPO3-binding agent that inhibits f-catenin sig 5 markers in the tumor cells, prevent metastasis of tumor cells, naling is antibody h131R011. decrease Survival of tumor cells, or modulate angiogenesis. In certain embodiments, the RSPO-binding agent (e.g., an In certain embodiments, the RSPO3-binding agents are antibody) inhibits activation of B-catenin. It is understood capable of inhibiting tumor growth. In certain embodiments, that a RSPO-binding agent that inhibits activation of the RSPO3-binding agents are capable of inhibiting tumor B-catenin may, in certain embodiments, inhibit activation of 10 growth in vivo (e.g., in a Xenograft mouse model, and/or in B-catenin by one or more receptors, but not necessarily a human having cancer). In certain embodiments, tumor inhibit activation of B-catenin by all receptors. In certain growth is inhibited at least about two-fold, about three-fold, alternative embodiments, activation of B-catenin by all about five-fold, about ten-fold, about 50-fold, about 100 human receptors may be inhibited. In certain embodiments, fold, or about 1000-fold as compared to a untreated tumor. activation of B-catenin by one or more receptors selected 15 In certain embodiments, the RSPO3-binding agents are from the group consisting of LGR4, LGR5, and LGR6 is capable of reducing the tumorigenicity of a tumor. In certain inhibited. In certain embodiments, the inhibition of activa embodiments, the RSPO3-binding agent or antibody is tion of B-catenin by a RSPO-binding agent is a reduction in capable of reducing the tumorigenicity of a tumor compris the level of activation of B-catenin of at least about 10%, at ing cancer Stem cells in an animal model. Such as a mouse least about 25%, at least about 50%, at least about 75%, at xenograft model. In certain embodiments, the RSPO3-bind least about 90%, or at least about 95%. In some embodi ing agent or antibody is capable of reducing the tumorige ments, a RSPO3-binding agent that inhibits activation of nicity of a tumor by decreasing the number or frequency of |B-catenin is antibody 131R002, antibody 131R003, or a cancer stem cells in the tumor. In certain embodiments, the variant of 131R003. In some embodiments, a RSPO3 number or frequency of cancer stem cells in a tumor is binding agent that inhibits activation of B-catenin is a 25 reduced by at least about two-fold, about three-fold, about humanized version of antibody 131 R002, antibody five-fold, about ten-fold, about 50-fold, about 100-fold, or 131R003, or a variant of 131R003. In some embodiments, a about 1000-fold. In certain embodiments, the reduction in RSPO3-binding agent that inhibits activation of B-catenin is the number or frequency of cancer stem cells is determined antibody h131R006A or antibody h131R006B. In some by limiting dilution assay using an animal model. Additional embodiments, a RSPO3-binding agent that inhibits activa 30 examples and guidance regarding the use of limiting dilution tion of B-catenin is antibody h131R005/131R007. In some assays to determine a reduction in the number or frequency embodiments, a RSPO3-binding agent that inhibits activa of cancer stem cells in a tumor can be found, e.g., in tion of B-catenin is antibody h131R008. In some embodi International Publication Number WO 2008/042236, U.S. ments, a RSPO3-binding agent that inhibits activation of Patent Publication No. 2008/0064049, and U.S. Patent Pub B-catenin is antibody h131R010. In some embodiments, a 35 lication No. 2008/0178305. RSPO3-binding agent that inhibits activation of B-catenin is In certain embodiments, the RSPO3-binding agents antibody h131R011. described herein have a circulating half-life in mice, cyno In vivo and in vitro assays for determining whether a molgus monkeys, or humans of at least about 2 hours, at RSPO-binding agent (or candidate RSPO-binding agent) least about 5 hours, at least about 10 hours, at least about 24 inhibits 3-catenin signaling are known in the art. For 40 hours, at least about 3 days, at least about 1 week, or at least example, cell-based, luciferase reporter assays utilizing a about 2 weeks. In certain embodiments, the RSPO3-binding TCF/Luc reporter vector containing multiple copies of the agent is an IgG (e.g., IgG1 or IgG2) antibody that has a TCF-binding domain upstream of a firefly luciferase reporter circulating half-life in mice, cynomolgus monkeys, or gene may be used to measure B-catenin signaling levels in humans of at least about 2 hours, at least about 5 hours, at vitro (Gazit et al., 1999, Oncogene, 18; 5.959-66; TOPflash, 45 least about 10 hours, at least about 24 hours, at least about Millipore, Billerica Mass.). The level of B-catenin signaling 3 days, at least about 1 week, or at least about 2 weeks. in the presence of one or more Wnts (e.g., Wnt(s) expressed Methods of increasing (or decreasing) the half-life of agents by transfected cells or provided by Wnt-conditioned media) Such as polypeptides and antibodies are known in the art. For with or without a RSPO protein or RSPO-conditioned media example, known methods of increasing the circulating half in the presence of a RSPO-binding agent is compared to the 50 life of IgG antibodies include the introduction of mutations level of signaling without the RSPO-binding agent present. in the Fc region which increase the pH-dependent binding of In addition to the TCF/Luc reporter assay, the effect of a the antibody to the neonatal Fc receptor (FcRn) at pH 6.0 RSPO-binding agent (or candidate agent) on B-catenin sig (see, e.g., U.S. Patent Publication Nos. 2005/0276799, 2007/ naling may be measured in vitro or in vivo by measuring the 0148164, and 2007/0122403). Known methods of increas effect of the agent on the level of expression of B-catenin 55 ing the circulating half-life of antibody fragments lacking regulated genes, such as c-myc (He et al., 1998, Science, the Fc region include such techniques as PEGylation. 281:1509-12), cyclin D1 (Tetsu et al., 1999, Nature, 398: In some embodiments, the RSPO3-binding agents are 422-6) and/or fibronectin (Gradlet al. 1999, Mol. Cell Biol., polyclonal antibodies. Polyclonal antibodies can be prepared 19:5576-87). In certain embodiments, the effect of a RSPO by any known method. In some embodiments, polyclonal binding agent on B-catenin signaling may also be assessed 60 antibodies are produced by immunizing an animal (e.g., a by measuring the effect of the agent on the phosphorylation rabbit, rat, mouse, goat, donkey) with an antigen of interest state of Dishevelled-1, Dishevelled-2, Dishevelled-3, LRP5, (e.g., a purified peptide fragment, full-length recombinant LRP6, and/or f3-catenin. protein, or fusion protein) using multiple Subcutaneous or In certain embodiments, the RSPO3-binding agents have intraperitoneal injections. The antigen can be optionally one or more of the following effects: inhibit proliferation of 65 conjugated to a carrier Such as keyhole limpet hemocyanin tumor cells, inhibit tumor growth, reduce the tumorigenicity (KLH) or serum albumin. The antigen (with or without a of a tumor, reduce the tumorigenicity of a tumor by reducing carrier protein) is diluted in sterile saline and usually com US 9,598,497 B2 55 56 bined with an adjuvant (e.g., Complete or Incomplete Fre In some embodiments, the constant regions are truncated or unds Adjuvant) to form a stable emulsion. After a sufficient removed to generate the desired antibody fragment of a period of time, polyclonal antibodies are recovered from the monoclonal antibody. Site-directed or high-density muta immunized animal, usually from blood or ascites. The genesis of the variable region can be used to optimize polyclonal antibodies can be purified from serum or ascites according to standard methods in the art including, but not specificity, affinity, etc. of a monoclonal antibody. limited to, affinity chromatography, ion-exchange chroma In Some embodiments, a monoclonal antibody against tography, gel electrophoresis, and dialysis. human RSPO3 is a humanized antibody. Typically, human In some embodiments, the RSPO3-binding agents are ized antibodies are human immunoglobulins in which resi monoclonal antibodies. Monoclonal antibodies can be pre dues from the CDRs are replaced by residues from a CDR pared using hybridoma methods known to one of skill in the 10 of a non-human species (e.g., mouse, rat, rabbit, hamster, art (see e.g., Kohler and Milstein, 1975, Nature, 256:495 etc.) that have the desired specificity, affinity, and/or binding 497). In some embodiments, using the hybridoma method, a capability using methods known to one skilled in the art. In mouse, hamster, or other appropriate host animal, is immu Some embodiments, the Fv framework region residues of a nized as described above to elicit from lymphocytes the human immunoglobulin are replaced with the corresponding production of antibodies that specifically bind the immuniz 15 ing antigen. In some embodiments, lymphocytes can be residues in an antibody from a non-human species that has immunized in vitro. In some embodiments, the immunizing the desired specificity, affinity, and/or binding capability. In antigen can be a human protein or a portion thereof. In some Some embodiments, a humanized antibody can be further embodiments, the immunizing antigen can be a mouse modified by the substitution of additional residues either in protein or a portion thereof. the Fv framework region and/or within the replaced non Following immunization, lymphocytes are isolated and human residues to refine and optimize antibody specificity, fused with a suitable myeloma cell line using, for example, affinity, and/or capability. In general, a humanized antibody polyethylene glycol. The hybridoma cells are selected using will comprise Substantially all of at least one, and typically specialized media as known in the art and unfused lympho two or three, variable domain regions containing all, or cytes and myeloma cells do not survive the selection pro 25 substantially all, of the CDRs that correspond to the non cess. Hybridomas that produce monoclonal antibodies human immunoglobulin whereas all, or Substantially all, of directed specifically against a chosen antigen may be iden the framework regions are those of a human immunoglobu tified by a variety of methods including, but not limited to, lin consensus sequence. In some embodiments, a humanized immunoprecipitation, immunoblotting, and in vitro binding antibody can also comprise at least a portion of an immu assays (e.g., flow cytometry, FACS, ELISA, and radioim 30 munoassay). The hybridomas can be propagated either in in noglobulin constant region or domain (Fc), typically that of vitro culture using standard methods (J. W. Goding, 1996, a human immunoglobulin. In certain embodiments, such Monoclonal Antibodies: Principles and Practice, 3" Edi humanized antibodies are used therapeutically because they tion, Academic Press, San Diego, Calif.) or in vivo as ascites may reduce antigenicity and HAMA (human anti-mouse tumors in an animal. The monoclonal antibodies can be 35 antibody) responses when administered to a human Subject. purified from the culture medium or ascites fluid according One skilled in the art would be able to obtain a functional to standard methods in the art including, but not limited to, humanized antibody with reduced immunogenicity follow affinity chromatography, ion-exchange chromatography, gel ing known techniques (see e.g., U.S. Pat. Nos. 5.225,539; electrophoresis, and dialysis. 5,585,089; 5,693,761; and 5,693,762). In certain embodiments, monoclonal antibodies can be 40 In certain embodiments, the RSPO3-binding agent is a made using recombinant DNA techniques as known to one human antibody. Human antibodies can be directly prepared skilled in the art. The polynucleotides encoding a monoclo using various techniques known in the art. In some embodi nal antibody are isolated from mature B-cells or hybridoma ments, human antibodies may be generated from immortal cells, such as by RT-PCR using oligonucleotide primers that ized human B lymphocytes immunized in vitro or from specifically amplify the genes encoding the heavy and light 45 lymphocytes isolated from an immunized individual. In chains of the antibody, and their sequence is determined either case, cells that produce an antibody directed against a using standard techniques. The isolated polynucleotides target antigen can be generated and isolated (see, e.g., Cole encoding the heavy and light chains are then cloned into et al., 1985, Monoclonal Antibodies and Cancer Therapy, Suitable expression vectors which produce the monoclonal Alan R. Liss, p. 77; Boemer et al., 1991, J. Immunol., antibodies when transfected into host cells such as E. coli, 50 147:86-95; and U.S. Pat. Nos. 5,750,373; 5,567,610 and simian COS cells, Chinese hamster ovary (CHO) cells, or 5,229.275). In some embodiments, the human antibody can myeloma cells that do not otherwise produce immunoglobu be selected from a phage library, where that phage library lin proteins. expresses human antibodies (Vaughan et al., 1996, Nature In certain other embodiments, recombinant monoclonal Biotechnology, 14:309-314; Sheets et al., 1998, PNAS, antibodies, or fragments thereof, can be isolated from phage 55 95:6157-6162; Hoogenboom and Winter, 1991, J. Mol. display libraries expressing variable domains or CDRs of a Biol., 227:381; Marks et al., 1991, J. Mol. Biol., 222:581). desired species (see e.g., McCafferty et al., 1990, Nature, Alternatively, phage display technology can be used to 348:552-554; Clackson et al., 1991, Nature, 352:624-628; produce human antibodies and antibody fragments in vitro, and Marks et al., 1991, J. Mol. Biol., 222:581-597). from immunoglobulin variable domain gene repertoires The polynucleotide(s) encoding a monoclonal antibody 60 from unimmunized donors. Techniques for the generation can be modified, for example, by using recombinant DNA and use of antibody phage libraries are also described in U.S. technology to generate alternative antibodies. In some Pat. Nos. 5,969,108; 6,172,197; 5,885,793; 6,521,404; embodiments, the constant domains of the light and heavy 6,544,731; 6,555,313; 6,582,915; 6,593,081; 6,300,064; chains of for example, a mouse monoclonal antibody can be 6,653,068; 6,706,484; and 7,264,963; and Rothe et al., 2008, Substituted for those regions of for example, a human 65 J. Mol. Bio., 376:1182-1200. Once antibodies are identified, antibody to generate a chimeric antibody, or for a non affinity maturation strategies known in the art, including but immunoglobulin polypeptide to generate a fusion antibody. not limited to, chain shuffling (Marks et al., 1992, Bio/ US 9,598,497 B2 57 58 Technology, 10:779-783) and site-directed mutagenesis, to cells which express a particular target antigen. These may be employed to generate high affinity human antibod antibodies possess an antigen-binding arm and an arm which 1CS binds a cytotoxic agent or a radionuclide chelator, Such as In some embodiments, human antibodies can be made in EOTUBE, DPTA, DOTA, or TETA. transgenic mice that contain human immunoglobulin loci. 5 Techniques for making bispecific antibodies are known by Upon immunization these mice are capable of producing the those skilled in the art, see for example, Millstein et al., full repertoire of human antibodies in the absence of endog 1983, Nature, 305:537-539; Brennan et al., 1985, Science, enous immunoglobulin production. This approach is 229:81; Suresh et al., 1986, Methods in Enzymol., 121:120; described in U.S. Pat. Nos. 5,545,807: 5,545,806; 5,569, Traunecker et al., 1991, EMBO.J., 10:3655-3659; Shalaby et 825; 5,625,126; 5,633,425; and 5,661.016. 10 This invention also encompasses bispecific antibodies al., 1992, J. Exp. Med., 175:217-225; Kostelny et al., 1992, that specifically recognize at least one human RSPO protein. J. Immunol., 148:1547-1553; Gruber et al., 1994, J. Immu Bispecific antibodies are capable of specifically recognizing mol., 152:5368; U.S. Pat. No. 5,731,168: International Pub and binding at least two different antigens or epitopes. The lication No. WO 2009/089004; and U.S. Patent Publication different epitopes can either be within the same molecule 15 No. 2011/0123532. In some embodiments, the bispecific (e.g., two epitopes on human RSPO3) or on different mol antibodies comprise heavy chain constant regions with ecules (e.g., one epitope on RSPO3 and one epitope on modifications in the amino acids which are part of the RSPO2). In some embodiments, a bispecific antibody has interface between the two heavy chains. In some embodi enhanced potency as compared to an individual antibody or ments, the bispecific antibodies can be generated using a to a combination of more than one antibody. In some “knobs-into-holes' strategy (see, e.g., U.S. Pat. No. 5,731, embodiments, a bispecific antibody has reduced toxicity as 168; Ridgway et al., 1996, Prot. Engin., 9:617-621). In compared to an individual antibody or to a combination of some cases, the “knobs' and “holes' terminology is replaced more than one antibody. It is known to those of skill in the with the terms “protuberances” and "cavities”. In some art that any binding agent (e.g., antibody) may have unique embodiments, the bispecific antibodies may comprise vari pharmacokinetics (PK) (e.g., circulating half-life). In some 25 ant hinge regions incapable of forming disulfide linkages embodiments, a bispecific antibody has the ability to syn between the heavy chains (see, e.g., WO 2006/028936). In chronize the PK of two active binding agents wherein the Some embodiments, the modifications may comprise two individual binding agents have different PK profiles. In changes in amino acids that result in altered electrostatic some embodiments, a bispecific antibody has the ability to interactions. In some embodiments, the modifications may concentrate the actions of two binding agents (e.g., 30 comprise changes in amino acids that result in altered antibodies) in a common area (e.g., a tumor and/or tumor hydrophobic/hydrophilic interactions. microenvironment). In some embodiments, a bispecific anti Bispecific antibodies can be intact antibodies or antibody body has the ability to concentrate the actions of two binding fragments comprising antigen-binding sites. Antibodies with agents (e.g., antibodies) to a common target (e.g., a tumor or more than two valencies are also contemplated. For a tumor cell). In some embodiments, a bispecific antibody 35 example, trispecific antibodies can be prepared (Tutt et al., has the ability to target the actions of two binding agents 1991, J. Immunol., 147:60). Thus, in certain embodiments (e.g., antibodies) to more than one biological pathway or the antibodies to RSPO3 are multispecific. function. In certain embodiments, the antibodies (or other polypep In certain embodiments, the bispecific antibody specifi tides) described herein may be monospecific. In certain cally binds RSPO3 and a second target. In certain embodi 40 embodiments, each of the one or more antigen-binding sites ments, the bispecific antibody specifically binds RSPO3 and that an antibody contains is capable of binding (or binds) a a second human RSPO (e.g., RSPO1, RSPO2, or RSPO4). homologous epitope on RSPO proteins. In certain embodi In certain embodiments, the bispecific antibody specifically ments, an antigen-binding site of a monospecific antibody binds RSPO3 and RSPO2. In some embodiments, the bispe described herein is capable of binding (or binds), for cific antibody is a monoclonal antibody. In some embodi 45 example, RSPO3 and RSPO2 (i.e., the same epitope is found ments, the bispecific antibody is a humanized antibody. In on both RSPO3 and RSPO2 proteins). Some embodiments, the bispecific antibody is a human In certain embodiments, the RSPO3-binding agent is an antibody. In some embodiments, the bispecific antibody is antibody fragment. Antibody fragments may have different an IgG1 antibody. In some embodiments, the bispecific functions or capabilities than intact antibodies; for example, antibody is an IgG2 antibody. In some embodiments, the 50 antibody fragments can have increased tumor penetration. bispecific antibody has decreased toxicity and/or side Various techniques are known for the production of antibody effects. In some embodiments, the bispecific antibody has fragments including, but not limited to, proteolytic digestion decreased toxicity and/or side effects as compared to a of intact antibodies. In some embodiments, antibody frag mixture of the two individual antibodies or the antibodies as ments include a F(ab')2 fragment produced by pepsin diges single agents. In some embodiments, the bispecific antibody 55 tion of an antibody molecule. In some embodiments, anti has an increased therapeutic index. In some embodiments, body fragments include a Fab fragment generated by the bispecific antibody has an increased therapeutic index as reducing the disulfide bridges of an F(ab')2 fragment. In compared to a mixture of the two individual antibodies or other embodiments, antibody fragments include a Fab frag the antibodies as single agents. ment generated by the treatment of the antibody molecule In some embodiments, the antibodies can specifically 60 with papain and a reducing agent. In certain embodiments, recognize and bind a first antigen target, (e.g., RSPO3) as antibody fragments are produced recombinantly. In some well as a second antigen target, such as an effector molecule embodiments, antibody fragments include Fv or single chain on a leukocyte (e.g., CD2, CD3, CD28, CTLA-4, CD80, or Fv (sclv) fragments. Fab, Fv, and scFv antibody fragments CD86) or a Fc receptor (e.g., CD64. CD32, or CD16) so as can be expressed in and secreted from E. coli or other host to focus cellular defense mechanisms to the cell expressing 65 cells, allowing for the production of large amounts of these and/or producing the first antigen target. In some embodi fragments. In some embodiments, antibody fragments are ments, the antibodies can be used to direct cytotoxic agents isolated from antibody phage libraries as discussed herein. US 9,598,497 B2 59 60 For example, methods can be used for the construction of Alterations to the variable region notwithstanding, those Fab expression libraries (Huse et al., 1989, Science, 246: skilled in the art will appreciate that the modified antibodies 1275-1281) to allow rapid and effective identification of of this invention will comprise antibodies (e.g., full-length monoclonal Fab fragments with the desired specificity for a antibodies or immunoreactive fragments thereof) in which at RSPO protein or derivatives, fragments, analogs or 5 least a fraction of one or more of the constant region homologs thereof. In some embodiments, antibody frag domains has been deleted or otherwise altered so as to ments are linear antibody fragments. In certain embodi provide desired biochemical characteristics such as ments, antibody fragments are monospecific or bispecific. In increased tumor localization or increased serum half-life certain embodiments, the RSPO3-binding agent is a schv. when compared with an antibody of approximately the same Various techniques can be used for the production of single 10 immunogenicity comprising a native or unaltered constant chain antibodies specific to one or more human RSPOs (see, region. In some embodiments, the constant region of the e.g., U.S. Pat. No. 4,946,778). modified antibodies will comprise a human constant region. It can further be desirable, especially in the case of Modifications to the constant region compatible with this antibody fragments, to modify an antibody in order to alter invention comprise additions, deletions or Substitutions of (e.g., increase or decrease) its serum half-life. This can be 15 one or more amino acids in one or more domains. The achieved, for example, by incorporation of a salvage recep modified antibodies disclosed herein may comprise altera tor binding epitope into the antibody fragment by mutation tions or modifications to one or more of the three heavy of the appropriate region in the antibody fragment or by chain constant domains (CH1, CH2 or CH3) and/or to the incorporating the epitope into a peptide tag that is then fused light chain constant domain (CL). In some embodiments, to the antibody fragment at either end or in the middle (e.g., one or more domains are partially or entirely deleted from by DNA or peptide synthesis). the constant regions of the modified antibodies. In some Heteroconjugate antibodies are also within the scope of embodiments, the modified antibodies will comprise domain the present invention. Heteroconjugate antibodies are com deleted constructs or variants wherein the entire CH2 posed of two covalently joined antibodies. Such antibodies domain has been removed (ACH2 constructs). In some have, for example, been proposed to target immune cells to 25 embodiments, the omitted constant region domain is unwanted cells (see, e.g., U.S. Pat. No. 4,676.980). It is also replaced by a short amino acid spacer (e.g., 10 amino acid contemplated that the heteroconjugate antibodies can be residues) that provides some of the molecular flexibility prepared in vitro using known methods in synthetic protein typically imparted by the absent constant region. chemistry, including those involving crosslinking agents. In some embodiments, the modified antibodies are engi For example, immunotoxins can be constructed using a 30 neered to fuse the CH3 domain directly to the hinge region disulfide exchange reaction or by forming a thioether bond. of the antibody. In other embodiments, a peptide spacer is Examples of suitable reagents for this purpose include inserted between the hinge region and the modified CH2 iminothiolate and methyl-4-mercaptobutyrimidate. and/or CH3 domains. For example, constructs may be For the purposes of the present invention, it should be expressed wherein the CH2 domain has been deleted and the appreciated that modified antibodies can comprise any type 35 remaining CH3 domain (modified or unmodified) is joined of variable region that provides for the association of the to the hinge region with a 5-20 amino acid spacer. Such a antibody with the target (i.e., human RSPO3). In this regard, spacer may be added to ensure that the regulatory elements the variable region may comprise or be derived from any of the constant domain remain free and accessible or that the type of mammal that can be induced to mount a humoral hinge region remains flexible. However, it should be noted response and generate immunoglobulins against the desired 40 that amino acid spacers may, in Some cases, prove to be antigen. As such, the variable region of the modified anti immunogenic and elicit an unwanted immune response bodies can be, for example, of human, murine, non-human against the construct. Accordingly, in certain embodiments, primate (e.g. cynomolgus monkeys, macaques, etc.) or rab any spacer added to the construct will be relatively non bit origin. In some embodiments, both the variable and immunogenic so as to maintain the desired biological quali constant regions of the modified immunoglobulins are 45 ties of the modified antibodies. human. In