WO2021206968A1 - High-potency agonists and antagonists of the wnt signaling pathway - Google Patents

Abstract

Engineered WNT signaling agonist or antagonist proteins are provided.

Description

HIGH-POTENCY AGONISTS AND ANTAGONISTS OF THE WNT SIGNALING PATHWAY

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit of and priority to U.S. Provisional Patent Application No. 63/007,057, filed April 8, 2020, the entire disclosure of which is hereby incorporated by reference herein in its entireties for all purposes.

BACKGROUND OF THE INVENTION

[0002] Wnt^-catenin signal transduction results in the cytoplasmic protein b-catenin entering the nucleus to modulate transcription. When the pathway is not activated, b-catenin is subject to a cycle of continual synthesis and destruction by the b-catenin destruction complex, comprised of the scaffold proteins Axin and APC and the kinases GSK3 and casein kinase 1 (CK1). Wnt signaling removes APC from the complex and relocalizes the other components to the plasma membrane via the adaptor Dsh, thus stabilizing b-catenin which enters the nucleus to mediate transcription.

[0003] R-spondins (RSPOs) are vertebrate-specific secreted proteins that amplify signaling through the WNT^-catenin pathway, a cell-cell communication system that regulates tissue patterning during embryonic development and regenerative responses in adults. Mutations that disrupt the RSPO signaling circuit cause structural birth defects, exemplified by limb truncations, lung hypoplasia, craniofacial malformations and cardiovascular defects seen in humans and mice carrying mutations in RSP02 and RSP03. In adults, RSPOs function as niche-derived signals required for the renewal of epithelial stem cells in multiple tissues, including the intestine, skin and bone. Elucidating the mechanisms that mediate the reception and transduction of RSPO signals will further our understanding of these fundamental developmental and homeostatic processes and is essential to harnessing the considerable therapeutic potential of this pathway to enhance regenerative responses.

[0004] Dickkopf proteins (DKKs) are secreted proteins that inhibit the WNT^-catenin pathway by binding to the WNT co-receptors LRP5 and LRP6. DKKs have been implicated in multiple diseases, including cancer, bone disease, heart disease, neurodegeneration, immune dysfunction and inflammation.

[0005] Development of R-spondin and DKK analogs and derivatives is of great interest for therapeutic and research purposes. SUMMARY OF THE INVENTION

[0006] Engineered WNT signaling agonists and antagonists are provided, including without limitation engineered Rspondin (RSPO) and Dickkopf (DKK) proteins; and methods for their use are provided. The engineered proteins comprise a protein sequence that modulates WNT activity, e.g. an RSPO protein sequence, a DKK sequence, etc.; operably linked to an antigen binding region (ABR) that selectively binds to heparan sulfate chains, e.g. the heparan sulfate chains present on heparan sulfate proteoglycan (HSPG), which ABR may be referred to as an anti-HSPG ABR. The anti-HSPG ABR enhances potency of the WNT modulating protein. In some embodiments the ABR binds to the heparan sulfate chains and not a protein component of the HSPG. In some embodiments the engineered WNT signaling agonist or antagonist enhances signaling potency of the modulating agent on cells that have low or no expression of one or more of leucine-rich repeat-containing G-protein coupled receptors (LGRs) 4, 5 and 6. Enhancement of activity may be assessed, relative to the corresponding native protein, e.g. native RSPO; DKK1c, etc. In some embodiments the engineered WNT signaling agonist or antagonist protein can enhance signaling potency of WNT proteins on cells that have low or no expression of each of LGR 4, 5 and 6, relative to the corresponding native RSPO protein. In some embodiments the engineered WNT signaling agonist or antagonist can enhance signaling potency of WNT proteins on cells that express LGR 4, 5, 6, relative to the corresponding native RSPO protein.

[0007] The anti-HSPG ABR can be fused or linked to the WNT signaling agonist or antagonist protein sequence. The anti-HSPG ABR and the WNT signaling agonist or antagonist sequence may be contiguously fused to each other, or separated by a linker, e.g. a polypeptide linker, or a non-peptidic linker, etc. The length of the linker, and therefore the spacing between the domains can be selected depending on the desired use of the protein. In some embodiments the linker is a rigid linker, in other embodiments the linker is a flexible linker. Where the linker is a peptide linker, it may be from about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acids in length, and may be of sufficient length and amino acid composition to enforce the distance between binding domains. In some embodiments the linker comprises or consists of one or more glycine and/or serine residues.