other embodiments, the variable regions of com In some embodiments, the modified antibodies may have patible antibodies (usually derived from a non-human only a partial deletion of a constant domain or Substitution Source) can be engineered or specifically tailored to improve of a few or even a single amino acid. For example, the the binding properties or reduce the immunogenicity of the mutation of a single amino acid in selected areas of the CH2 molecule. In this respect, variable regions useful in the 50 domain may be enough to Substantially reduce Fc binding present invention can be humanized or otherwise altered and thereby increase cancer cell localization and/or tumor through the inclusion of imported amino acid sequences. penetration. Similarly, it may be desirable to simply delete In certain embodiments, the variable domains in both the the part of one or more constant region domains that control heavy and light chains are altered by at least partial replace a specific effector function (e.g. complement C1q binding) ment of one or more CDRs and, if necessary, by partial 55 to be modulated. Such partial deletions of the constant framework region replacement and sequence modification regions may improve selected characteristics of the antibody and/or alteration. Although the CDRs may be derived from (serum half-life) while leaving other desirable functions an antibody of the same class or even Subclass as the associated with the Subject constant region domain intact. antibody from which the framework regions are derived, it Moreover, as alluded to above, the constant regions of the is envisaged that the CDRs may be derived from an antibody 60 disclosed antibodies may be modified through the mutation of different class and often from an antibody from a different or Substitution of one or more amino acids that enhances the species. It may not be necessary to replace all of the CDRs profile of the resulting construct. In this respect it may be with all of the CDRs from the donor variable region to possible to disrupt the activity provided by a conserved transfer the antigen binding capacity of one variable domain binding site (e.g., Fc binding) while Substantially maintain to another. Rather, it may only be necessary to transfer those 65 ing the configuration and immunogenic profile of the modi residues that are required to maintain the activity of the fied antibody. In certain embodiments, the modified anti antigen-binding site. bodies comprise the addition of one or more amino acids to US 9,598,497 B2 61 62 the constant region to enhance desirable characteristics Such method for producing an antibody that binds human RSPO3 as decreasing or increasing effector function or provide for is provided. In some embodiments, the method comprises more cytotoxin or carbohydrate attachment sites. using amino acids 22-272 of human RSPO3. In some It is known in the art that the constant region mediates embodiments, the method comprises using amino acids several effector functions. For example, binding of the C1 22-272 of SEQID NO:3. In some embodiments, the method component of complement to the Fc region of IgG or IgM of generating an antibody that binds RSPO3 comprises antibodies (bound to antigen) activates the complement screening a human phage library. The present invention system. Activation of complement is important in the further provides methods of identifying an antibody that opSonization and lysis of cell pathogens. The activation of binds RSPO3. In some embodiments, the antibody is iden complement also stimulates the inflammatory response and 10 tified by FACS screening for binding to RSPO3 or a portion can also be involved in autoimmune hypersensitivity. In thereof. In some embodiments, the antibody is identified by addition, the Fc region of an antibody can bind a cell FACS screening for binding to RSPO3 and a second RSPO expressing a Fc receptor (FcR). There are a number of Fc or a portion thereof. In some embodiments, the antibody is receptors which are specific for different classes of antibody, identified by FACS screening for binding to both RSPO3 including IgG (gamma receptors), IgE (epsilon receptors), 15 and RSPO2 or a portion thereof. In some embodiments, the IgA (alpha receptors) and IgM (mu receptors). Binding of antibody is identified by screening using ELISA for binding antibody to Fc receptors on cell Surfaces triggers a number to RSPO3. In some embodiments, the antibody is identified of important and diverse biological responses including by screening using ELISA for binding to RSPO3 and a engulfment and destruction of antibody-coated particles, second RSPO. In some embodiments, the antibody is iden clearance of immune complexes, lysis of antibody-coated tified by screening using ELISA for binding to both RSPO3 target cells by killer cells (called antibody-dependent cell and RSPO2. In some embodiments, the antibody is identi cytotoxicity or ADCC), release of inflammatory mediators, fied by screening by FACS for blocking of binding of placental transfer, and control of immunoglobulin produc RSPO3 to a human LGR protein. In some embodiments, the tion. antibody is identified by screening for inhibition or blocking In certain embodiments, the modified antibodies provide 25 of B-catenin signaling. for altered effector functions that, in turn, affect the biologi In some embodiments, a method of generating an anti cal profile of the administered antibody. For example, in body to human RSPO3 protein comprises immunizing a Some embodiments, the deletion or inactivation (through mammal with a polypeptide comprising amino acids 22-272 point mutations or other means) of a constant region domain of human RSPO3. In some embodiments, a method of may reduce Fc receptor binding of the circulating modified 30 generating an antibody to human RSPO3 protein comprises antibody thereby increasing cancer cell localization and/or immunizing a mammal with a polypeptide comprising at tumor penetration. In other embodiments, the constant least a portion of amino acids 22-272 of human RSPO3. In region modifications increase the serum half-life of the Some embodiments, the method further comprises isolating antibody. In other embodiments, the constant region modi antibodies or antibody-producing cells from the mammal. In fications reduce the serum half-life of the antibody. In some 35 Some embodiments, a method of generating a monoclonal embodiments, the constant region is modified to eliminate antibody which binds RSPO3 protein comprises: (a) immu disulfide linkages or oligosaccharide moieties. Modifica nizing a mammal with a polypeptide comprising at least a tions to the constant region in accordance with this invention portion of amino acids 22-272 of human RSPO3; (b) iso may easily be made using well known biochemical or lating antibody producing cells from the immunized mam molecular engineering techniques. 40 mal; (c) fusing the antibody-producing cells with cells of a In certain embodiments, a RSPO3-binding agent that is an myeloma cell line to form hybridoma cells. In some embodi antibody does not have one or more effector functions. For ments, the method further comprises (d) selecting a instance, in some embodiments, the antibody has no ADCC hybridoma cell expressing an antibody that binds RSPO3 activity, and/or no complement-dependent cytotoxicity protein. In some embodiments, the at least a portion of (CDC) activity. In certain embodiments, the antibody does 45 amino acids 22-272 of human RSPO3 is selected from the not bind an Fc receptor, and/or complement factors. In group consisting of SEQ ID NOS:5-8. In some embodi certain embodiments, the antibody has no effector function. ments, the at least a portion of amino acids 22-272 of human The present invention further embraces variants and RSPO3 is SEQID NO:5. In some embodiments, the at least equivalents which are substantially homologous to the chi a portion of amino acids 22-272 of human RSPO3 is SEQID meric, humanized, and human antibodies, or antibody frag 50 NO:6 or SEQ ID NO:7. In some embodiments, the at least ments thereof, set forth herein. These can contain, for a portion of amino acids 22-272 of human RSPO3 is SEQID example, conservative Substitution mutations, i.e. the Sub NO:6 and SEQ ID NO:7. In certain embodiments, the stitution of one or more amino acids by similar amino acids. mammal is a mouse. In some embodiments, the antibody is For example, conservative substitution refers to the substi selected using a polypeptide comprising at least a portion of tution of an amino acid with another amino acid within the 55 amino acid 22-272 of human RSPO3. In certain embodi same general class such as, for example, one acidic amino ments, the polypeptide used for selection comprising at least acid with another acidic amino acid, one basic amino acid a portion of amino acids 22-272 of human RSPO3 is selected with another basic amino acid or one neutral amino acid by from the group consisting of SEQ ID NOS:5-8. In some another neutral amino acid. What is intended by a conser embodiments, the antibody binds RSPO3 and at least one vative amino acid substitution is well known in the art and 60 other RSPO protein. In certain embodiments, the at least one described herein. other RSPO protein is selected from the group consisting of Thus, the present invention provides methods for produc RSPO1, RSPO2, and RSPO4. In certain embodiments, the ing an antibody that binds RSPO3, including bispecific antibody binds RSPO3 and RSPO1. In certain embodiments, antibodies that specifically bind both RSPO3 and a second the antibody binds RSPO3 and RSPO2. In certain embodi target (e.g., a human RSPO). In some embodiments, the 65 ments, the antibody binds RSPO3 and RSPO4. In certain method for producing an antibody that binds RSPO3 com embodiments, the antibody binds RSPO3, RSPO1, and prises using hybridoma techniques. In some embodiments, a RSPO2. In certain embodiments, the antibody binds RSPO3, US 9,598,497 B2 63 64 RSPO1, and RSPO4. In certain embodiments, the antibody The polypeptides, analogs and variants thereof, can be binds RSPO3, RSPO2, and RSPO4. In some embodiments, further modified to contain additional chemical moieties not the antibody binds both human RSPO3 and mouse RSPO3. normally part of the polypeptide. The derivatized moieties In Some embodiments, the antibody generated by the can improve or otherwise modulate the solubility, the bio methods described herein is a RSPO antagonist, particularly logical half-life, and/or absorption of the polypeptide. The a RSPO3 antagonist. In some embodiments, the antibody moieties can also reduce or eliminate undesirable side generated by the methods described herein inhibits B-catenin effects of the polypeptides and variants. An overview for signaling. chemical moieties can be found in Remington. The Science In some embodiments, a method of producing an antibody and Practice of Pharmacy, 22 Edition, 2012, Pharmaceu to at least one human RSPO protein comprises identifying 10 tical Press, London. an antibody using a membrane-bound heterodimeric mol The polypeptides described herein can be produced by ecule comprising a single antigen-binding site. In some any Suitable method known in the art. Such methods range non-limiting embodiments, the antibody is identified using from direct protein synthesis methods to constructing a methods and polypeptides described in International Publi DNA sequence encoding polypeptide sequences and cation WO 2011/100566. 15 expressing those sequences in a Suitable host. In some In some embodiments, a method of producing an antibody embodiments, a DNA sequence is constructed using recom to at least one human RSPO protein comprises screening an binant technology by isolating or synthesizing a DNA antibody-expressing library for antibodies that bind a human sequence encoding a wild-type protein of interest. Option RSPO protein. In some embodiments, the antibody-express ally, the sequence can be mutagenized by site-specific muta ing library is a phage library. In some embodiments, the genesis to provide functional analogs thereof. See, e.g., screening comprises panning. In some embodiments, the Zoeller et al., 1984, PNAS, 81:5662-5066 and U.S. Pat. No. antibody-expressing library is a phage library. In some 4,588,585. embodiments, the antibody-expressing library is a mamma In some embodiments, a DNA sequence encoding a lian cell library. In some embodiments, the antibody-ex polypeptide of interest may be constructed by chemical pressing library is screened using at least a portion of amino 25 synthesis using an oligonucleotide synthesizer. Oligonucle acids 22-272 of human RSPO3. In some embodiments, otides can be designed based on the amino acid sequence of antibodies identified in the first screening, are screened the desired polypeptide and selecting those codons that are again using a different RSPO protein thereby identifying an favored in the host cell in which the recombinant polypep antibody that binds RSPO3 and a second RSPO protein. In tide of interest will be produced. Standard methods can be certain embodiments, the polypeptide used for screening 30 applied to synthesize a polynucleotide sequence encoding an comprises at least a portion of amino acids 22-272 of human isolated polypeptide of interest. For example, a complete RSPO3 selected from the group consisting of SEQ ID amino acid sequence can be used to construct a back NOs:5-8. In some embodiments, the antibody identified in translated gene. Further, a DNA oligomer containing a the screening binds RSPO3 and at least one other RSPO nucleotide sequence coding for the particular isolated poly protein. In certain embodiments, the at least one other RSPO 35 peptide can be synthesized. For example, several Small protein is selected from the group consisting of RSPO1, oligonucleotides coding for portions of the desired polypep RSPO2, and RSPO4. In certain embodiments, the antibody tide can be synthesized and then ligated. The individual identified in the screening binds RSPO3 and RSPO1. In oligonucleotides typically contain 5' or 3' overhangs for certain embodiments, the antibody identified in the screen complementary assembly. ing binds RSPO3 and RSPO2. In certain embodiments, the 40 Once assembled (by synthesis, site-directed mutagenesis, antibody identified in the screening binds RSPO3 and or another method), the polynucleotide sequences encoding RSPO4. In some embodiments, the antibody identified in the a particular polypeptide of interest can be inserted into an screening binds both human RSPO3 and mouse RSPO3. In expression vector and operatively linked to an expression Some embodiments, the antibody identified in the screening control sequence appropriate for expression of the protein in is a RSPO3 antagonist. In some embodiments, the antibody 45 a desired host. Proper assembly can be confirmed by nucleo identified in the screening inhibits B-catenin signaling tide sequencing, restriction enzyme mapping, and/or expres induced by RSPO3. sion of a biologically active polypeptide in a Suitable host. In certain embodiments, the antibodies described herein As is well-known in the art, in order to obtain high expres are isolated. In certain embodiments, the antibodies sion levels of a transfected gene in a host, the gene must be described herein are substantially pure. 50 operatively linked to transcriptional and translational In some embodiments of the present invention, the expression control sequences that are functional in the RSPO3-binding agents are polypeptides. The polypeptides chosen expression host. can be recombinant polypeptides, natural polypeptides, or In certain embodiments, recombinant expression vectors synthetic polypeptides comprising an antibody, or fragment are used to amplify and express DNA encoding antibodies, thereof, that bind RSPO3. It will be recognized in the art that 55 or fragments thereof, against human RSPO3. For example, Some amino acid sequences of the invention can be varied recombinant expression vectors can be replicable DNA without significant effect of the structure or function of the constructs which have synthetic or cDNA-derived DNA protein. Thus, the invention further includes variations of the fragments encoding a polypeptide chain of a RSPO-binding polypeptides which show substantial activity or which agent, Such as an anti-RSPO antibody, or fragment thereof, include regions of an antibody, or fragment thereof, against 60 operatively linked to Suitable transcriptional and/or transla human RSPO3. In some embodiments, amino acid sequence tional regulatory elements derived from mammalian, micro variations of RSPO-binding polypeptides include deletions, bial, viral or insect genes. A transcriptional unit generally insertions, inversions, repeats, and/or other types of Substi comprises an assembly of (1) a genetic element or elements tutions. having a regulatory role in gene expression, for example, In certain embodiments, the polypeptides described 65 transcriptional promoters or enhancers, (2) a structural or herein are isolated. In certain embodiments, the polypeptides coding sequence which is transcribed into mRNA and trans described herein are substantially pure. lated into protein, and (3) appropriate transcription and US 9,598,497 B2 65 66 translation initiation and termination sequences. Regulatory Various mammalian culture systems may be used to elements can include an operator sequence to control tran express recombinant polypeptides. Expression of recombi scription. The ability to replicate in a host, usually conferred nant proteins in mammalian cells may be desirable because by an origin of replication, and a selection gene to facilitate these proteins are generally correctly folded, appropriately recognition of transformants can additionally be incorpo modified, and biologically functional. Examples of suitable rated. DNA regions are “operatively linked when they are mammalian host cell lines include, but are not limited to, functionally related to each other. For example, DNA for a COS-7 (monkey kidney-derived), L-929 (murine fibroblast signal peptide (secretory leader) is operatively linked to derived), C127 (murine mammary tumor-derived), 3T3 (mu DNA for a polypeptide if it is expressed as a precursor which rine fibroblast-derived), CHO (Chinese hamster ovary-de participates in the secretion of the polypeptide; a promoter 10 rived), HeLa (human cervical cancer-derived), BHK (hamster kidney fibroblast-derived), HEK-293 (human is operatively linked to a coding sequence if it controls the embryonic kidney-derived) cell lines and variants thereof. transcription of the sequence; or a ribosome binding site is Mammalian expression vectors can comprise non-tran operatively linked to a coding sequence if it is positioned so scribed elements such as an origin of replication, a Suitable as to permit translation. In some embodiments, structural 15 promoter and enhancer linked to the gene to be expressed, elements intended for use in yeast expression systems and other 5' or 3' flanking non-transcribed sequences, and 5' include a leader sequence enabling extracellular secretion of or 3' non-translated sequences, such as necessary ribosome translated protein by a host cell. In other embodiments, in binding sites, a polyadenylation site, splice donor and accep situations where recombinant protein is expressed without a tor sites, and transcriptional termination sequences. leader or transport sequence, it can include an N-terminal Expression of recombinant proteins in insect cell culture methionine residue. This residue can optionally be subse systems (e.g., baculovirus) also offers a robust method for quently cleaved from the expressed recombinant protein to producing correctly folded and biologically functional pro provide a final product. teins. Baculovirus systems for production of heterologous The choice of an expression control sequence and an proteins in insect cells are well-known to those of skill in the expression vector depends upon the choice of host. A wide 25 art (see, e.g., Luckow and Summers, 1988, Bio/Technology, variety of expression host/vector combinations can be 6:47). employed. Useful expression vectors for eukaryotic hosts Thus, the present invention provides cells comprising the include, for example, Vectors comprising expression control RSPO3-binding agents described herein. In some embodi sequences from SV40, bovine papilloma virus, adenovirus, ments, the cells produce the RSPO3-binding agents and cytomegalovirus. Useful expression vectors for bacterial 30 described herein. In certain embodiments, the cells produce hosts include known bacterial plasmids, such as plasmids an antibody. In some embodiments, the cells produce an from E. coli, including pCR1, pBR322, pMB9 and their antibody that binds human RSPO3. In certain embodiments, derivatives, and wider host range plasmids, such as M13 and the cells produce antibody 131 R002. In certain embodi other filamentous single-stranded DNA phages. ments, the cells produce antibody 131R003. In certain The RSPO-binding agents (e.g., polypeptides or 35 embodiments, the cells produce variants of antibody antibodies) of the present invention can be expressed from 131R003. In certain embodiments, the cells produce a one or more vectors. For example, in some embodiments, humanized version of antibody 131 R002, antibody one heavy chain polypeptide is expressed by one vector, a 131R003, or variants of antibody 131R003. In some second heavy chain polypeptide is expressed by a second embodiments, the cells produce a chimeric version of anti vector and a light chain polypeptide is expressed by a third 40 body 131R002, antibody 131R003, or variants of antibody vector. In some embodiments, a first heavy chain polypep 131R003. In some embodiments, the cells produce antibody tide and a light chain polypeptide is expressed by one vector h131R006A or antibody h131R006B. In some embodi and a second heavy chain polypeptide is expressed by a ments, the cells produce antibody h131R005/131R007. In second vector. In some embodiments, two heavy chain some embodiments, the cells produce antibody h131R008. polypeptides are expressed by one vector and a light chain 45 In some embodiments, the cells produce antibody polypeptide is expressed by a second vector. In some h131R010. In some embodiments, the cells produce anti embodiments, three polypeptides are expressed from one body h131R011. In some embodiments, the cells produce a vector. Thus, in Some embodiments, a first heavy chain bispecific antibody that binds RSPO3. In some embodi polypeptide, a second heavy chain polypeptide, and a light ments, the cells produce a bispecific antibody that binds chain polypeptide are expressed by a single vector. 50 RSPO3 and RSPO2. In some embodiments, the cell is a Suitable host cells for expression of a RSPO3-binding hybridoma cell. In some embodiments, the cell is a mam polypeptide or antibody (or a RSPO protein to use as an malian cell. In some embodiments, the cell is a prokaryotic antigen) include prokaryotes, yeast cells, insect cells, or cell. In some embodiments, the cell is an eukaryotic cell. higher eukaryotic cells under the control of appropriate The proteins produced by a transformed host can be promoters. Prokaryotes include gram-negative or gram 55 purified according to any suitable method. Standard methods positive organisms, for example E. coli or Bacillus. Higher include chromatography (e.g., ion exchange, affinity, and eukaryotic cells include established cell lines of mammalian sizing column chromatography), centrifugation, differential origin as described below. Cell-free translation systems may solubility, or by any other standard technique for protein also be employed. Appropriate cloning and expression vec purification. Affinity tags such as hexa-histidine, maltose tors for use with bacterial, fungal, yeast, and mammalian 60 binding domain, influenza coat sequence, and glutathione cellular hosts are described in Pouwels et al., 1985, Cloning S-transferase can be attached to the protein to allow easy Vectors: A Laboratory Manual, Elsevier, New York, N.Y. purification by passage over an appropriate affinity column. Additional information regarding methods of protein pro Affinity chromatography used for purifying immunoglobu duction, including antibody production, can be found, e.g., lins can include Protein A, Protein G, and Protein L chro in U.S. Patent Publication No. 2008/0187954, U.S. Pat. Nos. 65 matography. Isolated proteins can be physically character 6,413,746, 6,660,501; and International Patent Publication ized using Such techniques as proteolysis, size exclusion No. WO O4/OO9823. chromatography (SEC), mass spectrometry (MS), nuclear US 9,598,497 B2 67 68 magnetic resonance (NMR), isoelectric focusing (IEF), high 275:2677-83. In certain embodiments, phage or mammalian performance liquid chromatography (HPLC), and X-ray display technology may be used to produce and/or identify crystallography. The purity of isolated proteins can be a RSPO3-binding polypeptide. In certain embodiments, the determined using techniques known to those of skill in the polypeptide comprises a protein scaffold of a type selected art, including but not limited to, SDS-PAGE, SEC, capillary from the group consisting of protein A, protein G, a lipoca gel electrophoresis, IEF, and capillary isoelectric focusing lin, a fibronectin domain, an ankyrin consensus repeat (cIEF). domain, and thioredoxin. In some embodiments, Supernatants from expression sys In certain embodiments, the RSPO3-binding agents or tems which secrete recombinant protein into culture media antibodies can be used in any one of a number of conjugated can be first concentrated using a commercially available 10 (i.e. an immunoconjugate or radioconjugate) or non-conju protein concentration filter, for example, an Amicon or gated forms. In certain embodiments, the antibodies can be Millipore Pellicon ultrafiltration unit. Following the concen used in a non-conjugated form to harness the Subjects tration step, the concentrate can be applied to a Suitable natural defense mechanisms including complement-depen purification matrix. In some embodiments, an anion dent cytotoxicity and antibody dependent cellular toxicity to exchange resin can be employed, for example, a matrix or 15 eliminate malignant or cancer cells. substrate having pendant diethylaminoethyl (DEAE) In some embodiments, the RSPO3-binding agent (e.g., an groups. The matrices can be acrylamide, agarose, dextran, antibody or polypeptide) is conjugated to a cytotoxic agent. cellulose, or other types commonly employed in protein In some embodiments, the cytotoxic agent is a chemothera purification. In some embodiments, a cation exchange step peutic agent including, but not limited to, methotrexate, can be employed. Suitable cation exchangers include vari adriamicin, doxorubicin, melphalan, mitomycin C, chloram ous insoluble matrices comprising Sulfopropyl or carboxym bucil, daunorubicin or other intercalating agents. In some ethyl groups. In some embodiments, a hydroxyapatite media embodiments, the cytotoxic agent is an enzymatically active can be employed, including but not limited to, ceramic toxin of bacterial, fungal, plant, or animal origin, or frag hydroxyapatite (CHT). In certain embodiments, one or more ments thereof, including, but not limited to, diphtheria A reverse-phase HPLC steps employing hydrophobic 25 chain, non-binding active fragments of diphtheria toxin, RP-HPLC media, e.g., silica gel having pendant methyl or exotoxin A chain, ricin A chain, abrin A chain, modeccin A other aliphatic groups, can be employed to further purify a chain, alpha-sarcin, Aleurites fordii proteins, dianthin pro recombinant protein (e.g., a RSPO3-binding agent). Some or teins, Phytolaca americana proteins (PAPI, PAPII, and all of the foregoing purification steps, in various combina PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapa tions, can be employed to provide a homogeneous recom 30 onaria officinalis inhibitor, gelonin, mitogellin, restrictocin, binant protein. phenomycin, enomycin, and the tricothecenes. In some In some embodiments, heterodimeric proteins such as embodiments, the cytotoxic agent is a radioisotope to pro bispecific antibodies are purified according the any of the duce a radioconjugate or a radioconjugated antibody. A methods described herein. In some embodiments, anti variety of radionuclides are available for the production of RSPObispecific antibodies are isolated and/or purified using 35 radioconjugated antibodies including, but not limited to, at least one chromatography step. In some embodiments, the 90Y. 125I. 13 II, 123, In, 13 In, 103Rh, 'Sim, 67Cu, 7Ga, at least one chromatography step comprises affinity chro 'Ho, '77Lu, Re, Re and 'Bi. Conjugates of an matography. In some embodiments, the at least one chro antibody and one or more Small molecule toxins, such as matography step further comprises anion exchange chroma calicheamicins, maytansinoids, trichothenes, and CC 1065. tography. In some embodiments, the isolated and/or purified 40 and the derivatives of these toxins that have toxin activity, antibody product comprises at least 90% heterodimeric can also be used. Conjugates of an antibody and cytotoxic antibody. In some embodiments, the isolated and/or purified agent may be made using a variety of bifunctional protein antibody product comprises at least 95%, 96%, 97%, 98% or coupling agents such as N-Succinimidyl-3-(2-pyridyidithiol) 99% heterodimeric antibody. In some embodiments, the propionate (SPDP), iminothiolane (IT), bifunctional deriva isolated and/or purified antibody product comprises about 45 tives of imidoesters (such as dimethyl adipimidate HCl). 100% heterodimeric antibody. active esters (such as disuccinimidyl Suberate), aldehydes In some embodiments, recombinant protein produced in (such as glutareldehyde), bis-azido compounds (such as bacterial culture can be isolated, for example, by initial bis(p-azidobenzoyl) hexanediamine), bis-diazonium deriva extraction from cell pellets, followed by one or more con tives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), centration, salting-out, aqueous ion exchange, or size exclu 50 diisocyanates (such as toluene 2,6-diisocyanate), and bis sion chromatography steps. HPLC can be employed for final active fluorine compounds (such as 1,5-difluoro-2,4-dini purification steps. Microbial cells employed in expression of trobenzene). a recombinant protein can be disrupted by any convenient method, including freeze-thaw cycling, Sonication, III. POLYNUCLEOTIDES mechanical disruption, or use of cell lysing agents. 55 Methods known in the art for purifying antibodies and In certain embodiments, the invention encompasses poly other proteins also include, for example, those described in nucleotides comprising polynucleotides that encode a poly U.S. Patent Publication Nos. 2008/0312425, 2008/0177048, peptide (or a fragment of a polypeptide) that specifically and 2009/O187OO5. binds RSPO3. The term “polynucleotides that encode a In certain embodiments, the RSPO3-binding agent is a 60 polypeptide' encompasses a polynucleotide which includes polypeptide that is not an antibody. A variety of methods for only coding sequences for the polypeptide as well as a identifying and producing non-antibody polypeptides that polynucleotide which includes additional coding and/or bind with high affinity to a protein target are known in the non-coding sequences. For example, in some embodiments, art. See, e.g., Skerra, 2007, Curr: Opin. Biotechnol., 18:295 the invention provides a polynucleotide comprising a poly 304; Hosse et al., 2006, Protein Science, 15:14-27; Gill et 65 nucleotide sequence that encodes an antibody to human al., 2006, Curr. Opin. Biotechnol., 17:653-658; Nygren, RSPO3 or encodes a fragment of such an antibody (e.g., a 2008, FEBS J., 275:2668-76; and Skerra, 2008, FEBS J., fragment comprising the antigen-binding site). The poly US 9,598,497 B2 69 70 nucleotides of the invention can be in the form of RNA or comprises a polynucleotide comprising SEQ ID NO:77. In in the form of DNA. DNA includes clNA, genomic DNA, Some embodiments, a plasmid comprises a polynucleotide and synthetic DNA; and can be double-stranded or single comprising SEQ ID NO:84. In some embodiments, a plas Stranded, and if single Stranded can be the coding Strand or mid comprises a polynucleotide comprising SEQID NO:85. non-coding (anti-sense) Strand. In some embodiments, a plasmid comprises a polynucle In certain embodiments, the polynucleotide comprises a otide comprising SEQ ID NO:89. In some embodiments, a polynucleotide encoding a polypeptide comprising an amino plasmid comprises a polynucleotide comprising SEQ ID acid sequence selected from the group consisting of: SEQID NO:90. In some embodiments, a plasmid comprises a poly NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:21, nucleotide comprising SEQ ID NO:91. In some embodi SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:27, SEQ ID 10 ments, a plasmid comprises a polynucleotide comprising NO:28, SEQ ID NO:29, SEQ ID NO:36, SEQ ID NO:37, SEQID NO:92. In some embodiments, a plasmid comprises SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:41, SEQ ID a polynucleotide comprising SEQ ID NO:93. In some NO:42, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, embodiments, a plasmid comprises a polynucleotide com SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID prising SEQ ID NO:94. In some embodiments, a plasmid NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:68, 15 comprises a polynucleotide comprising SEQ ID NO:95. SEQ ID NO:69, SEQ ID NO:72, SEQ ID NO:73, SEQ ID In certain embodiments, the polynucleotide comprises a NO:74, SEQ ID NO:86, SEQ ID NO:87, and SEQ ID polynucleotide having a nucleotide sequence at least about NO:88. In some embodiments, the polynucleotide comprises 80% identical, at least about 85% identical, at least about a polynucleotide sequence selected from the group consist 90% identical, at least about 95% identical, and in some ing of: SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, embodiments, at least about 96%, 97%, 98% or 99% iden SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID tical to a polynucleotide comprising a sequence selected NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:40, from the group consisting of SEQ ID NO:18, SEQ ID SEQ ID NO:43, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:25, NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:26, SEQ ID NO:30, SEQ ID NO:31, SEQ ID SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID 25 NO:32, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:50, NO:70, SEQ ID NO:71, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID SEQ ID NO:77, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:65, SEQ ID NO:66, NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:67, SEQ ID NO:70, SEQ ID NO:71, SEQ ID SEQ ID NO:93, SEQ ID NO:94, and SEQ ID NO:95. NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:84, In some embodiments, a plasmid comprises a polynucle 30 SEQ ID NO:85, SEQ ID NO:89, SEQ ID NO:90, SEQ ID otide comprising SEQ ID NO:18. In some embodiments, a NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, plasmid comprises a polynucleotide comprising SEQ ID and SEQID NO:95. Also provided is a polynucleotide that NO:19. In some embodiments, a plasmid comprises a poly comprises a polynucleotide that hybridizes to SEQ ID nucleotide comprising SEQ ID NO:20. In some embodi NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:24, ments, a plasmid comprises a polynucleotide comprising 35 SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:30, SEQ ID SEQID NO:24. In some embodiments, a plasmid comprises NO:31, SEQ ID NO:32, SEQ ID NO:32, SEQ ID NO:40, a polynucleotide comprising SEQ ID NO:25. In some SEQ ID NO:43, SEQ ID NO:50, SEQ ID NO:51, SEQ ID embodiments, a plasmid comprises a polynucleotide com NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, prising SEQ ID NO:26. In some embodiments, a plasmid SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID comprises a polynucleotide comprising SEQ ID NO:30. In 40 NO:70, SEQ ID NO:71, SEQ ID NO:75, SEQ ID NO:76, Some embodiments, a plasmid comprises a polynucleotide SEQ ID NO:77, SEQ ID NO:84, SEQ ID NO:85, SEQ ID comprising SEQ ID NO:31. In some embodiments, a plas NO:89, SEQ ID NO:990, SEQ ID NO:91, SEQ ID NO:92, mid comprises a polynucleotide comprising SEQID NO:32. SEQ ID NO:93, SEQ ID NO:94, or SEQ ID NO:95. Also In some embodiments, a plasmid comprises a polynucle provided is a polynucleotide that comprises a polynucleotide otide comprising SEQ ID NO:40. In some embodiments, a 45 that hybridizes to the complement of SEQ ID NO:18, SEQ plasmid comprises a polynucleotide comprising SEQ ID ID NO:19, SEQID NO:20, SEQID NO:24, SEQID NO:25, NO:43. In some embodiments, a plasmid comprises a poly SEQ ID NO:26, SEQ ID NO:30, SEQ ID NO:31, SEQ ID nucleotide comprising SEQ ID NO:50. In some embodi NO:32, SEQ ID NO:32, SEQ ID NO:40, SEQ ID NO:43, ments, a plasmid comprises a polynucleotide comprising SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID SEQID NO:51. In some embodiments, a plasmid comprises 50 NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:65, a polynucleotide comprising SEQ ID NO:52. In some SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:70, SEQ ID embodiments, a plasmid comprises a polynucleotide com NO:71, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, prising SEQ ID NO:53. In some embodiments, a plasmid SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:89, SEQ ID comprises a polynucleotide comprising SEQ ID NO:54. In NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, Some embodiments, a plasmid comprises a polynucleotide 55 SEQ ID NO:94, or SEQID NO:95. In certain embodiments, comprising SEQ ID NO:55. In some embodiments, a plas the hybridization is under conditions of high Stringency. mid comprises a polynucleotide comprising SEQID NO:65. In some embodiments, an antibody is encoded by a In some embodiments, a plasmid comprises a polynucle polynucleotide comprising SEQ ID NO:18 and SEQ ID otide comprising SEQ ID NO:66. In some embodiments, a NO:20. In some embodiments, an antibody is encoded by a plasmid comprises a polynucleotide comprising SEQ ID 60 polynucleotide comprising SEQ ID NO:19 and SEQ ID NO:67. In some embodiments, a plasmid comprises a poly NO:20. In some embodiments, an antibody is encoded by a nucleotide comprising SEQ ID NO:70. In some embodi polynucleotide comprising SEQ ID NO:50 and SEQ ID ments, a plasmid comprises a polynucleotide comprising NO:20. In some embodiments, an antibody is encoded by a SEQID NO:71. In some embodiments, a plasmid comprises polynucleotide comprising SEQ ID NO:51 and SEQ ID a polynucleotide comprising SEQ ID NO:75. In some 65 NO:20. In some embodiments, an antibody is encoded by a embodiments, a plasmid comprises a polynucleotide com polynucleotide comprising SEQ ID NO:65 and SEQ ID prising SEQ ID NO:76. In some embodiments, a plasmid NO:20. In some embodiments, an antibody is encoded by a US 9,598,497 B2 71 72 polynucleotide comprising SEQ ID NO:18 and SEQ ID NO:77. In some embodiments, an antibody is encoded by a NO:75. In some embodiments, an antibody is encoded by a polynucleotide comprising SEQ ID NO:54 and SEQ ID polynucleotide comprising SEQ ID NO:19 and SEQ ID NO:77. In some embodiments, an antibody is encoded by a NO:75. In some embodiments, an antibody is encoded by a polynucleotide comprising SEQ ID NO:55 and SEQ ID polynucleotide comprising SEQ ID NO:50 and SEQ ID 5 NO:77. In some embodiments, an antibody is encoded by a NO:75. In some embodiments, an antibody is encoded by a polynucleotide comprising SEQ ID NO:67 and SEQ ID polynucleotide comprising SEQ ID NO:51 and SEQ ID NO:77. In some embodiments, an antibody is encoded by a NO:75. In some embodiments, an antibody is encoded by a polynucleotide comprising SEQ ID NO:71 and SEQ ID polynucleotide comprising SEQ ID NO:65 and SEQ ID NO:77. In some embodiments, an antibody is encoded by a NO:75. In some embodiments, an antibody is encoded by a 10 polynucleotide comprising SEQ ID NO:85 and SEQ ID polynucleotide comprising SEQ ID NO:95 and SEQ ID NO:91. In some embodiments, an antibody is encoded by a NO:89. In some embodiments, an antibody is encoded by a polynucleotide comprising SEQ ID NO:94 and SEQ ID polynucleotide comprising SEQ ID NO:92 and SEQ ID NO:91. NO:89. In certain embodiments, the polynucleotides comprise the In Some embodiments, an antibody is encoded by a 15 coding sequence for the mature polypeptide fused in the polynucleotide comprising SEQ ID NO:24 and SEQ ID same reading frame to a polynucleotide which aids, for NO:26. In some embodiments, an antibody is encoded by a example, in expression and secretion of a polypeptide from polynucleotide comprising SEQ ID NO:25 and SEQ ID a host cell (e.g., a leader sequence which functions as a NO:26. In some embodiments, an antibody is encoded by a secretory sequence for controlling transport of a polypeptide polynucleotide comprising SEQ ID NO:40 and SEQ ID from the cell). The polypeptide having a leader sequence is NO:26. In some embodiments, an antibody is encoded by a a preprotein and can have the leader sequence cleaved by the polynucleotide comprising SEQ ID NO:43 and SEQ ID host cell to form the mature form of the polypeptide. The NO:26. In some embodiments, an antibody is encoded by a polynucleotides can also encode for a proprotein which is polynucleotide comprising SEQ ID NO:52 and SEQ ID the mature protein plus additional 5' amino acid residues. A NO:26. In some embodiments, an antib ody is encoded by a 25 mature protein having a prosequence is a proprotein and is polynucleotide comprising SEQ ID NO:53 and SEQ ID an inactive form of the protein. Once the prosequence is NO:26. In some embodiments, an antib ody is encoded by a cleaved an active mature protein remains. polynucleotide comprising SEQ ID NO:66 and SEQ ID In certain embodiments, the polynucleotides comprise the NO:26. In some embodiments, an antib ody is encoded by a coding sequence for the mature polypeptide fused in the polynucleotide comprising SEQ ID NO:70 and SEQ ID 30 same reading frame to a marker sequence that allows, for NO:26. In some embodiments, an antib ody is encoded by a example, for purification of the encoded polypeptide. For polynucleotide comprising SEQ ID NO:24 and SEQ ID example, the marker sequence can be a hexa-histidine tag NO:76. In some embodiments, an antib ody is encoded by a supplied by a pCRE-9 vector to provide for purification of the polynucleotide comprising SEQ ID NO:25 and SEQ ID mature polypeptide fused to the marker in the case of a NO:76. In some embodiments, an antib ody is encoded by a 35 bacterial host, or the marker sequence can be a hemagglu polynucleotide comprising SEQ ID NO:40 and SEQ ID tinin (HA) tag derived from the influenza hemagglutinin NO:76. In some embodiments, an antib ody is encoded by a protein when a mammalian host (e.g., COS-7 cells) is used. polynucleotide comprising SEQ ID NO:43 and SEQ ID In some embodiments, the marker sequence is a FLAG-tag. NO:76. In some embodiments, an antib ody is encoded by a a peptide of sequence DYKDDDDK (SEQ ID NO:33) polynucleotide comprising SEQ ID NO:52 and SEQ ID 40 which can be used in conjunction with other affinity tags. NO:76. In some embodiments, an antib ody is encoded by a The present invention further relates to variants of the polynucleotide comprising SEQ ID NO:53 and SEQ ID hereinabove described polynucleotides encoding, for NO:76. In some embodiments, an antib ody is encoded by a example, fragments, analogs, and/or derivatives. polynucleotide comprising SEQ ID NO:66 and SEQ ID In certain embodiments, the present invention provides NO:76. In some embodiments, an antib ody is encoded by a 45 polynucleotides comprising polynucleotides having a polynucleotide comprising SEQ ID NO:70 and SEQ ID nucleotide sequence at least about 80% identical, at least NO:76. In some embodiments, an antib ody is encoded by a about 85% identical, at least about 90% identical, at least polynucleotide comprising SEQ ID NO:84 and SEQ ID about 95% identical, and in some embodiments, at least NO:90. In some embodiments, an antibody is encoded by a about 96%, 97%, 98% or 99% identical to a polynucleotide polynucleotide comprising SEQ ID NO:93 and SEQ ID 50 encoding a polypeptide comprising a RSPO3-binding agent NO:90. (e.g., an antibody), or fragment thereof, described herein. In Some embodiments, an antibody is encoded by a As used herein, the phrase a polynucleotide having a polynucleotide comprising SEQ ID NO:30 and SEQ ID nucleotide sequence at least, for example, 95% “identical to NO:32. In some embodiments, an antibody is encoded by a a reference nucleotide sequence is intended to mean that the polynucleotide comprising SEQ ID NO:31 and SEQ ID 55 nucleotide sequence of the polynucleotide is identical to the NO:32. In some embodiments, an antibody is encoded by a reference sequence except that the polynucleotide sequence polynucleotide comprising SEQ ID NO:54 and SEQ ID can include up to five point mutations per each 100 nucleo NO:32. In some embodiments, an antibody is encoded by a tides of the reference nucleotide sequence. In other words, to polynucleotide comprising SEQ ID NO:55 and SEQ ID obtain a polynucleotide having a nucleotide sequence at NO:32. In some embodiments, an antibody is encoded by a 60 least 95% identical to a reference nucleotide sequence, up to polynucleotide comprising SEQ ID NO:67 and SEQ ID 5% of the nucleotides in the reference sequence can be NO:32. In some embodiments, an antibody is encoded by a deleted or substituted with another nucleotide, or a number polynucleotide comprising SEQ ID NO:71 and SEQ ID of nucleotides up to 5% of the total nucleotides in the NO:32. In some embodiments, an antibody is encoded by a reference sequence can be inserted into the reference polynucleotide comprising SEQ ID NO:30 and SEQ ID 65 sequence. These mutations of the reference sequence can NO:77. In some embodiments, an antibody is encoded by a occur at the 5' or 3' terminal positions of the reference polynucleotide comprising SEQ ID NO:31 and SEQ ID nucleotide sequence or anywhere between those terminal US 9,598,497 B2 73 74 positions, interspersed either individually among nucleo vivo methods. In certain embodiments, a RSPO3-binding tides in the reference sequence or in one or more contiguous agent or polypeptide or antibody is an antagonist of human groups within the reference sequence. RSPO3. The polynucleotide variants can contain alterations in the In certain embodiments, the RSPO3-binding agents are coding regions, non-coding regions, or both. In some used in the treatment of a disease associated with activation embodiments, a polynucleotide variant contains alterations of B-catenin, increased B-catenin signaling, and/or aberrant which produce silent Substitutions, additions, or deletions, B-catenin signaling. In certain embodiments, the disease is a but does not alter the properties or activities of the encoded disease dependent upon B-catenin signaling. In certain polypeptide. In some embodiments, a polynucleotide variant embodiments, the RSPO3-binding agents are used in the comprises silent Substitutions that result in no change to the 10 treatment of disorders characterized by increased angiogen amino acid sequence of the polypeptide (due to the degen esis. In certain embodiments, the RSPO3-binding agents are eracy of the genetic code). In some embodiments, nucleotide used in the treatment of disorders characterized by increased variants comprise nucleotide sequences which result in levels of stem cells and/or progenitor cells. In some embodi expression differences (e.g., increased or decreased expres 15 ments, the methods comprise administering a therapeutically sion) at the transcript level. Polynucleotide variants can be effective amount of a RSPO3-binding agent (e.g., antibody) produced for a variety of reasons, for example, to optimize to a Subject. In some embodiments, the Subject is human. codon expression for a particular host (i.e., change codons in The present invention provides methods for inhibiting the human mRNA to those preferred by a bacterial host such growth of a tumor using the RSPO3-binding agents or as E. coli). In some embodiments, a polynucleotide variant antibodies described herein. In certain embodiments, the comprises at least one silent mutation in a non-coding or a method of inhibiting growth of a tumor comprises contact coding region of the sequence. ing a cell with a RSPO3-binding agent (e.g., an antibody) in In some embodiments, a polynucleotide variant is pro vitro. For example, an immortalized cell line or a cancer cell duced to modulate or alter expression (or expression levels) line is cultured in medium to which is added an anti-RSPO3 of the encoded polypeptide. In some embodiments, a poly 25 antibody or other agent to inhibit tumor growth. In some nucleotide variant is produced to increase expression of the embodiments, tumor cells are isolated from a patient sample encoded polypeptide. In some embodiments, a polynucle Such as, for example, a tissue biopsy, pleural effusion, or otide variant is produced to decrease expression of the blood sample and cultured in medium to which is added a encoded polypeptide. In some embodiments, a polynucle RSPO3-binding agent to inhibit tumor growth. otide variant has increased expression of the encoded poly 30 In some embodiments, the method of inhibiting growth of peptide as compared to a parental polynucleotide sequence. a tumor comprises contacting the tumor or tumor cells with In some embodiments, a polynucleotide variant has a RSPO3-binding agent (e.g., an antibody) in vivo. In certain decreased expression of the encoded polypeptide as com embodiments, contacting a tumor or tumor cell with a pared to a parental polynucleotide sequence. RSPO3-binding agent is undertaken in an animal model. For 35 example, a RSPO3-binding agent may be administered to In some embodiments, at least one polynucleotide variant immunocompromised mice (e.g. NOD/SCID mice) which is produced (without changing the amino acid sequence of have Xenografts. In some embodiments, cancer cells or the encoded polypeptide) to increase production of a het cancer stem cells are isolated from a patient sample Such as, eromultimeric molecule. In some embodiments, at least one for example, a tissue biopsy, pleural effusion, or blood polynucleotide variant is produced (without changing the 40 sample and injected into immunocompromised mice that are amino acid sequence of the encoded polypeptide) to increase then administered a RSPO3-binding agent to inhibit tumor production of a bispecific antibody. cell growth. In some embodiments, a RSPO3-binding agent In certain embodiments, the polynucleotides are isolated. is administered to the animal. In some embodiments, the In certain embodiments, the polynucleotides are Substan RSPO3-binding agent is administered at the same time or tially pure. 45 shortly after introduction of tumorigenic cells into the ani Vectors comprising the polynucleotides described herein mal to prevent tumor growth (“preventative model”). In are also provided. Cells comprising the polynucleotides some embodiments, the RSPO3-binding agent is adminis described herein are also provided. In some embodiments, tered as a therapeutic after tumors have grown to a specified an expression vector comprises a polynucleotide molecule. size (“therapeutic model). In some embodiments, the In some embodiments, a host cell comprises an expression 50 RSPO3-binding agent is an antibody. In some embodiments, vector comprising the polynucleotide molecule. In some the RSPO3-binding agent is an anti-RSPO3 antibody. In embodiments, a host cell comprises a polynucleotide mol some embodiments, the anti-RSPO3 antibody is antibody ecule. 