[0008] Compositions of interest are provided, including, without limitation, an effective dose of an engineered WNT signaling agonist or antagonist protein as described herein, in a pharmaceutically acceptable excipient. Compositions may comprise additional agents, e.g. adjuvants and the like. Engineered WNT signaling agonist or antagonist proteins may be produced synthetically; by various suitable recombinant methods, and the like, as known in the art. [0009] In some aspects of the invention, a method is provided for activating, increasing or enhancing Wnt signaling in a cell. In such methods, a cell expressing a receptor for Wnt, e.g. a frizzled receptor, is contacted with an effective amount of an engineered WNT signaling agonist or antagonist protein as described herein at a concentration that is effective to increase signaling, e.g. to increase signaling by 25%, 50%, 75%, 90%, 95%, or more, relative to the signaling in the absence of the engineered RSPO protein. In some embodiments the engineered RSPO protein is provided in combination with provision of a wnt signaling agonist, in other embodiments the engineered WNT signaling agonist or antagonist protein enhances or suppresses endogenous Wnt signaling. Such signaling activation may induce proliferation of the targeted cell, which cells include without limitation pluripotent stem cells, in vitro organoids, somatic stem cells and progenitors, etc. or may otherwise activate Wnt-signaling pathways in the targeted cell. In some methods, the receptor-expressing cell is contacted in vitro. In other embodiments, the receptor expressing cell is contacted in vivo. Cells of interest include a wide variety of Fzd-receptor expressing cells, as are known in the art, for example skin cells, intestinal cells, osteoblasts, stem cells, liver cells etc.

[0010] In some embodiments an engineered RSPO protein sequence comprises a native RSPO, full-length protein. In other embodiments, the RSPO protein sequence is truncated and lacks the native TSP/BR domain, or comprises targeted mutations to reduce binding of the native TSP/BR domain to HSPG. In some embodiments the RSPO protein sequence comprises the FU1 and FU2 domains of human RSP03, and lacks the native TSP/BR domain. In some such embodiments the engineered RSPO protein comprises the FU1 and FU2 domains of human RSP02 or human RSPO 3. In some such embodiments the RSPO sequence is SEQ ID NO:7, 9, 11 . In some embodiments the engineered protein is SEQ ID NO:13, 15, 17, 19, 20.

[0011] In other embodiments an engineered WNT signaling antagonist is provided, where an antagonist protein is conjugated to an ABR with an anti-heparan sulfate chain specificity. In some embodiments the antagonist is a DKK protein, including without limitation DKK1 , DKK1C, DKK2, DKK3, DKK4, Wnt inhibitory factor (Wif); etc. Conjugation of the anti-HSPG ABR to a WNT inhibitory protein enhances potency of signaling inhibition of WNT proteins relative to the native inhibitor. The polypeptide set forth in SEQ ID NO:26 is exemplary, where residues 39-381 are the mature DKK-linker-HS20 protein. In some aspects of the invention, a method is provided for treating or preventing a disease or disorder in a subject in need thereof, the method comprising providing to the subject an effective amount of an engineered WNT signaling agonist protein as described herein. Conditions for treatment include, without limitation, intestinal regeneration after chemotherapy or radiation, liver regeneration, skin regeneration, prevention of bone loss or promotion of bone growth, lung regeneration, hair growth, and the like. The engineered proteins are useful in repair of multiple organs after injury (e.g. chemotherapy, radiation, other toxins) or to enhance regeneration in age-related degeneration of tissues. In some embodiments the engineered WNT signaling agonist protein is provided in combination with provision of a wnt signaling agonist such as a WNT protein, etc., in other embodiments the engineered WNT signaling agonist protein enhances endogenous Wnt signaling. In particular embodiments, the subject has a disease or disorder associated with reduced wnt signaling.

[0012] In some aspects of the invention, a method is provided for enhancing wound healing and/or tissue generation in a subject in need thereof, the method comprising providing to the subject an effective amount of an engineered RSPO protein as described herein. In some embodiments the engineered RSPO protein is provided in combination with a wnt signaling agonist, in other embodiments the engineered RSPO protein enhances endogenous Wnt signaling.