131R002. In some embodiments, the anti-RSPO3 antibody is a humanized version of antibody 131 R002. In some IV. METHODS OF USE AND 55 embodiments, the anti-RSPO3 antibody is antibody PHARMACEUTICAL COMPOSITIONS 131R003. In some embodiments, the anti-RSPO3 antibody is a variant of antibody 131R003. In some embodiments, the The RSPO3-binding agents (including polypeptides and anti-RSPO3 antibody is a humanized version of antibody antibodies) of the invention are useful in a variety of 131R003. In some embodiments, the anti-RSPO3 antibody applications including, but not limited to, therapeutic treat 60 is a humanized version of a variant of antibody 131R003. In ment methods, such as the treatment of cancer. In certain some embodiments, the anti-RSPO3 antibody is antibody embodiments, the agents are useful for inhibiting B-catenin h131R006A or antibody h131R006B. In some embodi signaling, inhibiting tumor growth, modulating angiogen ments, the anti-RSPO3 antibody is antibody h131R005/ esis, inhibiting angiogenesis, inducing differentiation, 131R007. In some embodiments, the anti-RSPO3 antibody reducing tumor Volume, reducing the frequency of cancer 65 is antibody h131R008. In some embodiments, the anti stem cells in a tumor, and/or reducing the tumorigenicity of RSPO3 antibody is antibody h131R010. In some embodi a tumor. The methods of use may be in vitro, ex vivo, or in ments, the anti-RSPO3 antibody is antibody h131R011. US 9,598,497 B2 75 76 In certain embodiments, the method of inhibiting growth comprising AAS (SEQ ID NO:13), and a light chain CDR3 of a tumor comprises administering to a Subject a therapeu comprising QQSNEDPLT (SEQ ID NO:14). tically effective amount of a RSPO3-binding agent, wherein In certain embodiments, the method of inhibiting growth the RSPO3-binding agent comprises a heavy chain CDR1 of a tumor comprises administering to a subject a therapeu comprising KASGYTFTDYS (SEQ ID NO:9), KASGYT tically effective amount of a RSPO3-binding agent. In FTSYTF (SEQ ID NO:34), or DYSIH (SEQ ID NO:78), a certain embodiments, the Subject is a human. In certain heavy chain CDR2 comprising IYPSNGDS (SEQ ID embodiments, the Subject has a tumor or has had a tumor NO:10) or YTYPSNGDSGYNQKFK (SEQ ID NO:79), and which was removed. In some embodiments, the Subject has a heavy chain CDR3 comprising ATYFANYFDY (SEQ ID a tumor with an elevated expression level of at least one 10 RSPO protein (e.g., RSPO1, RSPO2, RSPO3, or RSPO4). In NO:11), ATYFANNFDY (SEQ ID NO:35), or TYFANNFD Some embodiments, the Subject has a tumor with a high (SEQ ID NO:80). In some embodiments of the method, the expression level of at least one RSPO protein (e.g., RSPO1, RSPO3-binding agent further comprises a light chain CDR1 RSPO2, RSPO3, or RSPO4). In some embodiments, the comprising QSVDYDGDSYM (SEQ ID NO:12) or RSPO-binding agent is a RSPO3-binding agent. In some KASQSVDYDGDSYMN (SEQ ID NO:81), a light chain 15 embodiments, the RSPO3-binding agent is an antibody. In CDR2 comprising AAS (SEQ ID NO:13) or AASNLES some embodiments, the RSPO3-binding agent is antibody (SEQ ID NO:82), and a light chain CDR3 comprising 131R002. In some embodiments, the anti-RSPO3 antibody QQSNEDPLT (SEQID NO:14) or QQSNEDPLTF (SEQID is antibody 131 R003. In some embodiments, the anti NO:83). In some embodiments, the RSPO3-binding agent RSPO3 antibody is a variant of antibody 131R003. In some comprises a heavy chain CDR1 comprising KASGYT embodiments, the anti-RSPO3 antibody is a humanized FTDYS (SEQ ID NO:9), a heavy chain CDR2 comprising version of antibody 131R003. In some embodiments, the IYPSNGDS (SEQ ID NO:10), and a heavy chain CDR3 anti-RSPO3 antibody is a humanized version of a variant of comprising ATYFANYFDY (SEQID NO:11), and/or a light antibody 131R003. In some embodiments, the RSPO3 chain CDR1 comprising QSVDYDGDSYM (SEQ ID binding agent is antibody h131R006A or antibody NO:12), a light chain CDR2 comprising AAS (SEQ ID 25 h131R006B. In some embodiments, the RSPO3-binding NO:13), and a light chain CDR3 comprising QQSNEDPLT agent is antibody h131R005/131R007. In some embodi (SEQ ID NO:14). In some embodiments, the RSPO3-bind ments, the RSPO3-binding agent is antibody h131R008. In ing agent comprises a heavy chain CDR1 comprising KAS some embodiments, the RSPO3-binding agent is antibody GYTFTDYS (SEQ ID NO:9), a heavy chain CDR2 com h131R010. In some embodiments, the RSPO3-binding agent prising IYPSNGDS (SEQ ID NO:10), and a heavy chain 30 is antibody h131R01. CDR3 comprising ATYFANNFDY (SEQID NO:35), and/or In certain embodiments, the tumor is a tumor in which a light chain CDR1 comprising QSVDYDGDSYM (SEQID B-catenin signaling is active. In some embodiments, the NO:12), a light chain CDR2 comprising AAS (SEQ ID tumor is a tumor in which B-catenin signaling is aberrant. In NO:13), and a light chain CDR3 comprising QQSNEDPLT certain embodiments, the tumor comprises an inactivating (SEQ ID NO:14). In some embodiments, the RSPO3-bind 35 mutation (e.g., a truncating mutation) in the APC tumor ing agent comprises a heavy chain CDR1 comprising KAS Suppressor gene. In certain embodiments, the tumor does not GYTFTSYTF (SEQ ID NO:34), a heavy chain CDR2 com comprise an inactivating mutation in the APC tumor Sup prising IYPSNGDS (SEQ ID NO:10), and a heavy chain pressor gene. In some embodiments, the tumor comprises a CDR3 comprising ATYFANNFDY (SEQID NO:35), and/or wild-type APC gene. In some embodiments, the tumor does a light chain CDR1 comprising QSVDYDGDSYM (SEQID 40 not comprise an activating mutation in the B-catenin gene. In NO:12), a light chain CDR2 comprising AAS (SEQ ID certain embodiments, a cancer for which a Subject is being NO:13), and a light chain CDR3 comprising QQSNEDPLT treated involves such a tumor. (SEQ ID NO:14). In some embodiments, the RSPO3-bind In some embodiments, the tumor comprises a RSPO gene ing agent comprises a heavy chain CDR1 comprising KAS fusion. In some embodiments, the tumor comprises a GYTFTDYS (SEQ ID NO:9) or DYSIH (SEQ ID NO:78), 45 RSPO2 gene fusion. In some embodiments, the tumor a heavy chain CDR2 comprising YTYPSNGDSGYNQKFK comprises a RSPO3 gene fusion. (SEQ ID NO:79), and a heavy chain CDR3 comprising In certain embodiments, the tumor expresses RSPO3 to TYFANNFD (SEQ ID NO:80), and/or a light chain CDR1 which a RSPO3-binding agent or antibody binds. In certain comprising KASQSVDYDGDSYMN (SEQ ID NO:81), a embodiments, the tumor has elevated expression levels of light chain CDR2 comprising AASNLES (SEQ ID NO:82), 50 RSPO1 or over-expresses RSPO1. In certain embodiments, and a light chain CDR3 comprising QQSNEDPLTF (SEQ the tumor has elevated expression levels of RSPO2 or ID NO:83). In some embodiments, the RSPO3-binding over-expresses RSPO2. In certain embodiments, the tumor agent comprises a heavy chain CDR1 comprising KASGYT has elevated expression levels of RSPO3 or over-expresses FTDYS (SEQ ID NO:9) or DYSIH (SEQ ID NO:78), a RSPO3. The phrase “a tumor has elevated expression levels heavy chain CDR2 comprising YTYPSNGDSGYNQKFK 55 of may refer to expression levels of a protein or expression (SEQ ID NO:79), and a heavy chain CDR3 comprising levels of a nucleic acid. In general, the phrase "a tumor has TYFANNFD (SEQ ID NO:80), and/or a light chain CDR1 elevated expression levels of a protein or a gene (or similar comprising KASQSVDYDGDSYMN (SEQ ID NO:81), a phrases) refers to expression levels of a protein or a gene in light chain CDR2 comprising AASNLES (SEQ ID NO:82), a tumor as compared to expression levels of the same protein and a light chain CDR3 comprising QQSNEDPLT (SEQ ID 60 or the same gene in a reference sample or to a pre NO:14). In some embodiments, the RSPO3-binding agent determined expression level. In some embodiments, the comprises a heavy chain CDR1 comprising KASGYT reference sample is normal tissue of the same tissue type. In FTDYS (SEQ ID NO:9) or DYSIH (SEQ ID NO:78), a Some embodiments, the reference sample is normal tissue of heavy chain CDR2 comprising IYPSNGDS (SEQ ID a group of tissue types. In some embodiments, the reference NO:10), and a heavy chain CDR3 comprising TYFANNFD 65 sample is a tumor or group of tumors of the same tissue type. (SEQ ID NO:80), and/or a light chain CDR1 comprising In some embodiments, the reference sample is a tumor or QSVDYDGDSYM (SEQ ID NO:12), a light chain CDR2 group of tumors of a different tissue type. Thus in some US 9,598,497 B2 77 78 embodiments, the expression levels of a protein or a gene in expression level of RSPO1. In some embodiments, the a tumor are “elevated' or “high as compared to the average tumor is a pancreatic tumor with an elevated expression expression level of the protein or the gene within a group of level of RSPO2. In some embodiments, the tumor is a colon tissue types. In some embodiments, the expression levels of tumor with an elevated expression level of RSPO2. In some a protein or a gene in a tumor are "elevated” or “high” as 5 embodiments, the tumor is a lung tumor with an elevated compared to the expression level of the protein or the gene expression level of RSPO2. In some embodiments, the in other tumors of the same tissue type or a different tissue tumor is a lung tumor with an elevated expression level of type. In some embodiments, the tumor expresses “elevated RSPO3. In some embodiments, the tumor is an ovarian or “high levels of RSPO1, RSPO2, RSPO3, and/or RSPO4 tumor with an elevated expression level of RSPO3. In some as compared to the RSPO levels expressed in normal tissue 10 of the same tissue type. In some embodiments, the tumor embodiments, the tumor is a breast tumor with an elevated expresses “elevated” or “high” levels of RSPO1, RSPO2, expression level of RSPO3. In some embodiments, the RSPO3, and/or RSPO4 as compared to a predetermined tumor is a colorectal tumor with an elevated expression level level. of RSPO3. In addition, the invention provides a method of inhibiting 15 The present invention further provides methods for treat growth of a tumor in a Subject, comprising administering a ing cancer comprising administering a therapeutically effec therapeutically effective amount of a RSPO3-binding agent tive amount of a RSPO3-binding agent to a subject. In to the Subject. In certain embodiments, the tumor comprises certain embodiments, the cancer is characterized by cells cancer stem cells. In certain embodiments, the frequency of expressing elevated levels of at least one RSPO protein as cancer stem cells in the tumor is reduced by administration compared to expression levels of the same RSPO protein in of the RSPO3-binding agent. The invention also provides a a reference sample. As used herein, a “reference sample method of reducing the frequency of cancer stem cells in a includes but is not limited to, normal tissue, non-cancerous tumor, comprising contacting the tumor with an effective tissue of the same tissue type, tumor tissue of the same tissue amount of a RSPO3-binding agent (e.g., an anti-RSPO3 type, and tumor tissue of a different tissue type. In certain antibody). In some embodiments, a method of reducing the 25 embodiments, the cancer is characterized by cells expressing frequency of cancer stem cells in a tumor in a subject, elevated levels of at least one RSPO protein as compared to comprising administering to the Subject a therapeutically a pre-determined level of the same RSPO protein. In some effective amount of a RSPO3-binding agent (e.g., an anti embodiments, determining the expression level of at least RSPO3 antibody) is provided. In some embodiments, the one RSPO is done prior to treatment. In some embodiments, RSPO3-binding agent is an antibody. In some embodiments, 30 determining the expression level of at least one RSPO is by the RSPO3-binding agent is an anti-RSPO3 antibody. In immunohistochemistry. Thus, in certain embodiments, the some embodiments, the anti-RSPO3 antibody is 131R002. cancer is characterized by cells expressing elevated levels of In some embodiments, the anti-RSPO3 antibody is antibody at least one RSPO protein as compared to expression levels 131R003. In some embodiments, the anti-RSPO3 antibody of the same RSPO protein in normal tissue. In certain is a variant of antibody 131R003. In some embodiments, the 35 embodiments, the cancer is characterized by cells over anti-RSPO3 antibody is a humanized version of antibody expressing RSPO1. In certain embodiments, the cancer is 131R003. In some embodiments, the anti-RSPO3 antibody characterized by cells over-expressing RSPO2. In certain is a humanized version of a variant of antibody 131R003. In embodiments, the cancer is characterized by cells over some embodiments, the anti-RSPO3 antibody is antibody expressing RSPO3. In certain embodiments, the cancer h131R006A or antibody h131R006B. In some embodi 40 over-expresses at least one RSPO protein selected from the ments, the anti-RSPO3 antibody is antibody h131R005/ group consisting of RSPO1, RSPO2, RSPO3, and/or 131R007. In some embodiments, the anti-RSPO3 antibody RSPO4. In certain embodiments, the cancer is characterized is antibody h131R008. In some embodiments, the anti by cells expressing B-catenin, wherein the RSPO3-binding RSPO3 antibody is antibody h131R010. In some embodi agent (e.g., an antibody) interferes with RSPO3-induced ments, the anti-RSPO3 antibody is antibody h131R011. 45 B-catenin signaling and/or activation. In some embodiments, the tumor is a Solid tumor. In In some embodiments, the RSPO-binding agent binds certain embodiments, the tumor is a tumor selected from the RSPO3, and inhibits or reduces growth of the cancer. In group consisting of colorectal tumor, pancreatic tumor, lung some embodiments, the RSPO-binding agent binds RSPO3, tumor, ovarian tumor, liver tumor, breast tumor, kidney interferes with RSPO3/LGR interactions, and inhibits or tumor, prostate tumor, gastrointestinal tumor, melanoma, 50 reduces growth of the cancer. In some embodiments, the cervical tumor, bladder tumor, glioblastoma, and head and RSPO-binding agent binds RSPO3, inhibits f-catenin acti neck tumor. As used herein, “lung cancer includes but is not Vation, and inhibits or reduces growth of the cancer. In some limited to, Small cell lung carcinoma and non-Small cell lung embodiments, the RSPO-binding agent binds RSPO3, and carcinoma (NSCLC). In certain embodiments, the tumor is reduces the frequency of cancer stem cells in the cancer. In a colorectal tumor. In certain embodiments, the tumor is an 55 some embodiments, the RSPO-binding agent is an antibody. ovarian tumor. In some embodiments, the tumor is a lung In some embodiments, the RSPO-binding agent is an anti tumor. In certain embodiments, the tumor is a pancreatic RSPO3 antibody. In some embodiments, the anti-RSPO3 tumor. In some embodiments, the tumor is a colorectal antibody is antibody 131R002. In some embodiments, the tumor that comprises an inactivating mutation in the APC anti-RSPO3 antibody is antibody 131R003. In some gene. In some embodiments, the tumor is a colorectal tumor 60 embodiments, the anti-RSPO3 antibody is a variant of that does not comprise an inactivating mutation in the APC antibody 131R003. In some embodiments, the anti-RSPO gene. In some embodiments, the tumor is a colorectal tumor antibody is a humanized version of antibody 131 R002. In that contains a RSPO gene fusion. In some embodiments, some embodiments, the anti-RSPO antibody is a humanized the tumor is a colorectal tumor that contains a RSPO2 gene version of antibody 131R003. In some embodiments, the fusion. In some embodiments, the tumor is a colorectal 65 anti-RSPO antibody is a humanized version of a variant of tumor that contains a RSPO3 gene fusion. In some embodi antibody 131R003. In some embodiments, the anti-RSPO3 ments, the tumor is an ovarian tumor with an elevated antibody is antibody h131R006A or antibody h131R006B. US 9,598,497 B2 79 80 In some embodiments, the anti-RSPO3 antibody is antibody AASNLES (SEQ ID NO:82), and a light chain CDR3 h131R005/131R007. In some embodiments, the anti-RSPO3 comprising QQSNEDPLT (SEQ ID NO:14). In some antibody is antibody h131R008. In some embodiments, the embodiments, the RSPO3-binding agent comprises a heavy anti-RSPO3 antibody is antibody h131R010. In some chain CDR1 comprising KASGYTFTDYS (SEQ ID NO:9) embodiments, the anti-RSPO3 antibody is antibody or DYSIH (SEQ ID NO:78), a heavy chain CDR2 compris h131 RO11. ing IYPSNGDS (SEQID NO:10), and a heavy chain CDR3 The present invention provides for methods of treating comprising TYFANNFD (SEQ ID NO:80), and/or a light cancer comprising administering a therapeutically effective chain CDR1 comprising QSVDYDGDSYM (SEQ ID amount of a RSPO3-binding agent to a subject (e.g., a NO:12), a light chain CDR2 comprising AAS (SEQ ID Subject in need of treatment). In certain embodiments, the 10 NO:13), and a light chain CDR3 comprising QQSNEDPLT method of treating cancer comprises administering to a (SEQ ID NO:14). In certain embodiments, the subject is a subject a therapeutically effective amount of a RSPO3 human. In certain embodiments, the Subject has a cancerous binding agent, wherein the RSPO3-binding agent comprises tumor. In certain embodiments, the Subject has had a tumor a heavy chain CDR1 comprising KASGYTFTDYS (SEQID removed. In some embodiments, a method of treating cancer NO:9), KASGYTFTSYTF (SEQ ID NO:34), or DYSIH 15 comprises administering a therapeutically effective amount (SEQ ID NO:78), a heavy chain CDR2 comprising of a RSPO3-binding agent to a subject, wherein the subject IYPSNGDS (SEQID NO:10) or YIYPSNGDSGYNQKFK has a tumor that has elevated expression of at least one (SEQ ID NO:79), and a heavy chain CDR3 comprising RSPO protein as compared to a reference sample or a ATYFANYFDY (SEQ ID NO:11), ATYFANNFDY (SEQ pre-determined level. In some embodiments, the subject has ID NO:35), or TYFANNFD (SEQ ID NO:80). In some a lung tumor that has elevated expression of RSPO3 and is embodiments of the method, the RSPO3-binding agent administered an anti-RSPO3 antibody. further comprises a light chain CDR1 comprising QSVDY The invention also provides a RSPO3-binding agent for DGDSYM (SEQ ID NO.12) or KASQSVDYDGDSYMN use in a method of treating cancer, wherein the RSPO3 (SEQ ID NO:81), a light chain CDR2 comprising AAS binding agent is an antibody described herein. The invention (SEQID NO:13) or AASNLES (SEQID NO:82), and a light 25 also provides the use of an RSPO3-binding agent (e.g., an chain CDR3 comprising QQSNEDPLT (SEQID NO:14) or antibody) described herein for the manufacture of a medi QQSNEDPLTF (SEQ ID NO:83). In some embodiments, cament for the treatment of cancer. the RSPO3-binding agent comprises a heavy chain CDR1 In certain embodiments, the cancer is a cancer selected comprising KASGYTFTDYS (SEQ ID NO:9), a heavy from the group consisting of colorectal cancer, pancreatic chain CDR2 comprising IYPSNGDS (SEQ ID NO:10), and 30 cancer, lung cancer, ovarian cancer, liver cancer, breast a heavy chain CDR3 comprising ATYFANYFDY (SEQ ID cancer, kidney cancer, prostate cancer, gastrointestinal can NO:11), and/or a light chain CDR1 comprising QSVDY cer, melanoma, cervical cancer, bladder cancer, glioblas DGDSYM (SEQ ID NO:12), a light chain CDR2 compris toma, and head and neck cancer. In certain embodiments, the ing AAS (SEQ ID NO:13), and a light chain CDR3 com cancer is pancreatic cancer. In certain embodiments, the prising QQSNEDPLT (SEQ ID NO:14). In some 35 cancer is ovarian cancer. In certain embodiments, the cancer embodiments, the RSPO3-binding agent comprises a heavy is colorectal cancer. In certain embodiments, the cancer is chain CDR1 comprising KASGYTFTDYS (SEQID NO:9), breast cancer. In certain embodiments, the cancer is prostate a heavy chain CDR2 comprising IYPSNGDS (SEQ ID cancer. In certain embodiments, the cancer is lung cancer. NO:10), and a heavy chain CDR3 comprising ATYFAN In addition, the invention provides a method of reducing NFDY (SEQ ID NO:35), and/or a light chain CDR1 com 40 the tumorigenicity of a tumor in a Subject, comprising prising QSVDYDGDSYM (SEQ ID NO:12), a light chain administering to a Subject a therapeutically effective amount CDR2 comprising AAS (SEQID NO:13), and a light chain of a RSPO3-binding agent. In certain embodiments, the CDR3 comprising QQSNEDPLT (SEQID NO:14). In some tumor comprises cancer stem cells. In some embodiments, embodiments, the RSPO3-binding agent comprises a heavy the tumorigenicity of a tumor is reduced by reducing the chain CDR1 comprising KASGYTFTSYTF (SEQ ID 45 frequency of cancer stem cells in the tumor. In some NO:34), a heavy chain CDR2 comprising IYPSNGDS (SEQ embodiments, the methods comprise using the RSPO3 ID NO:10), and a heavy chain CDR3 comprising ATYFAN binding agents described herein. In certain embodiments, the NFDY (SEQ ID NO:35), and/or a light chain CDR1 com frequency of cancer stem cells in the tumor is reduced by prising QSVDYDGDSYM (SEQ ID NO:12), a light chain administration of a RSPO3-binding agent. CDR2 comprising AAS (SEQID NO:13), and a light chain 50 In certain embodiments, the methods further comprise a CDR3 comprising QQSNEDPLT (SEQID NO:14). In some step of determining the expression level of at least one embodiments, the RSPO3-binding agent comprises a heavy RSPO (i.e., protein or nucleic acid) in the tumor or cancer. chain CDR1 comprising KASGYTFTDYS (SEQ ID NO:9) In some embodiments, the step of determining the expres or DYSIH (SEQ ID NO:78), a heavy chain CDR2 compris sion level of a RSPO in the tumor or cancer comprises ing YIYPSNGDSGYNQKFK (SEQID NO:79), and a heavy 55 determining the expression level of RSPO1, RSPO2, chain CDR3 comprising TYFANNFD (SEQ ID NO:80), RSPO3, and/or RSPO4. In some embodiments, the expres and/or a light chain CDR1 comprising KASQSVDYDGD sion level of RSPO1, RSPO2, RSPO3, and/or RSPO4 in a SYMN (SEQ ID NO:81), a light chain CDR2 comprising tumor or cancer is compared to the expression level of AASNLES (SEQ ID NO:82), and a light chain CDR3 RSPO1, RSPO2, RSPO3, and/or RSPO4 in a reference comprising QQSNEDPLTF (SEQ ID NO:83). In some 60 sample. In some embodiments, the expression level of embodiments, the RSPO3-binding agent comprises a heavy RSPO1, RSPO2, RSPO3, and/or RSPO4 in a tumor or chain CDR1 comprising KASGYTFTDYS (SEQ ID NO:9) cancer is compared to the expression level of RSPO1, or DYSIH (SEQ ID NO:78), a heavy chain CDR2 compris RSPO2, RSPO3, and/or RSPO4 in normal tissue. In some ing YIYPSNGDSGYNQKFK (SEQID NO:79), and a heavy embodiments, the level of expression of RSPO1, RSPO2, chain CDR3 comprising TYFANNFD (SEQ ID NO:80), 65 RSPO3, and/or RSPO4 in a tumor or cancer is compared to and/or a light chain CDR1 comprising KASQSVDYDGD a pre-determined level of expression of RSPO1, RSPO2, SYMN (SEQ ID NO:81), a light chain CDR2 comprising RSPO3, and/or RSPO4. In some embodiments, the level of US 9,598,497 B2 81 82 expression of RSPO1, RSPO2, RSPO3, and/or RSPO4 in a obtaining a tumor sample from the Subject, and determining tumor or cancer is compared to a pre-determined level of if the tumor has an elevated expression level of RSPO3 as expression of RSPO1, RSPO2, RSPO3, and/or RSPO4 in compared to a reference sample or a pre-determined level of normal tissue. In some embodiments, the tumor has a high RSPO3. In some embodiments, if the tumor has an elevated expression level of RSPO1. In some embodiments, the 5 expression level of RSPO3, the subject is selected for tumor has a high expression level of RSPO3. In general, the treatment with an antibody that specifically binds RSPO3. In expression level of a RSPO (i.e., protein or nucleic acid) is some embodiments, if selected for treatment, the subject is compared to the expression level of the RSPO (i.e., protein administered a RSPO3-binding agent or antibody describe or nucleic acid) in normal tissue of the same tissue type. herein. In some embodiments, if the tumor has an elevated However, in some embodiments, the expression level of a 10 expression level of more than one RSPO (i.e., protein or RSPO (i.e., protein or nucleic acid) is compared to the nucleic acid), the subject is administered a RSPO-binding average expression level of the RSPO (i.e., protein or agent that binds the RSPO with the highest level of expres nucleic acid) within a group of tissue types. In some Sion. In certain embodiments, the Subject has had a tumor embodiments, the expression levels of a RSPO (i.e., protein removed. For example, in Some embodiments, the expres or nucleic acid) in a tumor is compared to the expression 15 sion level of RSPO1, RSPO2, RSPO3, and/or RSPO4 in a level of the RSPO (i.e., protein or nucleic acid) in other tumor is determined, if the tumor has an elevated level of tumors of the same tissue type or a different tissue type. RSPO3 expression as compared to the level of RSPO3 in In some embodiments, determining the level of RSPO normal tissue, the subject is selected for treatment with an expression is done prior to treatment. In some embodiments, antibody that specifically binds RSPO3. If selected for the subject is administered a RSPO3-binding agent or anti treatment, the subject is administered an anti-RSPO3 anti body describe herein if the tumor or cancer has an elevated body describe herein. In some embodiments, the RSPO3 expression level of RSPO as compared to the expression binding agent is antibody 131 R002. In some embodiments, level of the same RSPO in a reference sample (e.g., normal the RSPO3-binding agent is antibody 131R003. In some tissue) or a pre-determined level. For example, in some embodiments, the RSPO3-binding agent is a variant of embodiments, the subject is administered a RSPO3-binding 25 antibody 131R003. In some embodiments, the RSPO3 agent (e.g., anti-RSPO3 antibody) if the tumor or cancer has binding agent is a humanized form of antibody 131R003. In an elevated expression level of RSPO3 (i.e., protein or some embodiments, the RSPO3-binding agent is a human nucleic acid) as compared to the expression level of RSPO3 ized form of a variant of antibody 131R003. In some in normal or control tissue. embodiments, the RSPO3-binding agent is antibody In certain embodiments, the methods further comprise a 30 h131R006A or antibody h131R006B. In some embodi step of determining if the tumor or cancer has an inactivating ments, the RSPO3-binding agent is antibody h131R005/ mutation in the APC gene. In some embodiments, the 131R007. In some embodiments, the RSPO3-binding agent methods further comprise a step of determining if the tumor is antibody h131R008. In some embodiments, the RSPO3 or cancer has an activating mutation in the B-catenin gene. binding agent is antibody h131R010. In some embodiments, In some embodiments, the methods further comprise a step 35 the RSPO3-binding agent is antibody h131R011. of determining if the tumor or cancer has a RSPO gene The present invention provides methods of selecting a fusion. human subject for treatment with a RSPO-binding agent, In addition, the invention provides a method of modulat comprising determining if the Subject has a tumor that has an ing angiogenesis, comprising administering to a Subject a elevated expression level of at least one RSPO (i.e., protein therapeutically effective amount of a RSPO3-binding agent. 40 or nucleic acid), as compared to expression of the same In some embodiments, the modulating angiogenesis com RSPO in normal tissue or as compared to a predetermined prises inhibiting angiogenesis. In some embodiments, the level, wherein if the tumor has an elevated expression level methods comprise using the RSPO3-binding agents of at least one RSPO, the subject is selected for treatment described herein. In certain embodiments, the RSPO3-bind with an antibody that specifically binds the RSPO with the ing agent binds RSPO3 and inhibits or reduces angiogenesis. 45 elevated expression level. In some embodiments, if selected In certain embodiments, the inhibition and/or reduction of for treatment, the subject is administered a RSPO-binding angiogenesis inhibits or reduces growth of a tumor or cancer. agent or antibody describe herein. In some embodiments, a In some embodiments, the RSPO3-binding agent binds method of selecting a human Subject for treatment with an RSPO3 and promotes aberrant angiogenesis. In some antibody that specifically binds RSPO3 comprises: deter embodiments, the RSPO3-binding agent binds RSPO3 and 50 mining if the Subject has a tumor that has an elevated promotes unproductive angiogenesis. In certain embodi expression level of RSPO3 as compared to a reference ments, the aberrant angiogenesis or the unproductive angio sample or a pre-determined level of RSPO3. In some genesis inhibits or reduces growth of a tumor or cancer. embodiments, a method of selecting a human Subject for In addition, the present invention provides methods of treatment with an antibody that specifically binds RSPO3 identifying a human subject for treatment with a RSPO 55 comprises obtaining a tumor sample from the Subject, and binding agent, comprising determining if the Subject has a determining if the tumor has an elevated expression level of tumor that has an elevated expression level of RSPO (i.e., RSPO3 as compared to a reference sample or a pre-deter protein or nucleic acid) as compared to expression of the mined level of RSPO3. In some embodiments, a method of same RSPO (i.e., protein or nucleic acid) in normal tissue, selecting a human Subject for treatment with an antibody in a reference sample, or to a pre-determined level of the 60 that specifically binds RSPO3, comprises determining if the RSPO protein. In some embodiments, a method of identi Subject has a tumor that has an elevated expression level of fying a human subject for treatment with a RSPO3-binding RSPO3 as compared to a reference sample or a pre-deter agent comprises determining if the Subject has a tumor that mined level of RSPO3, wherein if the tumor has an elevated has an elevated expression level of RSPO3 as compared to expression level of RSPO3 the subject is selected for treat a reference sample or a pre-determined level of RSPO3. In 65 ment with the antibody. In some embodiments, a method of Some embodiments, a method of identifying a human Sub selecting a human Subject for treatment with an antibody ject for treatment with a RSPO3-binding agent comprises: that specifically binds RSPO3, comprises obtaining a tumor US 9,598,497 B2 83 84 sample from the Subject, and determining if the tumor has an mining if the tumor has a RSPO gene fusion, wherein if the elevated expression level of RSPO3 as compared to a tumor has a RSPO gene fusion, then the subject is selected reference sample or a pre-determined level of RSPO3, for treatment with the antibody. In some embodiments, a wherein if the tumor has an elevated expression level of method of selecting a human Subject for treatment with an RSPO3 the subject is selected for treatment with the anti antibody that specifically binds RSPO3, comprises deter body. In certain embodiments, the Subject has had a tumor mining if the subject has a tumor that has a RSPO gene removed. In some embodiments, the RSPO-binding agent is fusion. a RSPO3-binding agent. In some embodiments, the RSPO3 The present invention also provides methods of selecting binding agent is an anti-RSPO3 antibody. In some embodi a human subject for treatment with a RSPO-binding agent, ments, the anti-RSPO3 antibody is antibody 131R002. In 10 comprising determining if the Subject has a tumor that has a some embodiments, the anti-RSPO3 antibody is antibody RSPO gene fusion, wherein if the tumor has a RSPO gene 131R003. In some embodiments, the anti-RSPO3 antibody fusion, the subject is selected for treatment with an antibody is a variant of antibody 131R003. In some embodiments, the that specifically binds a RSPO protein. In some embodi anti-RSPO3 antibody is a humanized version of antibody ments, a method of selecting a human Subject for treatment 131R002. In some embodiments, the anti-RSPO3 antibody 15 with an antibody that specifically binds RSPO3 comprises is a humanized version of antibody 131 R003. In some determining if the subject has a tumor that has a RSPO gene embodiments, the anti-RSPO3 antibody is a humanized fusion. In some embodiments, a method of selecting a version of a variant of antibody 131 R003. In some embodi human subject for treatment with an antibody that specifi ments, the anti-RSPO3 antibody is antibody h131R006A or cally binds RSPO3, comprises obtaining a tumor sample h131R006B. In some embodiments, the anti-RSPO3 anti from the subject, and determining if the tumor has a RSPO body is antibody h131R005/131R007. In some embodi gene fusion. In some embodiments, a method of selecting a ments, the anti-RSPO3 antibody is antibody h131R008. In human subject for treatment with an antibody that specifi some embodiments, the anti-RSPO3 antibody is antibody cally binds RSPO3, comprises determining if the subject has h131R010. In some embodiments, the anti-RSPO3 antibody a tumor that has a RSPO gene fusion, wherein if the tumor is antibody h131R011. 25 has a RSPO gene fusion the subject is selected for treatment The present invention also provides methods of treating with the antibody. In some embodiments, a method of cancer in a human Subject, comprising: (a) selecting a selecting a human Subject for treatment with an antibody Subject for treatment based, at least in part, on the Subject that specifically binds RSPO3, comprises obtaining a tumor having a cancer that has an elevated level of a RSPO, and (b) sample from the Subject, and determining if the tumor has a administering to the subject a therapeutically effective 30 RSPO gene fusion, wherein if the tumor has a RSPO gene amount of a RSPO3-binding agent described herein. In some fusion the subject is selected for treatment with the antibody. embodiments, the RSPO3-binding agent is antibody In some embodiments, the RSPO gene fusion is a RSPO2 131R002. In some embodiments, the RSPO3-binding agent gene fusion. In some embodiments, the RSPO gene fusion is is antibody 131R003. In some embodiments, the RSPO3 a RSPO3 gene fusion. In some embodiments, if selected for binding agent is a variant of antibody 131R003. In some 35 treatment, the subject is administered a RSPO-binding agent embodiments, the anti-RSPO3 antibody is a humanized or antibody describe herein. In certain embodiments, the version of antibody 131R002. In some embodiments, the Subject has had a tumor removed. In some embodiments, the anti-RSPO3 antibody is a humanized version of antibody RSPO-binding agent is a RSPO3-binding agent. In some 131R003. In some embodiments, the anti-RSPO3 antibody embodiments, the RSPO3-binding agent is an anti-RSPO3 is a humanized version of a variant of antibody 131R003. In 40 antibody. In some embodiments, the anti-RSPO3 antibody is some embodiments, the anti-RSPO3 antibody is antibody antibody 131R002. In some embodiments, the anti-RSPO3 h131R006A or h131R006B. In some embodiments, the antibody is antibody 131R003. In some embodiments, the anti-RSPO3 antibody is antibody h131R005/131R007. In anti-RSPO3 antibody is a variant of antibody 131R003. In some embodiments, the anti-RSPO3 antibody is antibody some embodiments, the anti-RSPO3 antibody is a human h131R008. In some embodiments, the anti-RSPO3 antibody 45 ized version of antibody 131R002. In some embodiments, is antibody h131R010. In some embodiments, the anti the anti-RSPO3 antibody is a humanized version of antibody RSPO3 antibody is antibody h131R011. 131R003. In some embodiments, the anti-RSPO3 antibody Methods for determining the level of RSPO expression in is a humanized version of a variant of antibody 131R003. In a cell, tumor or cancer are known by those of skill in the art. some embodiments, the anti-RSPO3 antibody is antibody For nucleic acid expression these methods include, but are 50 h131R006A or h131R006B. In some embodiments, the not limited to, PCR-based assays, microarray analyses and anti-RSPO3 antibody is antibody h131R005/131R007. In nucleotide sequencing (e.g., NextGen sequencing). For pro some embodiments, the anti-RSPO3 antibody is antibody tein expression these methods include, but are not limited to, h131R008. In some embodiments, the anti-RSPO3 antibody Western blot analysis, protein arrays, ELISAs, immunohis is antibody h131R010. In some embodiments, the anti tochemistry (IHC) assays, and FACS. 55 RSPO3 antibody is antibody h131R011. The present invention provides methods of identifying a The present invention also provides methods of treating human subject for treatment with a RSPO3-binding agent, cancer in a human Subject, comprising: (a) selecting a comprising obtaining a tumor sample from the Subject, and Subject for treatment based, at least in part, on the Subject determining if the tumor has a RSPO gene fusion. In some having a cancer that has a RSPO gene fusion, and (b) embodiments, a method of identifying a human Subject for 60 administering to the subject a therapeutically effective treatment with a RSPO3-binding agent comprises: determin amount of a RSPO3-binding agent described herein. In some ing if the subject has a tumor that has a RSPO gene fusion, embodiments, the RSPO3-binding agent is antibody wherein if the tumor has a RSPO gene fusion, then the 131R002. In some embodiments, the RSPO3-binding agent subject is selected for treatment with the antibody. In some is antibody 131R003. In some embodiments, the RSPO3 embodiments, a method of identifying a human Subject for 65 binding agent is a variant of antibody 131R003. In some treatment with a RSPO3-binding agent comprises: (a) embodiments, the anti-RSPO3 antibody is a humanized obtaining a tumor sample from the Subject, and (b) deter version of antibody 131R002. In some embodiments, the US 9,598,497 B2 85 86 anti-RSPO3 antibody is a humanized version of antibody and/or LGR6. In certain embodiments, the LGR is LGR4. In 131R003. In some embodiments, the anti-RSPO3 antibody certain embodiments, the LGR is LGR5. In certain embodi is a humanized version of a variant of antibody 131R003. In ments, the LGR is LGR6. In some embodiments, the RSPO some embodiments, the anti-RSPO3 antibody is antibody binding agent is a RSPO3-binding agent. In some embodi h131R006A or h131R006B. In some embodiments, the 5 ments, the RSPO3-binding agent is an antibody. In some anti-RSPO3 antibody is antibody h131R005/131R007. In embodiments, the RSPO3-binding agent is antibody some embodiments, the anti-RSPO3 antibody is antibody 131R002. In some embodiments, the RSPO3-binding agent h131R008. In some embodiments, the anti-RSPO3 antibody is antibody 131R003. In some embodiments, the RSPO3 is antibody h131R010. In some embodiments, the anti binding agent is a variant of antibody 131R003. In some RSPO3 antibody is antibody h131R011. 10 embodiments, the RSPO3-binding agent is a humanized Methods for determining whether a tumor has a RSPO version of antibody 131R002. In some embodiments, the gene fusion are known by those of skill in the art. Methods RSPO3-binding agent is a humanized version of antibody may include but are not limited to, PCR-based assays, 131R003. In some embodiments, the RSPO3-binding agent microarray analyses, and nucleotide sequencing (e.g., Nex is a humanized version of a variant of antibody 131R003. In tGen sequencing, whole-genome sequencing (WGS)). 15 some embodiments, the RSPO3-binding agent is antibody Methods for determining whether a tumor or cancer has h131R006A or antibody h131R006B. In some embodi an elevated level of RSPO expression or has a RSPO gene ments, the RSPO3-binding agent is antibody h131R005/ fusion can use a variety of samples. In some embodiments, 131R007. In some embodiments, the RSPO3-binding agent the sample is taken from a subject having a tumor or cancer. is antibody h131R008. In some embodiments, the RSPO3 In some embodiments, the sample is a fresh tumor/cancer binding agent is antibody h131R010. In some embodiments, sample. In some embodiments, the sample is a frozen the RSPO3-binding agent is antibody h131R011. tumor/cancer sample. In some embodiments, the sample is a The use of the RSPO-binding agents, polypeptides, or formalin-fixed paraffin-embedded sample. In some embodi antibodies described herein to induce the differentiation of ments, the sample is processed to a cell lysate. In some cells, including, but not limited to tumor cells, is also embodiments, the sample is processed to DNA or RNA. 25 provided. In some embodiments, methods of inducing cells Methods of treating a disease or disorder in a Subject, to differentiate comprise contacting the cells with an effec wherein the disease or disorder is associated with aberrant tive amount of a RSPO-binding agent (e.g., an anti-RSPO (e.g., increased levels) f-catenin signaling are further pro antibody) described herein. In certain embodiments, meth vided. Methods of treating a disease or disorder in a subject, ods of inducing cells in a tumor in a Subject to differentiate wherein the disease or disorder is characterized by an 30 comprise administering a therapeutically effective amount increased level of stem cells and/or progenitor cells are of a RSPO-binding agent, polypeptide, or antibody to the further provided. In some embodiments, the treatment meth subject. In some embodiments, methods for inducing dif ods comprise administering a therapeutically effective ferentiation markers on tumor cells comprise administering amount of a RSPO-binding agent, polypeptide, or antibody a therapeutically effective amount of a RSPO-binding agent, to the subject. In some embodiments, the RSPO-binding 35 polypeptide, or antibody. In some embodiments, the tumor agent is a RSPO3-binding agent. In some embodiments, the is a Solid tumor. In some embodiments, the tumor is selected RSPO3-binding agent is an antibody. In some embodiments, from the group consisting of colorectal tumor, pancreatic the RSPO3-binding agent is antibody 131R002. In some tumor, lung tumor, ovarian tumor, liver tumor, breast tumor, embodiments, the RSPO3-binding agent is antibody kidney tumor, prostate tumor, gastrointestinal tumor, mela 131R003. In some embodiments, the RSPO3-binding agent 40 noma, cervical tumor, bladder tumor, glioblastoma, and head is a variant of antibody 131R003. In some embodiments, the and neck tumor. In certain embodiments, the tumor is an RSPO3-binding agent is a humanized version of antibody ovarian tumor. In certain other embodiments, the tumor is a 131R002. In some embodiments, the RSPO3-binding agent colon tumor. In some embodiments, the tumor is a lung is a humanized version of antibody 131 R003. In some tumor. In certain embodiments, the method is an in vivo embodiments, the RSPO3-binding agent is a humanized 45 method. In certain embodiments, the method is an in vitro version of a variant of antibody 131 R003. In some embodi method. In some embodiments, the RSPO-binding agent is ments, the RSPO3-binding agent is antibody h131R006A or a RSPO3-binding agent. In some embodiments, the RSPO3 antibody h131R006B. In some embodiments, the RSPO3 binding agent is an antibody. In some embodiments, the binding agent is antibody h131R005/131R007. In some RSPO3-binding agent is antibody 131R002. In some embodiments, the RSPO3-binding agent is antibody 50 embodiments, the RSPO3-binding agent is antibody h131R008. In some embodiments, the RSPO3-binding agent 131R003. In some embodiments, the RSPO3-binding agent is antibody h131R010. In some embodiments, the RSPO3 is a variant of antibody 131R003. In some embodiments, the binding agent is antibody h131R011. RSPO3-binding agent is a humanized version of antibody The invention also provides a method of inhibiting 131R002. In some embodiments, the RSPO3-binding agent B-catenin signaling in a cell comprising contacting the cell 55 is a humanized version of antibody 131 R003. In some with an effective amount of a RSPO-binding agent. In embodiments, the RSPO3-binding agent is a humanized certain embodiments, the cell is a tumor cell. In certain version of a variant of antibody 131 R003. In some embodi embodiments, the method is an in vivo method wherein the ments, the RSPO3-binding agent is antibody h131R006A or step of contacting the cell with the RSPO3-binding agent antibody h131R006B. In some embodiments, the RSPO3 comprises administering a therapeutically effective amount 60 binding agent is antibody h131R005/131R007. In some of the RSPO3-binding agent to the subject. In some embodi embodiments, the RSPO3-binding agent is antibody ments, the method is an in vitro or ex vivo method. In certain h131R008. In some embodiments, the RSPO3-binding agent embodiments, the RSPO-binding agent inhibits B-catenin is antibody h131R010. In some embodiments, the RSPO3 signaling. In some embodiments, the RSPO-binding agent binding agent is antibody h131R011. inhibits activation of B-catenin. In certain embodiments, the 65 The invention further provides methods of differentiating RSPO-binding agent interferes with a RSPO/LGR interac tumorigenic cells into non-tumorigenic cells comprising tion. In certain embodiments, the LGR is LGR4, LGR5, contacting the tumorigenic cells with a RSPO-binding agent. US 9,598,497 B2 87 88 In some embodiments, the method comprises administering nontoxic buffers such as phosphate, citrate, and other the RSPO-binding agent to a subject that has a tumor organic acids; salts such as sodium chloride; antioxidants comprising tumorigenic cells or that has had such a tumor including ascorbic acid and methionine; preservatives Such removed. In certain embodiments, the tumorigenic cells are as octadecyldimethylbenzyl ammonium chloride, hexam ovarian tumor cells. In certain embodiments, the tumori ethonium chloride, benzalkonium chloride, benzethonium genic cells are colon tumor cells. In some embodiments, the chloride, phenol, butyl or benzyl alcohol, alkyl parabens, tumorigenic cells are lung tumor cells. In some embodi Such as methyl or propyl paraben, catechol, resorcinol, ments, the RSPO-binding agent is a RSPO3-binding agent. cyclohexanol, 3-pentanol, and m-cresol; low molecular In some embodiments, the RSPO3-binding agent is an weight polypeptides (e.g., less than about 10 amino acid antibody. In some embodiments, the RSPO3-binding agent 10 is antibody 131R002. In some embodiments, the RSPO3 residues); proteins such as serum albumin, gelatin, or immu binding agent is antibody 131 R003. In some embodiments, noglobulins; hydrophilic polymers such as polyvinylpyrroli the RSPO3-binding agent is a variant of antibody 131R003. done; amino acids such as glycine, glutamine, asparagine, In some embodiments, the RSPO3-binding agent is a histidine, arginine, or lysine; carbohydrates Such as mono humanized version of antibody 131R002. In some embodi 15 saccharides, disaccharides, glucose, mannose, or dextrins; ments, the RSPO3-binding agent is a humanized version of chelating agents such as EDTA; Sugars such as Sucrose, antibody 131R003. In some embodiments, the RSPO3 mannitol, trehalose or Sorbitol; salt-forming counter-ions binding agent is a humanized version of a variant of anti Such as sodium; metal complexes such as Zn-protein com body 131R003. In some embodiments, the RSPO3-binding plexes; and non-ionic surfactants such as TWEEN or poly agent is antibody h131R006A or antibody h131R006B. In ethylene glycol (PEG). (Remington. The Science and Prac some embodiments, the RSPO3-binding agent is antibody tice of Pharmacy, 22 Edition, 2012, Pharmaceutical Press, h131R005/131R007. In some embodiments, the RSPO3 London.) binding agent is antibody h131R008. In some embodiments, The pharmaceutical compositions of the present invention the RSPO3-binding agent is antibody h131R010. In some can be administered in any number of ways for either local embodiments, the RSPO3-binding agent is antibody 25 or systemic treatment. Administration can be topical by h131 RO11. epidermal or transdermal patches, ointments, lotions, In certain embodiments, the disease treated with the creams, gels, drops, suppositories, sprays, liquids and pow RSPO3-binding agents described herein is not a cancer. For ders; pulmonary by inhalation or insufflation of powders or example, the disease may be a metabolic disorder Such as aerosols, including by nebulizer, intratracheal, and intrana obesity or diabetes (e.g., type II diabetes) (Jin T., 2008, 30 sal; oral; or parenteral including intravenous, intraarterial, Diabetologia, 51:1771-80). Alternatively, the disease may intratumoral, Subcutaneous, intraperitoneal, intramuscular be a bone disorder such as osteoporosis, osteoarthritis, or (e.g., injection or infusion), or intracranial (e.g., intrathecal rheumatoid arthritis (Corr M., 2008, Nat. Clin. Pract. Rheu or intraventricular). matoll., 4:550-6: Day et al., 2008, Bone Joint Surg. Am., 90 The therapeutic formulation can be in unit dosage form. Suppl 1:19-24). The disease may also be a kidney disorder, 35 Such formulations include tablets, pills, capsules, powders, such as a polycystic kidney disease (Harris et al., 2009, Ann. granules, Solutions or Suspensions in water or non-aqueous Rev. Med., 60:321-337; Schmidt-Ott et al., 2008, Kidney media, or Suppositories. In solid compositions such as Int., 74: 1004-8; Benzing et al., 2007, J. Am. Soc. Nephrol., tablets the principal active ingredient is mixed with a 18:1389-98). Alternatively, eye disorders including, but not pharmaceutical carrier. Conventional tableting ingredients limited to, macular degeneration and familial exudative 40 include corn starch, lactose, Sucrose, Sorbitol, talc, Stearic vitreoretinopathy may be treated (Lad et al., 2009, Stem acid, magnesium Stearate, dicalcium phosphate or gums, and Cells Dev., 18:7-16). Cardiovascular disorders, including diluents (e.g., water). These can be used to form a solid myocardial infarction, atherosclerosis, and valve disorders, pre-formulation composition containing a homogeneous may also be treated (Al-Aly Z., 2008, Transl. Res., 151:233 mixture of a compound of the present invention, or a 9; Kobayashi et al., 2009, Nat. Cell Biol., 11:46-55; van Gijn 45 non-toxic pharmaceutically acceptable Salt thereof. The et al., 2002, Cardiovasc. Res., 55:16-24; Christman et al., solid pre-formulation composition is then subdivided into 2008, Am. J. Physiol. Heart Circ. Physiol., 294:H2864-70). unit dosage forms of a type described above. The tablets, In some embodiments, the disease is a pulmonary disorder pills, etc. of the formulation or composition can be coated or Such as idiopathic pulmonary arterial hypertension or pull otherwise compounded to provide a dosage form affording monary fibrosis (Laumanns et al., 2008, Am. J. Respir: Cell 50 the advantage of prolonged action. For example, the tablet or Mol. Biol., 2009, 40.683-691; Konigshoffet al., 2008, PLoS pill can comprise an inner composition covered by an outer ONE 3:e2142). In some embodiments, the disease treated component. Furthermore, the two components can be sepa with the RSPO3-binding agent is a liver disease, such as rated by an enteric layer that serves to resist disintegration cirrhosis or liver fibrosis (Cheng et al., 2008, Am. J. Physiol. and permits the inner component to pass intact through the Gastrointest. Liver Physiol., 294:G39-49). 55 stomach or to be delayed in release. A variety of materials The present invention further provides pharmaceutical can be used for Such enteric layers or coatings, such mate compositions comprising the RSPO3-binding agents rials include a number of polymeric acids and mixtures of described herein. In certain embodiments, the pharmaceuti polymeric acids with Such materials as shellac, cetyl alcohol cal compositions further comprise a pharmaceutically and cellulose acetate. acceptable vehicle. In some embodiments, these pharma 60 The RSPO3-binding agents or antibodies described herein ceutical compositions find use in inhibiting tumor growth can also be entrapped in microcapsules. Such microcapsules and treating cancer in a subject (e.g., a human patient). are prepared, for example, by coacervation techniques or by In certain embodiments, formulations are prepared for interfacial polymerization, for example, hydroxymethylcel storage and use by combining a purified antibody or agent of lulose or gelatin-microcapsules and poly-(methylmethacry the present invention with a pharmaceutically acceptable 65 late) microcapsules, respectively, in colloidal drug delivery vehicle (e.g., a carrier or excipient). Suitable pharmaceuti systems (for example, liposomes, albumin microspheres, cally acceptable vehicles include, but are not limited to, microemulsions, nanoparticles and nanocapsules) or in mac US 9,598,497 B2 89 90 roemulsions as described in Remington. The Science and complexes such as cisplatin, mono(platinum), bis(platinum) Practice of Pharmacy, 22 Edition, 2012, Pharmaceutical and tri-nuclear platinum complexes and carboplatin), anthra Press, London. cyclines, antibiotics, antifolates, antimetabolites, chemo In certain embodiments, pharmaceutical formulations therapy sensitizers, duocarmycins, etoposides, fluorinated include a RSPO3-binding agent (e.g., an antibody) of the pyrimidines, ionophores, lexitropsins, nitrosoureas, plati present invention complexed with liposomes. Methods to nols, purine antimetabolites, puromycins, radiation sensitiz produce liposomes are known to those of skill in the art. For ers, steroids, taxanes, topoisomerase inhibitors, Vinca alka example, Some liposomes can be generated by reverse phase loids, or the like. In certain embodiments, the second evaporation with a lipid composition comprising phospha therapeutic agent is an alkylating agent, an antimetabolite, tidylcholine, cholesterol, and PEG-derivatized phosphatidy 10 an antimitotic, a topoisomerase inhibitor, or an angiogenesis lethanolamine (PEG-PE). Liposomes can be extruded inhibitor. In some embodiments, the second therapeutic through filters of defined pore size to yield liposomes with agent is a platinum complex such as carboplatin or cisplatin. the desired diameter. In some embodiments, the additional therapeutic agent is a In certain embodiments, Sustained-release preparations platinum complex in combination with a taxane. can be produced. Suitable examples of Sustained-release 15 Therapeutic agents that may be administered in combi preparations include semi-permeable matrices of Solid nation with the RSPO3-binding agents include chemothera hydrophobic polymers containing a RSPO3-binding agent peutic agents. Thus, in some embodiments, the method or (e.g., an antibody), where the matrices are in the form of treatment involves the administration of a RSPO3-binding shaped articles (e.g., films or microcapsules). Examples of agent or antibody of the present invention in combination Sustained-release matrices include polyesters, hydrogels with a chemotherapeutic agent or cocktail of multiple dif Such as poly(2-hydroxyethyl-methacrylate) or poly(vinyl ferent chemotherapeutic agents. Treatment with a RSPO3 alcohol), polylactides, copolymers of L-glutamic acid and 7 binding agent (e.g., an antibody) can occur prior to, concur ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, rently with, or Subsequent to administration of degradable lactic acid-glycolic acid copolymers such as the chemotherapies. Combined administration can include co LUPRON DEPOTTM (injectable microspheres composed of 25 administration, either in a single pharmaceutical formulation lactic acid-glycolic acid copolymer and leuprolide acetate), or using separate formulations, or consecutive administra Sucrose acetate isobutyrate, and poly-D-(-)-3-hydroxybu tion in either order but generally within a time period such tyric acid. that all active agents can exert their biological activities In certain embodiments, in addition to administering a simultaneously. Preparation and dosing schedules for Such RSPO3-binding agent (e.g., an antibody), the method or 30 chemotherapeutic agents can be used according to manu treatment further comprises administering at least one addi facturers instructions or as determined empirically by the tional therapeutic agent. An additional therapeutic agent can skilled practitioner. Preparation and dosing schedules for be administered prior to, concurrently with, and/or Subse such chemotherapy are also described in The Chemotherapy quently to, administration of the RSPO3-binding agent. Source Book, 4" Edition, 2008, M. C. Perry, Editor, Lippin Pharmaceutical compositions comprising a RSPO3-binding 35 cott, Williams & Wilkins, Philadelphia, Pa. agent and the additional therapeutic agent(s) are also pro Chemotherapeutic agents useful in the instant invention vided. In some embodiments, the at least one additional include, but are not