[0013] In some aspects of the invention, a method is provided for treating or preventing a disease or disorder in a subject in need thereof, the method comprising providing to the subject an effective amount of an engineered WNT signaling antagonist (inhibitor) protein as described herein. Conditions for treatment include, without limitation, cancer, e.g. carcinomas such as colorectal carcinoma. Wnt activity is associated with tumorigenesis, cell invasion and metastasis in cancer. Wnt signaling-dependent cancers include those that harbor mutations in components of the pathway, and those that have a dysregulation of Wnt signaling due to epigenetically driven up- or downregulation in expression levels of the pathway components. For example, mutations in Wnt pathway suppressor APC are associated with familial adenomatous polyposis (FAP) and colorectal carcinomas.

[0014] Wnt Inhibition is also useful in treating cancer for chemoresistance and cancer stem cell (CSC) propagation. Wnt pathway is active in CSC, and upregulates transcription of genes necessary for proliferation. The role of active Wnt signaling in chemo- and radio-resistance is linked to the survival of CSCs: being relatively dormant, they can better withstand the therapy to repopulate the shrunken tumor, which results in tumor recurrence. There is also a separate mechanism of involvement of the Wnt pathway in cancer chemoresistance and radioresistance. Wnt pathway inhibition for cancer treatment is therefore beneficial at multiple levels: inhibition of tumor growth and survival with minimal effects on somatic cells, inhibition of CSC maintenance, and prevention of the development of tumor resistance to chemo- and radio-therapy.

[0015] In further embodiments, methods and compositions are provided for targeting proteins to desired receptors and cells, by conjugating the protein to an ABR with an anti-heparan sulfate chain specificity. Fusion of the FISPG ABR can serve as a general strategy to target proteins to desired receptors and cells with high affinity. For example, it is shown that conjugation of the anti- HSPG ABR to a WNT inhibitory protein (DKK1c or Dickkopf WNT signaling pathway inhibitor 1) can dramatically improve the ability of the WNT inhibitor to block the WNT signaling pathway.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The invention is best understood from the following detailed description when read in conjunction with the accompanying drawings. It is emphasized that, according to common practice, the various features of the drawings are not to-scale. On the contrary, the dimensions of the various features are arbitrarily expanded or reduced for clarity. Included in the drawings are the following figures.

[0017] Figure 1 . Mutations in the putative HSPG-binding surface of the RSP03 TSP/BR domain impair its ability to amplify WNT signaling. (A) Homology model of the isolated TSP/BR domain of human RSP03 (residues valine 146 - isoleucine 232; UniprotKB Q9BXY4). Left panel: cartoon representation of the protein backbone, with side-chains shown for lysine (K) and arginine (R) residues. N- and C-termini are circled and disulfide bonds are labelled with roman numerals. Two positively charged HS-binding grooves, revealed by the electrostatic potential map (middle panel), are labeled Site-1 and Site-2. K and R residues lining Site-1 and Site-2 are colored purple and orange, respectively, and residues mutated to glutamic acid (E) in the RSP03 TSP/BR (K/R E) mutant are labeled. Middle panel: the electrostatic potential map calculated with the Adaptive Poisson-Boltzmann Solver (Jurrus et al., 2018) is displayed from -10 kT/e to +10 kT/e (blue: positively charged; red: negatively charged). Two virtually docked 8-mer heparin chains (estimated free energies are -13.9 and -14.8 kcal/mol for Site-1 and Site-2, respectively) are depicted in stick representation (red: oxygen, blue: nitrogen, gold: Sulphur, green: carbon of heparin-1 , yellow: carbon of heparin-2). Right panel: electrostatic potential map of the RSP03 TSP/BR (K/R E) mutant. The orientation of the middle and right panels is 90° rotated clockwise around the y-axis relative to the left panel. (B) Schematic representation of WT and mutant RSP03 variants showing the domains and mutations present in these proteins. The N-terminal HA and the C-terminal Fc and 1 D4 tags present in these constructs are not shown. Amino acid numbers for human RSP03 (UniProtKB Q9BXY4) are indicated below. Green and red arrows show K E and R E mutations, respectively, introduced into the TSP/BR domain of the RSP03 TSP/BR (K/R E) mutant. Polypeptide lengths are drawn to scale. See Supplementary file 2 for the nucleotide sequences of these constructs. (C) Coomassie-stained polyacrylamide gels showing equal volumes of the three purified RSP03 proteins used in (D) and (E). Molecular weight standards in kilodaltons (kDa) are indicated to the right. (D) and (E) Dose-response curves for the indicated purified RSP03 variants in WT HAP1-7TGP (D) and LGR4/5/6^KO (E) cells, in the presence of 1 .43% WNT3A CM. Each circle represents the median WNT reporter fluorescence from 2,500 cells. Dose-response curves were fitted to the data using non-linear regression as described in Materials and methods. EC50 values for RSP03 (WT) in WT HAP1-7TGP and LGR4/5/6KO cells are 0.048 ± 0.005 nM and 0.092 ± 0.007 nM, respectively. EC50 in LGR4/5/6KO cells could not be calculated. EC50 for RSP03 ATSP/BR in WT HAP1-7TGP cells is 3.93 ± 1.0 nM (EC50 in LGR4/5/6KO cells could not be calculated).