limited to, alkylating agents such as therapeutic agent comprises 1, 2, 3, or more additional thiotepa and cyclosphosphamide (CYTOXAN); alkyl sul therapeutic agents. fonates such as buSulfan, improSulfan and piposulfan, aziri Combination therapy with two or more therapeutic agents 40 dines Such as benzodopa, carboquone, meturedopa, and often uses agents that work by different mechanisms of uredopa; ethylenimines and methylamelamines including action, although this is not required. Combination therapy altretamine, triethylenemelamine, trietylenephosphoramide, using agents with different mechanisms of action may result triethylenethiophosphaoramide and trimethylolomelamime; in additive or synergetic effects. Combination therapy may nitrogen mustards such as chlorambucil, chlornaphazine, allow for a lower dose of each agent than is used in 45 cholophosphamide, estramustine, ifosfamide, mechlore monotherapy, thereby reducing toxic side effects and/or thamine, mechlorethamine oxide hydrochloride, melphalan, increasing the therapeutic index of the agent(s). Combina novembichin, phenesterine, prednimustine, trofosfamide, tion therapy may decrease the likelihood that resistant uracil mustard; nitroSureas such as carmustine, chlorozoto cancer cells will develop. In some embodiments, combina cin, fotemustine, lomustine, nimustine, ranimustine; antibi tion therapy comprises a therapeutic agent that affects (e.g., 50 otics such as aclacinomysins, actinomycin, authramycin, inhibits or kills) non-tumorigenic cells and a therapeutic aZaserine, bleomycins, cactinomycin, calicheamicin, carabi agent that affects (e.g., inhibits or kills) tumorigenic CSCs. cin, caminomycin, carzinophilin, chromomycins, dactino In some embodiments, the combination of a RSPO3 mycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleu binding agent and at least one additional therapeutic agent cine, doxorubicin, epirubicin, esorubicin, idarubicin, results in additive or synergistic results. In some embodi 55 marcellomycin, mitomycins, mycophenolic acid, nogalamy ments, the combination therapy results in an increase in the cin, olivomycins, peplomycin, potfiromycin, puromycin, therapeutic index of the RSPO3-binding agent. In some quelamycin, rodorubicin, streptonigrin, Streptozocin, tuber embodiments, the combination therapy results in an increase cidin, ubenimex, Zinostatin, Zorubicin; anti-metabolites Such in the therapeutic index of the additional agent(s). In some as methotrexate and 5-fluorouracil (5-FU); folic acid ana embodiments, the combination therapy results in a decrease 60 logues such as denopterin, methotrexate, pteropterin, trime in the toxicity and/or side effects of the RSPO3-binding trexate; purine analogs such as fludarabine, 6-mercaptopu agent. In some embodiments, the combination therapy rine, thiamiprine, thioguanine; pyrimidine analogs such as results in a decrease in the toxicity and/or side effects of the ancitabine, azacitidine, 6-aZauridine, carmofur, cytosine ara additional agent(s). binoside, dideoxyuridine, doxifluridine, enocitabine, floxu Useful classes of therapeutic agents include, for example, 65 ridine, 5-FU; androgens such as calusterone, dromo antitubulin agents, auristatins, DNA minor groove binders, stanolone propionate, epitiostanol, mepitioStane, DNA replication inhibitors, alkylating agents (e.g., platinum testolactone; anti-adrenals such as aminoglutethimide, mito US 9,598,497 B2 91 92 tane, triloStane; folic acid replenishers such as folinic acid; the additional therapeutic agent is gemcitabine. Thus, in aceglatone; aldophosphamide glycoside; aminolevulinic Some embodiments, a method comprises administering a acid; amsacrine; bestrabucil; bisantrene, edatraxate; defo RSPO3-binding agent in combination with an anti-metabo famine; demecolcine; diaziquone; elformithine; elliptinium lite. In some embodiments, a method comprises administer acetate; etoglucid: gallium nitrate; hydroxyurea; lentinan; ing anti-RSPO3 antibody 131R002, 131R003, a variant of lonidamine; mitoguaZone, mitoxantrone; mopidamol; nitra 131R003, a humanized version of 131R003, h131R006A, crine; pentostatin; phenamet, pirarubicin; podophyllinic h131R006B, h131R005/131R007, h131R008, h131R010, or acid; 2-ethylhydrazide; procarbazine; PSK, razoxane: sizo h131R011 in combination with gemcitabine. In some furan; Spirogermanium; tenuaZonic acid; triaziquone; 2.2, embodiments, a method comprises administering anti 2"-trichlorotriethylamine; urethan; vindesine; dacarbazine: 10 RSPO3 antibody 131R002, 131R003, a variant of 131R003, mannomustine, mitobronitol; mitolactol; pipobroman; gacy a humanized version of 131R003, h131R006A, h131R006B, tosine; arabinoside (Ara-C); taxoids, e.g. paclitaxel h131R005/131R007, h131R008, h131R010, or hl31RO11 in (TAXOL) and docetaxel (TAXOTERE); chlorambucil; gem combination with pemetrexed. citabine; 6-thioguanine; mercaptopurine; platinum analogs In certain embodiments, the chemotherapeutic agent is an Such as cisplatin and carboplatin: vinblastine; platinum; 15 antimitotic agent, including, but not limited to, agents that etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone: bind tubulin. In some embodiments, the agent is a taxane. In Vincristine; Vinorelbine; navelbine; novantrone; teniposide; certain embodiments, the agent is paclitaxel or docetaxel, or daunomycin; aminopterin; ibandronate; CPT11: topoi a pharmaceutically acceptable salt, acid, or derivative of somerase inhibitor RFS 2000; difluoromethylornithine paclitaxel or docetaxel. In certain embodiments, the agent is (DMFO); retinoic acid; esperamicins; capecitabine (XE paclitaxel (TAXOL), docetaxel (TAXOTERE), albumin LODA); and pharmaceutically acceptable salts, acids or bound paclitaxel (nab-paclitaxel; ABRAXANE), DHA-pa derivatives of any of the above. Chemotherapeutic agents clitaxel, or PG-paclitaxel. In certain alternative embodi also include anti-hormonal agents that act to regulate or ments, the antimitotic agent comprises a Vinca alkaloid, Such inhibit hormone action on tumors such as anti-estrogens as Vincristine, binblastine, Vinorelbine, or vindesine, or including for example tamoxifen, raloxifene, aromatase 25 pharmaceutically acceptable salts, acids, or derivatives inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, thereof. In some embodiments, the antimitotic agent is an keoxifene, LY 1 17018, onapristone, and toremifene inhibitor of kinesin Eg5 or an inhibitor of a mitotic kinase (FARESTON); and anti-androgens such as flutamide, nilu such as Aurora A or Plk1. In certain embodiments, where the tamide, bicalutamide, leuprolide, and goserelin; and phar chemotherapeutic agent administered in combination with a maceutically acceptable salts, acids or derivatives of any of 30 RSPO-binding agent is an anti-mitotic agent, the cancer or the above. In certain embodiments, the additional therapeu tumor being treated is breast cancer or a breast tumor. In tic agent is cisplatin. In certain embodiments, the additional some embodiments, a method comprises administering anti therapeutic agent is carboplatin. In certain embodiments, the RSPO3 antibody 131R002, 131R003, a variant of 131R003, additional therapeutic agent is paclitaxel (taxol). In some a humanized version of 131R003, h131R006A, h131R006B, embodiments, a method comprises administering anti 35 h131R005/131R007, h131R008, h131R010, or hl31RO11 in RSPO3 antibody 131R002, 131R003, a variant of 131R003, combination with paclitaxel. In some embodiments, a 131R006A, 131R006B, 131R005/131R007, or 131R008 in method comprises administering anti-RSPO3 antibody combination with cisplatin. 131R002, 131R003, a variant of 131R003, a humanized In certain embodiments, the chemotherapeutic agent is a version of 131 R003, h131R006A, h131R006B, h131R005/ topoisomerase inhibitor. Topoisomerase inhibitors are che 40 131R007, h131R008, h131R010, or hl31R011 in combina motherapy agents that interfere with the action of a topoi tion with nab-paclitaxel (ABRAXANE). In some embodi Somerase enzyme (e.g., topoisomerase I or II). Topoi ments, a method comprises administering anti-RSPO3 somerase inhibitors include, but are not limited to, antibody 131R002, 131R003, a variant of 131R003, a doxorubicin HCl, daunorubicin citrate, mitoxantrone HCl, humanized version of 131R003, h131R006A, h131R006B, actinomycin D, etoposide, topotecan HCl, teniposide (VM 45 h131R005/131R007, h131R008, h131R010, or hl31RO11 in 26), and irinotecan, as well as pharmaceutically acceptable combination with gemcitabine and nab-paclitaxel (ABRAX salts, acids, or derivatives of any of these. In some embodi ANE). ments, the additional therapeutic agent is irinotecan. Thus, in In some embodiments, an additional therapeutic agent Some embodiments, a method comprises administering a comprises an agent Such as a small molecule. For example, RSPO3-binding agent in combination with a topoisomerase 50 treatment can involve the combined administration of a inhibitor. In some embodiments, a method comprises admin RSPO3-binding agent (e.g. an antibody) of the present istering anti-RSPO3 antibody 131R002, 131R003, a variant invention with a small molecule that acts as an inhibitor of 131R003, a humanized version of 131R003, h131R006A, against additional tumor-associated antigens including, but h131R006B, h131R005/131R007, h131R008, h131R010, or not limited to, EGFR, ErbB2, HER2, and/or VEGF. In h131R011 in combination with irinotecan. 55 certain embodiments, the additional therapeutic agent is a In certain embodiments, the chemotherapeutic agent is an Small molecule that inhibits a cancer stem cell pathway. In anti-metabolite. An anti-metabolite is a chemical with a Some embodiments, the additional therapeutic agent is an structure that is similar to a metabolite required for normal inhibitor of the Notch pathway. In some embodiments, the biochemical reactions, yet different enough to interfere with additional therapeutic agent is an inhibitor of the Wnt one or more normal functions of cells, such as cell division. 60 pathway. In some embodiments, the additional therapeutic Anti-metabolites include, but are not limited to, gemcit agent is an inhibitor of the BMP pathway. In some embodi abine, fluorouracil, capecitabine, methotrexate sodium, rali ments, the additional therapeutic agent is a molecule that trexed, pemetrexed, tegafur, cytosine arabinoside, thiogua inhibits f-catenin signaling. nine, 5-azacytidine, 6-mercaptopurine, azathioprine, In some embodiments, an additional therapeutic agent 6-thioguanine, pentostatin, fludarabine phosphate, and 65 comprises a biological molecule. Such as an antibody. For cladribine, as well as pharmaceutically acceptable salts, example, treatment can involve the combined administration acids, or derivatives of any of these. In certain embodiments, of a RSPO3-binding agent (e.g. an antibody) of the present US 9,598,497 B2 93 94 invention with other antibodies against additional tumor RSPO3 antibodies 131R002, 131R003, a variant of associated antigens including, but not limited to, antibodies 131R003, a humanized version of 131R003, h131R006A, that bind EGFR, ErbB2, HER2, and/or VEGF. In some h131R006B, h131R005/131R007, h131R008, h131R010, or embodiments, the additional therapeutic agent is an antibody h131R011 in combination with a FZD-Fc soluble receptor. that binds a second RSPO, e.g., RSPO1, RSPO2, and/or 5 In some embodiments, a method comprises administering RSPO4. In some embodiments, the additional therapeutic anti-RSPO3 antibodies 131R002, 131R003, a variant of agent is an anti-RSPO2 antibody. In some embodiments, the 131R003, a humanized version of 131R003, h131R006A, additional therapeutic agent is an anti-RSPO1 antibody. In h131R006B, h131R005/131R007, h131R008, h131R010, or certain embodiments, the additional therapeutic agent is an h131R011 in combination with a FZD8-Fc soluble receptor. antibody specific for an anti-cancer stem cell marker. In 10 In some embodiments, the methods described herein Some embodiments, the additional therapeutic agent is an comprise administering a therapeutically effective amount antibody that binds a component of the Notch pathway. In of a RSPO-binding agent in combination with more than one Some embodiments, the additional therapeutic agent is an additional therapeutic agent. Thus, in Some embodiments, a antibody that binds a component of the Wnt pathway. In method comprises administering a RSPO-binding agent in certain embodiments, the additional therapeutic agent is an 15 combination with a chemotherapeutic agent and a Wnt antibody that inhibits a cancer stem cell pathway. In some pathway inhibitor. In some embodiments, a method com embodiments, the additional therapeutic agent is an inhibitor prises administering a RSPO3-binding agent in combination of the Notch pathway. In some embodiments, the additional with a chemotherapeutic agent and a Wnt pathway inhibitor. therapeutic agent is an inhibitor of the Wnt pathway. In some In some embodiments, a method comprises administering a embodiments, the additional therapeutic agent is an inhibitor RSPO3-binding agent in combination with a chemothera of the BMP pathway. In some embodiments, the additional peutic agent and anti-FZD antibody 18R5. In some embodi therapeutic agent is an antibody that inhibits B-catenin ments, a method comprises administering a RSPO3-binding signaling. In certain embodiments, the additional therapeutic agent in combination with a chemotherapeutic agent and a agent is an antibody that is an angiogenesis inhibitor (e.g., FZD8-Fc soluble receptor. In some embodiments, a method an anti-VEGF or VEGF receptor antibody). In certain 25 comprises administering a RSPO3-binding agent in combi embodiments, the additional therapeutic agent is bevaci nation with gemcitabine and a Wnt pathway inhibitor. In Zumab (AVASTIN), trastuzumab (HERCEPTIN), panitu Some embodiments, a method comprises administering anti mumab (VECTIBIX), or cetuximab (ERBITUX). RSPO3 antibodies 131R002, 131R003, a variant of In some embodiments, the methods described herein 131R003, a humanized version of 131R003, h131R006A, comprise administering a therapeutically effective amount 30 h131R006B, h131R005/131R007, h131R008, h131R010, or of a RSPO3-binding agent in combination with Wnt path h131R011 in combination with gemcitabine and anti-FZD way inhibitors. In some embodiments, the Wnt pathway antibody 18R5. In some embodiments, a method comprises inhibitors are frizzled (FZD) protein binding agents, “FZD administering anti-RSPO3 antibodies 131R002, 131R003, a binding agents'. Non-limiting examples of FZD-binding variant of 131R003, a humanized version of 131R003, agents can be found in U.S. Pat. No. 7,982,013, which is 35 h131R006A, h131R006B, h131R005/131R007, h131R008, incorporated by reference herein in its entirety. FZD-binding h131R010, or h131R011 in combination with gemcitabine agents may include, but are not limited to, anti-FZD anti and FZD8-Fc soluble receptor. bodies. In some embodiments, a method comprises admin Furthermore, treatment with a RSPO3-binding agent istering a RSPO-binding agent in combination with an described herein can include combination treatment with anti-FZD antibody. In some embodiments, a method com 40 other biologic molecules, such as one or more cytokines prises administering a RSPO-binding agent in combination (e.g., lymphokines, interleukins, tumor necrosis factors, with the anti-FZD antibody 18R5. In some embodiments, and/or growth factors) or can be accompanied by Surgical the Wnt pathway inhibitors are Wnt protein binding agents, removal of tumors, cancer cells or any other therapy deemed “Wnt-binding agents'. Nonlimiting examples of Wnt-bind necessary by a treating physician. ing agents can be found in U.S. Pat. Nos. 7,723,477 and 45 In certain embodiments, the treatment involves the 7,947,277; and International Publications WO 2011/088127 administration of a RSPO3-binding agent (e.g. an antibody) and WO 2011/088123, which are incorporated by reference of the present invention in combination with radiation herein in their entirety. Wnt-binding agents may include, but therapy. Treatment with a RSPO3-binding agent can occur are not limited to, anti-Wnt antibodies and FZD-Fc soluble prior to, concurrently with, or Subsequent to administration receptors. In some embodiments, a method comprises 50 of radiation therapy. Dosing schedules for Such radiation administering a RSPO3-binding agent in combination with therapy can be determined by the skilled medical practitio a FZD-Fc soluble receptor. In some embodiments, a method . comprises administering a RSPO3-binding agent in combi Combined administration can include co-administration, nation with a FZD8-Fc soluble receptor. In some embodi either in a single pharmaceutical formulation or using sepa ments, a method comprises administering a RSPO3-binding 55 rate formulations, or consecutive administration in either agent in combination with an anti-FZD antibody. In some order but generally within a time period such that all active embodiments, a method comprises administering anti agents can exert their biological activities simultaneously. RSPO3 antibodies 131R002, 131R003, a variant of It will be appreciated that the combination of a RSPO3 131R003, a humanized version of 131R003, h131R006A, binding agent and at least one additional therapeutic agent h131R006B, h131R005/131R007, h131R008, h131R010, or 60 may be administered in any order or concurrently. In some h131R011 in combination with an anti-FZD antibody. In embodiments, the RSPO3-binding agent will be adminis Some embodiments, a method comprises administering anti tered to patients that have previously undergone treatment RSPO3 antibodies 131R002, 131R003, a variant of with a second therapeutic agent. In certain other embodi 131R003, a humanized version of 131R003, h131R006A, ments, the RSPO3-binding agent and a second therapeutic h131R006B, h131R005/131R007, h131R008, h131R010, or 65 agent will be administered Substantially simultaneously or h131R011 in combination with anti-FZD antibody 18R5. In concurrently. For example, a subject may be given a RSPO3 Some embodiments, a method comprises administering anti binding agent (e.g., an antibody) while undergoing a course US 9,598,497 B2 95 96 of treatment with a second therapeutic agent (e.g., chemo at a therapeutically effective dose. In some cases, drug therapy). In certain embodiments, a RSPO3-binding agent therapy must be discontinued, and other agents may be tried. will be administered within 1 year of the treatment with a However, many agents in the same therapeutic class often second therapeutic agent. In certain alternative embodi display similar side effects and/or toxicities, meaning that ments, a RSPO3-binding agent will be administered within the patient either has to stop therapy, or if possible, suffer 10, 8, 6, 4, or 2 months of any treatment with a second from the unpleasant side effects associated with the thera therapeutic agent. In certain other embodiments, a RSPO3 peutic agent. binding agent will be administered within 4, 3, 2, or 1 weeks Thus, the present invention provides methods of treating of any treatment with a second therapeutic agent. In some cancer in a Subject comprising using an intermittent dosing embodiments, a RSPO3-binding agent will be administered 10 strategy for administering one or more agents, which may within 5, 4, 3, 2, or 1 days of any treatment with a second therapeutic agent. It will further be appreciated that the two reduce side effects and/or toxicities associated with admin (or more) agents or treatments may be administered to the istration of a RSPO3-binding agent, chemotherapeutic Subject within a matter of hours or minutes (i.e., Substan agent, etc. In some embodiments, a method for treating tially simultaneously). 15 cancer in a human Subject comprises administering to the For the treatment of a disease, the appropriate dosage of subject a therapeutically effective dose of a RSPO3-binding an RSPO3-binding agent (e.g., an antibody) of the present agent in combination with a therapeutically effective dose of invention depends on the type of disease to be treated, the a chemotherapeutic agent, wherein one or both of the agents severity and course of the disease, the responsiveness of the are administered according to an intermittent dosing strat disease, whether the RSPO3-binding agent or antibody is egy. In some embodiments, the intermittent dosing strategy administered for therapeutic or preventative purposes, pre comprises administering an initial dose of a RSPO3-binding vious therapy, the patient's clinical history, and so on, all at agent to the Subject, and administering Subsequent doses of the discretion of the treating physician. The RSPO3-binding the RSPO3-binding agent about once every 2 weeks. In agent or antibody can be administered one time or over a Some embodiments, the intermittent dosing strategy com series of treatments lasting from several days to several 25 prises administering an initial dose of a RSPO3-binding months, or until a cure is effected or a diminution of the agent to the Subject, and administering Subsequent doses of disease state is achieved (e.g., reduction in tumor size). the RSPO3-binding agent about once every 3 weeks. In Optimal dosing schedules can be calculated from measure Some embodiments, the intermittent dosing strategy com ments of drug accumulation in the body of the patient and prises administering an initial dose of a RSPO3-binding will vary depending on the relative potency of an individual 30 antibody or agent. The administering physician can easily agent to the Subject, and administering Subsequent doses of determine optimum dosages, dosing methodologies, and the RSPO3-binding agent about once every 4 weeks. In repetition rates. In certain embodiments, dosage is from 0.01 some embodiments, the RSPO3-binding agent is adminis ug to 100 mg/kg of body weight, from 0.1 g to 100 mg/kg tered using an intermittent dosing strategy and the chemo of body weight, from 1 Jug to 100 mg/kg of body weight, 35 therapeutic agent is administered weekly. from 1 mg to 100 mg/kg of body weight, 1 mg to 80 mg/kg of body weight from 10 mg to 100 mg/kg of body weight, V. KITS COMPRISING RSPO-BINDING from 10 mg to 75 mg/kg of body weight, or from 10 mg to AGENTS 50 mg/kg of body weight. In certain embodiments, the dosage of the antibody or other RSPO3-binding agent is 40 The present invention provides kits that comprise the from about 0.1 mg to about 20 mg/kg of body weight. In RSPO3-binding agents (e.g., antibodies) described herein certain embodiments, dosage can be given once or more and that can be used to perform the methods described daily, weekly, monthly, or yearly. In certain embodiments, herein. In certain embodiments, a kit comprises at least one the antibody or other RSPO3-binding agent is given once purified antibody against at least one human RSPO protein every week, once every two weeks or once every three 45 in one or more containers. In some embodiments, the kits weeks. contain all of the components necessary and/or Sufficient to In some embodiments, a RSPO3-binding agent (e.g., an perform a detection assay, including all controls, directions antibody) may be administered at an initial higher “loading for performing assays, and any necessary Software for dose, followed by one or more lower doses. In some analysis and presentation of results. One skilled in the art embodiments, the frequency of administration may also 50 will readily recognize that the disclosed RSPO3-binding change. In some embodiments, a dosing regimen may com agents of the present invention can be readily incorporated prise administering an initial dose, followed by additional into one of the established kit formats which are well known doses (or “maintenance' doses) once a week, once every two in the art. weeks, once every three weeks, or once every month. For Further provided are kits comprising a RSPO3-binding example, a dosing regimen may comprise administering an 55 initial loading dose, followed by a weekly maintenance dose agent (e.g., an anti-RSPO3 antibody), as well as at least one of for example, one-half of the initial dose. Or a dosing additional therapeutic agent. In certain embodiments, the regimen may comprise administering an initial loading dose, second (or more) therapeutic agent is a chemotherapeutic followed by maintenance doses of, for example one-half of agent. In certain embodiments, the second (or more) thera the initial dose every other week. Or a dosing regimen may 60 peutic agent is a Wnt pathway inhibitor. In certain embodi comprise administering three initial doses for 3 weeks, ments, the second (or more) therapeutic agent is an angio followed by maintenance doses of for example, the same genesis inhibitor. amount every other week. Embodiments of the present disclosure can be further As is known to those of skill in the art, administration of defined by reference to the following non-limiting examples, any therapeutic agent may lead to side effects and/or tox 65 which describe in detail preparation of certain antibodies of icities. In some cases, the side effects and/or toxicities are so the present disclosure and methods for using antibodies of severe as to preclude administration of the particular agent the present disclosure. It will be apparent to those skilled in US 9,598,497 B2 97 98 the art that many modifications, both to materials and hybridized to Affymetrix HG-U133 plus 2.0 microarrays methods, may be practiced without departing from the scope (Affymetrix, Santa Clara, Calif.) as outlined in the manu of the present disclosure. facturer's technical manuals. After hybridization, the microarrays were washed, scanned, and analyzed. Scanned EXAMPLES array background adjustment and signal intensity normal ization were performed using the GCRMA algorithm (Bio Example 1 conductor, www.bioconductor.org). Expression of RSPO and LGR in Human Tumors Particular human RSPOs and human LGRs were evalu 10 ated RSPO1 (241450 at), RSPO2 (1554012 at), RSPO3 mRNA from normal tissue, benign tumor and malignant (228.186 s at), RSPO4 (237423 at), LGR4 (218326 s at), tumor samples of a large number of human patients was LGR5 (210393 at) and LGR6 (227819 at). Microarray analyzed by microarray analysis (Genelogic BioExpress analysis showed that, while LGR4 and LGR6 were broadly Datasuite). This data revealed elevated expression levels of expressed in almost all tumors, many tumors were found to RSPO1 in malignant tissue relative to normal tissue in 15 greatly over-express only particular RSPO family members several tumor types including kidney, endometrial, and and LGR5 (Table 2), although these expression levels were ovarian. RSPO1 was noted to be frequently over-expressed not compared to expression levels in normal tissue. Gener in ovarian cancer (FIG. 1A and FIG. 1B). In addition, this ally there is only a single RSPO family member that is data suggested elevated expression levels of RSPO3 in highly expressed in a given tumor, Suggesting that there may malignant tissue relative to normal tissue in several tumor be functional redundancy within the RSPO family. TABLE 2

Tumor RSPO1 RSPO2 RSPO3 RSPO4 LGR4 LGRS LGR6

Breast tumor

B34 4.79 4.93 3O3.31 4.41 B39 20.59 588.88 22.60 4.40 B60 4.6O 4.92 10.89 64.79 BO2 4.6O 4.92 692.34 4.41 2678.95 4.28 SO.88 BO3 5.56 4.89 1870.42 4.41 686.47 30.78 73.49 BO6 4.6O 4.91 4.51 120.72 274.54 4.26 20.77 B59 4.6O 4.91 4.53 1158.11 200.48 4.26 6467.15 Colon tumors

C11 4.63 4.98 4.56 443 3852.26 6.22 11.31 C17 4.64 S.OO 4.57 4.44 2822.46 62.34 4394 C18 4.63 4.95 13.83 4.42 2454.15 4.29 723.15 C27 6.66 980.49 4.75 4.40 SO83.84 4.30 20.82 Lung tumors

LUO2 4.62. 15190.40 4.SS 4.43 13.95 4.29 14.56 LU11 4.6O 4.92 453 4.41 999.55 4.27 146.67 LU2S 4.64 S.S6 11123.06 4.44 1208.92 4.29 41089 LU33 4.64 S.O1 12.O2 62.98 329.62 4.30 20.96 LU45 4.64 4.99 4.62 4.44 3877.47 4.29 4.86 Melanoma tumors

MO6 4.73 21.8O 4.65 4...SO 1077.93 4.34 3.90 Ovarian tumors

OV12 4.72 S.12 4.64 46040 S383.63 1152.73 115.04 OV19 96.O.19 4.74 69.77 20.90 494.67 5.72 4302.78 OV22 4.66 S.10 132.85 37.43 3743.91 482.33 812.OS OV27 4.SS 4.86 125.78 4.92 OV38 9.19 4.83 3439.88 16.35 1528.12 4.24 1949 Pancreatic tumors

PNO7 4.58 689.52 4.51 4.40 6777.41 4.28 746.38 PN18 4.72 2508.47 4.65 4.50 6750.73 51.15 564.94

55 types including ovarian, pancreas, and lung (FIG. 1E and Example 2 FIG. 1F). In addition, it was found that LGR5 and LGR6 were over-expressed in malignant breast tumors, colon Binding of RSPO Proteins to LGR5 tumors, lung tumors, and ovarian tumors relative to normal tissue, while LGR4 was over-expressed in lung tumors. 60 A cell Surface LGR5 protein was generated by ligating LGR5 and LGR6 over-expression appeared to be restricted amino acids 22-564 of human LGR5 to an N-terminal FLAG to triple-negative (ER"PR'HER2") breast tumors rela tag and to the transmembrane domain of CD4 and a C-ter tive to other breast tumor subtypes. minal GFP protein tag using standard recombinant DNA RNA was isolated from a series of human tumors grown techniques (FLAG-LGR5-CD4TM-GFP). RSPO-Fc con in murine Xenografts. The RNA samples were prepared and 65 structs were generated using standard recombinant DNA processed using established Affymetrix protocols for the techniques. Specifically, full-length human RSPO1, RSPO2, generation of labeled cRNA. The processed RNA was RSPO3 and RSPO4 were ligated in-frame to a human Fc US 9,598,497 B2 99 100 region and the recombinant RSPO-Fc proteins were The antibodies were serially diluted 2-fold from 100 nM to expressed in insect cells using baculovirus. The fusion 0.78 nM in HBS-P (0.01M HEPES pH 7.4, 0.15M NaCl, proteins were purified from the insect medium using protein 0.005% v/v Surfactant P20) and were injected over the chip A chromatography. surface. Kinetic data were collected over time and were fit HEK-293 cells were transiently transfected with the using the simultaneous global fit equation to yield affinity FLAG-LGR5-CD4TM-GFP construct. After 48 hours, constants (K. values) for each antibody. transfected cells were suspended in ice cold PBS containing Antibody 131R002 had an affinity constant (K) for 2% FBS and heparin and incubated on ice in the presence of human RSPO3 of 8.2 nM and antibody 131R003 had a K, 10 g/ml RSPO1-Fc, RSPO2-Fc, RSPO3-Fc, RSPO4-Fc, or for human RSPO3 of 7.3 nM. FZD8-Fc fusion proteins for 15 minutes. A second incuba 10 tion with 100 ul PE-conjugated anti-human Fc secondary Example 4 antibody was performed to detect cells bound by the Fc fusion proteins. Cells were incubated with an anti-FLAG In Vitro Testing for Inhibition of B-Catenin Activity antibody (Sigma-Aldrich, St. Louis, Mo.) as a positive by Anti-RSPO3 Antibodies control and with an anti-PE antibody as a negative control. 15 The cells were analyzed on a FACSCalibur instrument (BD HEK-293 cells were transfected with a 6XTCF-luciferase Biosciences, San Jose, Calif.) and the data was processed reporter vector (TOPflash, Millipore, Billerica, Mass.). After using FlowJo software. 24-48 hrs, the transfected HEK-293 cells were incubated As shown in FIG. 2, RSPO1, RSPO2, RSPO3 and RSPO4 with a combination of WNT3a (5 ng/ml) and human RSPO3 all bound to LGR5 expressed on the surface of the HEK-293 (10 ng/ml, R&D BioSystems, Minneapolis, Minn.) in the cells, while FZD8, the negative control, did not bind LGR5. presence of anti-RSPO3 antibodies 131R002 and 131R003. Binding affinities between RSPO proteins and LGR5 were Antibodies 131 R002 and 131 R003 were added to the cells in analyzed by surface plasmon resonance. A soluble LGR5-Fc 4-fold serial dilutions from 20 ug/ml to 0.02 ug/ml. As construct was generated using standard recombinant DNA controls, cells were incubated with a combination of WNT3a techniques. Specifically, amino acids 1-564 of human LGR5 25 and RSPO3, WNT3a only, RSPO3 only, or with no addition. were ligated in frame to human Fc and the recombinant The cells were incubated for 16 hours and luciferase activity LGR5-Fc fusion protein was expressed in insect cells using was measured using Steady-Glo R. Luciferase Assay System baculovirus. The LGR5-Fc fusion protein was purified from according to the manufacturers instructions (Promega, the insect medium using protein A chromatography. Cleav Madison, Wis.). age of the LGR5 signal sequence results in a mature LGR5 30 As shown in FIG. 3, anti-RSPO3 antibodies 131R002 and Fc fusion protein containing amino acids 22-564 of LGR5. 131R003 each reduced RSPO3-induced f-catenin signaling Recombinant RSPO1-Fc, RSPO2-Fc, RSPO3-Fc and in a dose-dependent manner. These results demonstrated that RSPO4-Fc fusion proteins were immobilized on CM5 chips antibodies 131R002 and 131R003 are specific inhibitors of using standard amine-based chemistry (NHS/EDC). Two RSPO3 and are capable of reducing and/or blocking fold dilutions of soluble LGR5-Fc were injected over the 35 RSPO3-induced f-catenin signaling. chip surface (100 nM to 0.78 nM). Kinetic data were collected over time using a Biacore 2000 system from Example 5 Biacore Life Sciences (GE Healthcare) and the data were fit using the simultaneous global fit equation to yield affinity Affinity Maturation and Humanization of RSPO3 constants (K, values) for each RSPO protein (Table 3). 40 Antibodies

TABLE 3 Anti-RSPO3 antibody 131R003 was affinity matured and several variants were identified. One 131R003 variant had LGR5 (nM) an altered heavy chain CDR1 (SEQID NO:34) as compared RSPO1 110 45 to parental 131R003 antibody. A second variant had an RSPO2 14 altered heavy chain CDR3 (SEQID NO:35) as compared to RSPO3 <1.0 parental 131R003. An additional variant was generated that RSPO4 73 comprised both the altered heavy chain CDR1 and CDR3 as compared to parental 131R003. Human RSPO1, RSPO2, RSPO3 and RSPO4 all bound to 50 HEK-293 cells were transfected with a 6XTCF-luciferase LGR5, demonstrating that RSPO proteins may be ligands reporter vector (TOPflash, Millipore, Billerica, Mass.). After for LGR proteins. 24-48 hrs, the transfected HEK-293 cells were incubated with a combination of WNT3a and human RSPO3 in the Example 3 presence of anti-RSPO3 antibodies 131R003, 131R003 55 CDR1 variant and 131 R003 CDR3 variant. 131 R003, Identification of Anti-RSPO3 Antibodies 131R003 CDR1 variant, and 131R003 CDR3 variant were added to the cells in 5-fold serial dilutions from 20 ug/ml to A mammalian cell antibody library was screened and two 0.006 ug/ml. As controls, cells were incubated with a anti-RSPO3 antibodies, 131R002 and 131R003, were iden combination of WNT3a and RSPO3, WNT3a only, RSPO3 tified. Sequence data Subsequently demonstrated that anti 60 only, a control antibody, or with no addition. The cells were bodies 131R002 and 131R003 have the same light chain incubated for 16 hours and luciferase activity was measured sequence but different heavy chain sequences. using Steady-Glo R. Luciferase Assay System according to The Ks of antibodies 131R002 and 131R003 were the manufacturer's instructions (Promega, Madison, Wis.). determined using a Biacore 2000 system from Biacore As shown in FIG. 4, anti-RSPO3 antibodies 131R003 LifeSciences (GE Healthcare). Recombinant human RSPO3 65 CDR1 variant and 131R003 CDR3 variant each reduced protein was biotinylated and captured on Streptavidin-coated RSPO3-induced B-catenin signaling in a dose-dependent chips (GE Healthcare) with coating densities of 400-700 ru. manner and at lower concentrations than parental 131R003. US 9,598,497 B2 101 102 These results demonstrated that the 131R003 variants combination of 89M5 and 131R002, a combination of retained the characteristics of parental 131R003, i.e., they 89M5, 131R002 and taxol, or a control antibody. Antibodies were specific inhibitors of RSPO3 and were capable of were dosed at 20 mg/kg once a week, and taxol was dosed reducing and/or blocking RSPO3-induced B-catenin signal at 15 mg/ml once a week through day 46 and Subsequently ing. In addition, these results demonstrated that the 131R003 dosed at 7.5 mg/kg. Administration of the antibodies and variants had better activity than parental 131R003. taxol was performed via injection into the intraperitoneal Humanized forms of 131R003 variants were generated cavity. Tumor growth was monitored and tumor Volumes using standard techniques. Humanized antibodies were measured with electronic calipers at the indicated time h131R005, h131R007, h131R008, h131R010, h131R011 points. Data are expressed as meantS.E.M. comprise an altered heavy chain CDR3 as compared to 10 As shown in FIG. 6, a combination of anti-RSPO1 parental 131R003 antibody. Humanized 131R006B com antibody 89M5 and anti-RSPO3 antibody 131R002 inhib prises an altered heavy chain CDR3 as compared to parental ited OMP-OV38 ovarian tumor growth as compared to 131R003 antibody. Antibodies h131R005/131R007, control antibody. Combinations of anti-RSPO1 antibody h131R010, and h131R011 comprise several amino acid 89M5 and taxolor anti-RSPO3 antibody 131R002 and taxol Substitutions in framework region 3 as compared to antibody 15 had no effect relative to taxol alone. However, surprisingly 131R006B. Antibodies hl31 R005/131R007, h131R006, and a combination of anti-RSPO189M5, anti-RSPO3 antibody h131R011 are IgG2 antibodies. Antibodies h131R008 and 131R002, and taxol showed activity that was greater than h131R010 are IgG1 antibodies. Antibodies h131R005/ taxol alone. 131R007, h131R010, and h131R011 comprise the same heavy chain variable region. Antibodies h131R010 and Example 7 h131R011 comprise the same light chain variable region, which is different than the light chain variable region of Inhibition of Lung Tumor Growth. In Vivo by h131 ROOS/13.1 ROOT. Anti-RSPO3 Antibodies A plasmid encoding the heavy chain of the 131R010 antibody was deposited with American Type Culture Col 25 In OMP-LU45 non-small cell lung tumors, it has been lection (ATCC), 10801 University Boulevard, Manassas, observed that CD201" cells are more tumorigenic than Va., USA, under the conditions of the Budapest Treaty on CD201 cells. Furthermore, RSPO3 was found to be highly Jun. 18, 2013, and assigned ATCC deposit designation expressed in the CD201" cell population. Dissociated and number PTA-120420. A plasmid encoding the light chain of sorted OMP-LU45 CD44"CD201" lung tumor cells (5x10' the 131R010 antibody was deposited with ATCC, 10801 30 cells) were injected into 6-8 week old NOD/SCID mice. University Boulevard, Manassas, Va., USA, under the con Tumors were allowed to grow for 38 days until they reached ditions of the Budapest Treaty on Jun. 18, 2013, and an average volume of 140 mm. The mice were randomized assigned ATCC deposit designation number PTA-120421. (n=10 per group) and treated with anti-RSPO3 antibody 131R002 or a control antibody. Antibodies were dosed at 25 Example 6 35 mg/kg once a week and administration of the antibodies was performed via injection into the intraperitoneal cavity. Inhibition of Ovarian Tumor Growth. In Vivo by Tumor growth was monitored and tumor Volumes were Anti-RSPO Antibodies measured with electronic calipers at the indicated time points. Data are expressed as meantS.E.M. Dissociated OMP-OV38 ovarian tumor cells (1x10 cells) 40 In a study with a second lung tumor, dissociated OMP were injected in to 6-8 week old NOD/SCID mice. Tumors LU25 lung tumor cells (5x10" cells) were injected into 6-8 were allowed to grow for 39 days until they reached an week old NOD/SCID mice. Tumors were allowed to grow average volume of 150 mm. The mice were randomized for 48 days until they reached an average volume of 110 (n=8 per group) and treated with a combination of anti mm. The mice were randomized (n=9 per group) and RSPO1 antibody 89M5 and anti-RSPO3 antibody 131R003, 45 treated with anti-RSPO3 antibody 131R002 or a control a combination of anti-RSPO1 antibody 89M5, anti-RSPO3 antibody. Antibodies were dosed at 25 mg/kg once a week antibody 131R003, and taxol, taxol as a single agent, or a and administration of the antibodies was performed via control antibody. Antibodies were dosed at 20 mg/kg once a injection into the intraperitoneal cavity. Tumor growth was week, and taxol was dosed at 15 mg/ml once a week. monitored and tumor Volumes were measured with elec Administration of the antibodies and taxol was performed 50 tronic calipers at the indicated time points. Data are via injection into the intraperitoneal cavity. Tumor growth expressed as meantS.E.M. was monitored and tumor Volumes were measured with As shown in FIGS. 7A and 7B, anti-RSPO antibody electronic calipers at the indicated time points. Data are 131R002 inhibited growth of both lung tumors OMP-LU45 expressed as meantS.E.M. and OMP-LU25 as compared to a control antibody. As shown in FIG. 5, a combination of anti-RSPO1 and 55 anti-RSPO3 antibodies inhibited OMP-OV38 ovarian tumor Example 8 growth. Surprisingly, a combination of anti-RSPO1 anti body 89M5, anti-RSPO3 antibody 131R002, and taxol Inhibition of B-Catenin Activity by Anti-RSPO3 inhibited tumor growth to a significantly greater level than Antibodies taxol alone or the antibody combination alone. 60 Dissociated OMP-OV38 ovarian tumor cells (1x10 cells) HEK-293 cells were transfected with a 6XTCF-luciferase were injected in to 6-8 week old NOD/SCID mice. Tumors reporter vector (TOPflash, Millipore, Billerica, Mass.). After were allowed to grow for 35 days until they reached an 24-48 hrs, the transfected HEK-293 cells were incubated average volume of 140 mm. The mice were randomized with a combination of WNT3a conditioned medium (5 (n=10 per group) and treated with anti-RSPO3 antibody 65 ng/ml) and human RSPO3 (10 ng/ml, R&D BioSystems) in 131R002, anti-RSPO1 antibody 89M5, taxol, a combination the presence of anti-RSPO3 antibodies 131R002, of 89M5 and taxol, a combination of 131R002 and taxol, a 131R006B, or 131 R007. Antibodies 131R002, 131 R006 or US 9,598,497 B2 103 131R007 were added to the cells in 5-fold Serial dilutions TABLE 4 from 20 lug/ml to 0.0064 ug/ml. As controls, cells were incubated with WNT3a conditioned medium alone, a com RSPO3 Antibody KD bination of WNT3a conditioned medium and human 131 R002 (IgG2) 1.3 nM RSPO3, or with no addition to cells. The cells were incu 5 131 R003 (IgG2) 1.9 nM 131 R003 CDR3 variant (IgG2) 1.7 nM bated for 16 hours and luciferase activity was measured h131 R007 (IgG2) 654 pM using Steady-Glo R. Luciferase Assay System according to h131 R008 (IgG1) 876 pM the manufacturer's instructions (Promega, Madison, Wis.). h131 RO10 (IgG1) ND h131 RO11 (IgG2) 686 pM As shown in FIG. 8, all three anti-RSPO3 antibodies 10 reduced WNT3a/RSPO3-induced f-catenin signaling in a dose-dependent manner. The humanized antibodies In additional experiments, antibody h131R008 was shown 131R006B and 131R007 appeared to have a greater ability to have a K, as low as 448 pM for human RSPO3, no to inhibit B-catenin activity than antibody 131R002. These detectable binding to human RSPO1 or RSPO2, and weak results demonstrated that humanized antibodies 131R006B 15 binding to human RSPO4. Antibody h131R008 was shown and 131R007 are stronger inhibitors of RSPO3 than to have a K, of 248 pM for murine RSPO3, no detectable 131R002 and are capable of reducing and/or blocking binding to murine RSPO1 or RSPO2 and weak binding to WNT3a/RSPO3-induced B-catenin signaling. murine RSPO4. Example 11 Example 9 Inhibition of Lung Tumor Growth. In Vivo by Inhibition of RSPO3 Binding to LGR5 Anti-RSPO3 Antibodies 25 The non-small cell lung cancer (NSCLC) cell line NCI HEK-293T cells were transfected with a cDNA expres H2030 was selected for testing based on a high level of sion vector that encoded the extracellular domain of human RSPO3 expression in microarray data. NCI-H2030 cells LGR5 (FLAG-LGR5-CD4TM-GFP). Transfected cells were (1x10) were injected into NOD-SCID mice. Tumors were incubated with recombinant RSPO3-biotin fusion protein in allowed to grow for approximately 60 days until they the presence of anti-RSPO3 antibodies 131R006B or 30 reached an average volume of 100 mm. Tumor-bearing 131R007. Cells were incubated without antibody as a con mice were randomized into 4 groups (n=7-9 per group). trol. Cells were washed in PBS and binding of RSPO3 to Tumor-bearing mice were treated with anti-RSPO3 antibody LGR5-expressing transfected cells was determined by addi 131R002, carboplatin, a combination of anti-RSPO3 anti tion of PE-conjugated streptavidin and analysis by flow body 131R002 and carboplatin, or a control antibody. Anti cytometry. 35 bodies were dosed at 25 mg/kg once a week. Carboplatin was dosed at 50 mg/kg once a week. Tumor growth was As shown in FIG. 9, anti-RSPO3 antibodies 131R006B monitored and tumor Volumes were measured with elec and 131R007 were highly effective in blocking binding of tronic calipers on the indicated days post-treatment. RSPO3 to LGR5-expressing cells. As shown in FIG. 10, treatment with anti-RSPO3 anti 40 body in combination with carboplatin inhibited NCI-H2030 Example 10 tumor growth better than carboplatin alone or the antibody alone. OMP-LU102 is a patient-derived non-small cell lung Binding Affinities of RSPO3 Antibodies cancer (NSCLC) xenograft that was selected for testing 45 based on a high level of RSPO3 expression in microarray The K of RSPO3 antibodies 131R002, 131R003, data. OMP-LU102 lung tumor cells (1x10) were injected 131R003 CDR3 variant, h131R007, h131R008, and into NOD-SCID mice. Tumors were allowed to grow for 22 h131R011 were determined using a Biacore 2000 system days until they reached an average volume of 90 mm. from Biacore LifeSciences (GE Healthcare). The method Tumor-bearing mice were randomized into 4 groups (n=10 used was different than described in Example 3. A goat 50 per group). Tumor-bearing mice were treated with anti anti-human IgG antibody was coupled to a carboxymethyl RSPO3 antibody 131R002, carboplatin, a combination of dextran (CM5) SPR chip using standard amine-based chem anti-RSPO3 antibody 131R002 and carboplatin, or a control istry (NHS/EDC) and blocked with ethanolamine. Antibod antibody. Antibodies were dosed at 25 mg/kg once a week. ies (purified antibody or culture supernatant) were diluted to Carboplatin was dosed at 50 mg/kg once a week. Tumor a concentration of 10 g/ml in HBS-P-BSA (0.01M HEPES 55 growth was monitored and tumor Volumes were measured pH7.4, 0.15M NaCl, 0.005% v/v Polysorbate 20, 100 ug/ml with electronic calipers on the indicated days post-treatment. BSA) and captured onto the chip via the anti-human IgG As shown in FIG. 11A, treatment with anti-RSPO3 anti antibody. Human RSPO3 (R&D Systems) was serially body inhibited OMP-LU102 lung tumor growth as a single diluted 2-fold from 300 nM to 37.5 nM in HBS-P-BSA and agent but had much greater effect in combination with injected sequentially over the captured anti-RSPO3 antibod 60 carboplatin. ies. RSPO3 association and dissociation was measured at RNA was prepared from tumors from each of the four each concentration. After each antigen injection 5 Jul of 100 experimental groups following the treatment. Gene expres mM HP0 was injected to remove the antigen-antibody sion was characterized by microarray analysis. Gene set complex and a Subsequent injection performed. Kinetic data enrichment analysis indicated that anti-RSPO3 antibody were collected over time and were fit using the simultaneous 65 treatment (either as a single agent or in combination with global fit equation to yield affinity constants (K. values) for carboplatin) inhibited the expression of various gene sets each antibody (Table 4). characteristic of normal stem cells or cancer stem cells as US 9,598,497 B2 105 106 shown in FIG. 11B. Treatment with carboplatin alone did not OMP-LU25 tumor cells (5x10) were injected into NOD have this effect on gene expression. SCID mice. Tumors were allowed to grow for 33 days until they reached an average volume of 120 mm. Tumor-bearing Example 12 mice were randomized into 4 groups (n=9 per group). Tumor-bearing mice were treated with either control anti Inhibition of Pancreatic Tumor Growth. In Vivo by body, anti-RSPO3 antibody 131R008, paclitaxel, or the Anti-RSPO3 Antibodies combination of anti-RSPO3 antibody 131R008 and pacli taxel. Antibodies were dosed weekly at 20 mg/kg. Paclitaxel OMP-PN35 is patient-derived pancreatic ductal adeno was dosed weekly at 15 mg/kg. Tumor growth was moni caricoma (PDAC) xenograft that was selected for testing 10 tored and tumor volumes were measured with electronic based on high level of RSPO3 expression in microarray data. calipers on the indicated days post-treatment. OMP-PN35 (1x10) tumor cells were injected into NOD SCID mice. Tumors were allowed to grow for 30 days until As shown in FIG. 14, anti-RSPO3 antibody 131R008 they reached an average volume of 90 mm. Tumor-bearing inhibited OMP-LU25 tumor growth as a single agent and in mice were randomized into 4 groups (n=10 per group). 15 combination with chemotherapy. Furthermore, the combi Tumor-bearing mice were treated with anti-RSPO3 antibody nation of anti-RSPO3 antibody 131R008 with paclitaxelled 131R002, gemcitabine plus nab-paclitaxel (ABRAXANE), to tumor regression. a combination of anti-RSPO3 antibody and gemcitabine and It is understood that the examples and embodiments nab-paclitaxel (ABRAXANE). Antibodies were dosed at 25 described herein are for illustrative purposes only and that mg/kg once a week. Gemcitabine was dosed at 20 mg/kg various modifications or changes in light thereof will be once a week and nab-paclitaxel (ABRAXANE) was dosed at Suggested to person skilled in the art and are to be included 30 mg/kg once a week. Tumor growth was monitored and within the spirit and purview of this application. tumor Volumes were measured with electronic calipers on the indicated days post-treatment. All publications, patents, patent applications, internet In FIG. 12A the results from all four treatment groups are 25 sites, and accession numbers/database sequences including shown and in FIG. 12B only the combination treatments are both polynucleotide and polypeptide sequences cited herein shown on an expanded scale. FIGS. 12A and 12B show that are hereby incorporated by reference herein in their entirety anti-RSPO3 antibody in combination with gemcitabine and for all purposes to the same extent as if each individual nab-paclitaxel (ABRAXANE) inhibited OMP-PN35 pan publication, patent, patent application, internet site, or acces creatic tumor growth better than gemcitabine and nab 30 sion number/database sequence were specifically and indi paclitaxel (ABRAXANE) alone. vidually indicated to be so incorporated by reference. Example 13 The sequences disclosed in the application are:

Inhibition of B-Catenin Activity by Anti-RSPO3 35 Human RSPO1 protein sequence with signal sequence Antibodies (SEQ ID NO: 1) MRLGLCVVALVLSWTHLTISSRGIKGKRORRISAEGSOACAKGCELCSEV HEK-293 cells were transfected with a 6XTCF-luciferase reporter vector (TOPflash, Millipore, Billerica, Mass.). After NGCLKCSPKLFILLERNDIROVGVCLPSCPPGYFDARNPDMNKCIKCKIE 24-48 hrs, the transfected HEK-293 cells were incubated 40 with a combination of WNT3a conditioned medium (5 HCEACFSHNFCTKCKEGLYLHKGRCYPACPEGSSAANGTMECSSPAOCEM ng/ml) and human RSPO3 (2 ng/ml, R&D BioSystems) in the presence of anti-RSPO3 antibodies h131R007 or SEWSPWGPCSKKOOLCGFRRGSEERTRRVLHAPVGDHAACSDTKETRRCT h131R010. Antibodies h131R007 or h131R010 were added WRRVPCPEGOKRRKGGOGRRENANRNLARKESKEAGAGSRRRKGQQQQQQ to the cells in 5-fold serial dilutions from 20 ug/ml to 0.0064 45 ug/ml. As controls, cells were incubated with WNT3a con QGTWGPLTSAGPA ditioned medium alone, a combination of WNT3a condi tioned medium and human RSPO3, or with no addition to Human RSPO2 protein sequence with signal sequence cells. The cells were incubated for 16 hours and luciferase (SEQ ID NO: 2) activity was measured using Steady-Glo R. Luciferase Assay 50 MOFRLFSFALIILNCMDYSHCOGNRWRRSKRASYVSNPICKGCLSCSKDN System according to the manufacturers instructions (Pro mega, Madison, Wis.). GCSRCOOKLFFFLRREGMROYGECLHSCPSGYYGHRAPDMNRCARCRIEN As shown in FIG. 13, antibody h131R010 reduced CDSCFSKDFCTKCKWGFYLHRGRCFDECPDGFAPLEETMECWEGCEWGHW WNT3a/RSPO3-induced f-catenin signaling in a dose-de pendent manner and to a similar extent as h131R007. Since 55 SEWGTCSRNNRTCGFKWGLETRTROIWKKPVKDTIPCPTIAESRRCKMTM h131R010 inhibited f-catenin signaling to the same extent as hl31R007, it is clear that activity of the anti-RSPO3 RHCPGGKRTPKAKEKRNKKKKRKLIERAOEOHSVFLATDRANO antibody was not affected by conversion to an IgG1 isotype. Human RSPO3 protein sequence with signal sequence Example 14 60 (SEQ ID NO : 3) MHLRLISWLFIILNFMEYIGSONASRGRRORRMHPNVSOGCOGGCATCSD Inhibition of Lung Tumor Growth. In Vivo by Anti-RSPO3 Antibodies YNGCLSCKPRLFFALERIGMKOIGVCLSSCPSGYYGTRYPDINKCTKCKA DCDTCFNKNFCTKCKSGFYLHLGKCLDNCPEGLEANNHTMECWSIWHCEW OMP-LU25 is a patient-derived non small cell lung 65 cancer (NSCLC) xenograft that was selected for testing SEWNPWSPCTKKGKTCGFKRGTETRVREIIOHPSAKGNLCPPTNETRKCT based on high level of RSPO3 expression in microarray data.

US 9,598,497 B2 135 136 - Continued

Lys Glu Gly Lell Tyr Luell His Gly Arg Cys Pro Ala 115 12 O 125

Pro Glu Gly Ser Ser Ala Ala Asn Gly Thir Met Glu Ser Ser 13 O 135 14 O

Pro Ala Glin Glu Met Ser Glu Trp Ser Pro Trp Gly Pro Ser 145 150 155 160

Glin Glin Lell Gly Phe Arg Arg Gly Ser Glu Glu Arg Thir 1.65

Arg Wall Luell His Ala Pro Wall Gly Asp His Ala Ala Cys Ser Asp 18O 185 19 O

Thir Glu Thir Arg Arg Thir Wall Arg Arg Wall Pro Pro Glu 195

Gly Glin Arg Arg Gly Gly Glin Gly Arg Arg Glu Asn Ala Asn 21 O 215

Arg Asn Luell Ala Arg Lys Glu Ser Glu Ala Gly Ala Gly Ser Arg 225 23 O 235 24 O

Arg Arg Gly Glin Glin Glin Glin Glin Glin Glin Gly Thir Wall Gly Pro 245 250 255

Lell Thir Ser Ala Gly Pro Ala 26 O

<210s, SEQ ID NO 2 &211s LENGTH: 243 212. TYPE : PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 2

Met Glin Phe Arg Lell Phe Ser Phe Ala Luell Ile Ile Lell Asn Cys Met 1. 5 1O 15

Asp Ser His Cys Glin Gly Asn Arg Trp Arg Arg Ser Lys Arg Ala 2O 25

Ser Wall Ser Asn Pro Ile Cys Gly Cys Lell Ser Ser 35 4 O 45

Asp Asn Gly Ser Arg Cys Glin Glin Luell Phe Phe Phe Luell Arg SO 55 6 O

Arg Glu Gly Met Arg Glin Gly Glu Luell His Ser Pro Ser 65 70 8O

Gly Tyr Gly His Arg Ala Pro Asp Met ASn Arg Ala Arg 85 90 95

Arg Ile Glu Asn Asp Ser Phe Ser Asp Phe Cys Thir 105 11 O

Wall Gly Phe Tyr Lell His Arg Gly Arg Phe Asp Glu 115 12 O 125

Pro Asp Gly Phe Ala Pro Lell Glu Glu Thir Met Glu Wall Glu Gly 13 O 135 14 O

Cys Glu Wall Gly His Trp Ser Glu Trp Gly Thir Ser Arg Asn Asn 145 150 155 160

Arg Thir Gly Phe Trp Gly Luell Glu Thir Thir Arg Glin Ile 1.65 17O 17s

Wall Pro Wall Lys Asp Thir Ile Pro Pro Thir Ile Ala Glu 18O 185 19 O

Ser Arg Arg Met Thir Met Arg His Pro Gly Gly Arg 195 2O5

Thir Pro Ala Glu Arg Asn Lys Arg Luell US 9,598,497 B2 137 138 - Continued

21 O 215 22O Ile Glu Arg Ala Glin Glu Gln His Ser Val Phe Lieu Ala Thr Asp Arg 225 23 O 235 24 O

Ala Asn. Glin

<210s, SEQ ID NO 3 &211s LENGTH: 272 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 3 Met His Lieu. Arg Lieu. Ile Ser Trp Leu Phe Ile Ile Lieu. Asn Phe Met 1. 5 1O 15 Glu Tyr Ile Gly Ser Glin Asn Ala Ser Arg Gly Arg Arg Glin Arg Arg 2O 25 3O Met His Pro Asn Val Ser Glin Gly Cys Glin Gly Gly Cys Ala Thr Cys 35 4 O 45 Ser Asp Tyr Asn Gly Cys Lieu. Ser Cys Llys Pro Arg Lieu. Phe Phe Ala SO 55 6 O Lieu. Glu Arg Ile Gly Met Lys Glin Ile Gly Val Cys Lieu. Ser Ser Cys 65 70 7s 8O Pro Ser Gly Tyr Tyr Gly Thr Arg Tyr Pro Asp Ile Asn Lys Cys Thr 85 90 95 Lys Cys Lys Ala Asp Cys Asp Thr Cys Phe Asn Lys Asn. Phe Cys Thr 1OO 105 11 O Lys Cys Llys Ser Gly Phe Tyr Lieu. His Lieu. Gly Lys Cys Lieu. Asp ASn 115 12 O 125 Cys Pro Glu Gly Lieu. Glu Ala Asn Asn His Thr Met Glu. Cys Val Ser 13 O 135 14 O Ile Val His Cys Glu Val Ser Glu Trp Asn Pro Trp Ser Pro Cys Thr 145 150 155 160 Llys Lys Gly Lys Thr Cys Gly Phe Lys Arg Gly Thr Glu Thir Arg Val 1.65 17O 17s Arg Glu Ile Ile Gln His Pro Ser Ala Lys Gly Asn Lieu. Cys Pro Pro 18O 185 19 O Thir Asn. Glu Thir Arg Lys Cys Thr Val Glin Arg Llys Llys Cys Glin Lys 195 2OO 2O5 Gly Glu Arg Gly Llys Lys Gly Arg Glu Arg Lys Arg Llys Llys Pro Asn 21 O 215 22O Lys Gly Glu Ser Lys Glu Ala Ile Pro Asp Ser Lys Ser Lieu. Glu Ser 225 23 O 235 24 O Ser Lys Glu Ile Pro Glu Glin Arg Glu Asn Lys Glin Glin Gln Lys Llys 245 250 255 Arg Llys Val Glin Asp Llys Glin Llys Ser Val Ser Val Ser Thr Val His 26 O 265 27 O

<210s, SEQ ID NO 4 &211s LENGTH: 272 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 4 Met His Lieu. Arg Lieu. Ile Ser Trp Leu Phe Ile Ile Lieu. Asn Phe Met 1. 5 1O 15

Glu Tyr Ile Gly Ser Glin Asn Ala Ser Arg Gly Arg Arg Glin Arg Arg 2O 25 3O US 9,598,497 B2 139 140 - Continued

Met His Pro Asn Wall Ser Glin Gly Glin Gly Gly Cys Ala Thir 35 4 O 45

Ser Asp Asn Gly Lell Ser Pro Arg Lell Phe Phe Ala SO 55 6 O

Lell Glu Arg Ile Gly Met Glin Ile Gly Wall Lell Ser Ser Cys 65 70

Pro Ser Gly Tyr Tyr Gly Thir Arg Pro Asp Ile Asn Cys Thir 85 90 95

Ala Asp Asp Thir Cys Phe ASn Asn Phe Thir 105 11 O

Lys Ser Gly Phe Luell His Luell Gly Cys Luell Asp Asn 115 12 O 125

Pro Glu Gly Lell Glu Ala Asn Asn His Thir Met Glu Wall Ser 13 O 135 14 O

Ile Wall His Glu Wall Ser Glu Trp Asn Pro Trp Ser Pro Thir 145 150 155 160

Gly Thir Gly Phe Arg Gly Thir Glu Thir Arg Wall 1.65 17s

Arg Glu Ile Ile Glin His Pro Ser Ala Gly Asn Lell Cys Pro Pro 18O 185 19 O

Thir Asn Glu Thir Arg Thir Wall Glin Arg Lys Glin 195

Gly Glu Arg Gly Gly Arg Glu Arg Arg Pro Asn 21 O 215

Lys Gly Glu Ser Glu Ala Ile Pro Asp Ser Ser Luell Glu Ser 225 23 O 235 24 O

Ser Glu Ile Pro Glu Glin Arg Glu Asn Lys Glin Glin Glin Lys 245 250 255

Arg Wall Glin Asp Glin Ser Wall Ser Wall Ser Thir Wall His 26 O 265 27 O

<210s, SEQ ID NO 5 &211s LENGTH: 251 212. TYPE : PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 5

Glin Asn Ala Ser Arg Gly Arg Arg Glin Arg Arg Met His Pro Asn Wall 1. 5 1O 15

Glin Gly Cys Glin Gly Gly Ala Thir Ser Asp Tyr Asn Gly 2O 25 3O

Luell Ser Cys Pro Arg Luell Phe Phe Ala Lell Glu Arg Ile Gly 35 4 O 45

Met Lys Glin Ile Gly Wall Cys Luell Ser Ser Pro Ser Gly SO 55 6 O

Gly Thir Arg Pro Asp Ile Asn Thir Ala Asp 65 70

Asp Thir Phe Asn Asn Phe Cys Thir Ser Gly 85 90 95

Phe Luell His Lell Gly Luell Asp ASn Pro Glu Gly Luell 1OO 105 11 O

Glu Ala Asn Asn His Thir Met Glu Wall Ser Ile Wall His Glu 115 12 O 125

Wall Ser Glu Trp Asn Pro Trp Ser Pro Thir Gly Thir US 9,598,497 B2 141 142 - Continued

13 O 135 14 O

Cys Gly Phe Arg Gly Thir Glu Thr Arg Val Arg Glu Ile Ile Glin 145 150 155 160

His Pro Ser Ala Lys Gly Asn Luell Cys Pro Pro Thir Asn Glu Thir Arg 1.65 17O 17s

Thir Wall Glin Arg Cys Glin Lys Gly Glu Arg Gly 18O 185 19 O

Gly Arg Glu Arg Arg Lys Llys Pro Asn Gly Glu Ser 195 2OO 2O5

Glu Ala Ile Pro Asp Ser Lys Ser Lieu. Glu Ser Ser Glu Ile Pro 21 O 215 22O

Glu Glin Arg Glu Asn Lys Glin Glin Gln Lys Llys Arg Wall Glin Asp 225 23 O 235 24 O

Glin Ser Wall Ser Wall Ser Thir Wal His 245 250

<210s, SEQ ID NO 6 &211s LENGTH: 52 212. TYPE : PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 6

Pro Asn. Wal Ser Gln Gly Cys Glin Gly Gly Cys Ala Thir Cys Ser Asp 1. 5 1O 15

Tyr Asn Gly Cys Lieu. Ser Cys Llys Pro Arg Lieu. Phe Phe Ala Lieu. Glu 25 3O

Arg Ile Gly Met Lys Glin Ile Gly Val Cys Lieu. Ser Ser Cys Pro Ser 35 4 O 45 Gly Tyr Tyr Gly SO

<210s, SEQ ID NO 7 &211s LENGTH: 44 212. TYPE : PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 7 Ile Asn Lys Cys Thr Lys Cys Lys Ala Asp Cys Asp Thr Cys Phe Asn 1. 5 15 Lys Asn. Phe Cys Thr Lys Cys Llys Ser Gly Phe Tyr Lieu. His Leu Gly 25 Lys Cys Lieu. Asp Asn. Cys Pro Glu Gly Lieu. Glu Ala 35 4 O

SEQ ID NO 8 LENGTH: 61 TYPE : PRT ORGANISM: Homo sapiens

< 4 OOs SEQUENCE: 8

His Cys Glu Wall Ser Glu Trp Asn Pro Trp Ser Pro Cys Thr Lys Lys 1. 1O 15

Gly Lys Thir Cys Gly Phe Arg Gly Thr Glu Thir Arg Val Arg Glu 25 3O

Ile Ile Glin His Pro Ser Ala Lys Gly Asn Lieu. Cys Pro Pro Thr Asn 35 4 O 45

Glu Thir Arg Lys Cys Thir Wall Glin Arg Llys Llys Cys Glin SO 55 6 O US 9,598,497 B2 143 144 - Continued

<210s, SEQ ID NO 9 &211s LENGTH: 11 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: 131ROO2/131R003 Heavy chain CDR1 <4 OOs, SEQUENCE: 9 Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr Ser 1. 5 1O

<210s, SEQ ID NO 10 &211s LENGTH: 8 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: 131ROO2/131R003 Heavy chain CDR2 <4 OOs, SEQUENCE: 10 Ile Tyr Pro Ser Asn Gly Asp Ser 1. 5

<210s, SEQ ID NO 11 &211s LENGTH: 10 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: 131ROO2/131R003 Heavy chain CDR3 <4 OOs, SEQUENCE: 11 Ala Thr Tyr Phe Ala Asn Tyr Phe Asp Tyr 1. 5 1O

<210s, SEQ ID NO 12 &211s LENGTH: 11 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: 131ROO2/131ROO3 Light chain CDR1 <4 OOs, SEQUENCE: 12 Glin Ser Val Asp Tyr Asp Gly Asp Ser Tyr Met 1. 5 1O

<210s, SEQ ID NO 13 &211s LENGTH: 3 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: 131ROO2/131ROO3 Light chain CDR2 <4 OOs, SEQUENCE: 13

Ala Ala Ser 1.

<210s, SEQ ID NO 14 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: 131ROO2/131ROO3 Light chain CDR3

<4 OOs, SEQUENCE: 14 Glin Glin Ser Asn. Glu Asp Pro Lieu. Thr 1. 5 US 9,598,497 B2 145 146 - Continued

SEO ID NO 15 LENGTH: 120 TYPE PRT ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: 131Roo2 Heavy chain variable region SEQUENCE: 15 Glin Val Glin Leu Gln Glu Ser Gly Pro Glu Luell Wall Pro Gly Ala 1. 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thir Phe Thir Asp 2O 25 3O

Ser Ile His Trp Val Lys Glin Asn His Gly Lys Ser Lell Asp Trp Ile 35 4 O 45

Gly Tyr Ile Tyr Pro Ser Asn Gly Asp Ser Gly Tyr Asn Glin Phe SO 55 6 O

Lys Asn Arg Ala Thr Lieu. Thr Val Asp Thr Ser Ser Ser Thir Ala Tyr 65 70

Lieu. Glu Val Arg Arg Lieu. Thir Phe Glu Asp Ser Ala Wall Tyr 85 90 95

Ala Thr Tyr Phe Ala Asn Tyr Phe Asp Tyr Trp Gly Glin Gly Thir Thir 1OO 105 11 O

Lieu. Thir Wal Ser Ser Ala Ser Thr 115 12 O

SEQ ID NO 16 LENGTH: 120 TYPE PRT ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: 131Roo3 Heavy chain variable region SEQUENCE: 16

Glin Val Glin Lieu Lys Glin Ser Gly Pro Glu Luell Wall Pro Gly Ala 1. 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thir Phe Thir Asp 2O 25 3O

Ser Ile His Trp Val Lys Glin Asn His Gly Lys Ser Lell Asp Trp Ile 35 4 O 45

Gly Tyr Ile Tyr Pro Ser Asn Gly Asp Ser Gly Tyr Asn Glin Phe SO 55 6 O

Lys Asn Arg Ala Thr Lieu. Thr Val Asp Thr Ser Tyr Ser Thir Ala Tyr 65 70

Lieu. Glu Val Arg Arg Lieu. Thir Phe Glu Asp Ser Ala Wall Tyr 85 90 95

Ala Thr Tyr Phe Ala Asn Tyr Phe Asp Tyr Trp Gly Glin Gly Thir Thir 1OO 105 11 O

Lieu. Thir Wal Ser Ser Ala Ser Thr 115 12 O

SEO ID NO 17 LENGTH: 112 TYPE PRT ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: 131ROO2/131ROO3 Light chain variable region

SEQUENCE: 17 Asp Ile Val Lieu. Thr Glin Ser Pro Ala Ser Lieu Ala Val Ser Lieu. Gly