[0018] Figure 2. The TSP/BR domain of RSP03 can be replaced by HS20, a scFv that binds FIS chains, without compromising activity. (A) Cartoons depicting synthetic proteins in which the FU1 and FU2 domains of RSP03 were fused to HS20, a scFv that binds to FIS chains. In two control proteins designed to abolish FIS binding, the CDR3 loop of HS20 was replaced with a glycine- serine (GS) or a poly-alanine (A) linker. In two other proteins, we introduced mutations that diminish binding of the FU1 domain to ZNRF3/RNF43 (R67A/Q72A) or of the FU2 domain to LGRs (F106E/F110E). (B) Coomassie-stained polyacrylamide gels showing equal volumes of the five purified RSP03 proteins used in (C-H). Molecular weight standards in kDa are indicated to the right of each gel. (C-H) Dose-response curves for the indicated purified RSP03 variants in WT HAP1-7TGP (C, E and G) and LGR4/5/6^KO (D, F and H) cells, in the presence of 1.43% WNT3A CM. Each circle represents the median WNT reporter fluorescence from 2,500 cells. Dose-response curves were fitted to the data using non-linear regression as described in Materials and methods. EC50 for RSP03 (WT) and RSP03 ATSP/BR HS20 are 0.046 ± 0.004 nM and 0.032 ± 0.002 nM in C and 0.11 ± 0.015 nM and 0.11 ± 0.013 nM in D. EC50 for RSP03 ATSP/BR HS20 is 0.035 ± 0.003 nM in E and 0.18 ± 0.03 nM in E. EC₅₀s for RSP03 ATSP/BR HS20, RSP03 ATSP/BR HS20 (R67A/Q72A) and RSP03 ATSP/BR HS20 (F106E/F110E) are 0.20 ± 0.017 nM, 2.0 ± 0.26 nM and 0.68 ± 0.014 nM respectively in G. EC50S for RSP03 ATSP/BR HS20 and RSP03 ATSP/BR HS20 (F106E/F110E) are 1.1 ± 0.12 nM and 0.49 ± 0.07 nM respectively in H.

[0019] Figure 3. Signaling by WT RSP03 and RSP03 ATSP/BR HS20 causes internalization and degradation of RNF43 in the presence and absence of LGRs. Cell-surface RNF43-Flag (captured by surface biotinylation; see Materials and methods), total RNF43-Flag and total actin were visualized by immunoblotting after treatment of WT FIAP1-7TGP (A) or LGR4/5/6^KO (B) cells with the indicated RSP03 variants.

[0020] Figure 4. RSP03 ATSP/BR HS20 engages the FIS chains of multiple HSPGs. (A) Binding of HS20 to GPC3 or GPC4. An ELISA plate was coated with Fc-tagged GPC3, GPC4, or mutant versions lacking FIS chains (GPC3AHS or GPC4AHS). HS20 scFv was added at increasing concentrations (x-axis), followed by a goat anti-human IgG horseradish peroxidase conjugate. Bound HS20 was quantified with 3,3',5,5'-tetramethylbenzidine detection reagent by measuring absorbance at 450 nm (y-axis). An irrelevant Fc fusion protein (CD276-hFc, labeled “Control”) was used as an antigen control. (B-C) Fold change in the EC₅o of WT RSP03 (B) or RSP03 ATSP/BR HS20 (C) in LGR4/5/6^KO cells and clonal derivatives in which different HSPGs were depleted through the indicated gene disruptions (see Results and Materials and methods for details). Since deletion of some GPCs in HAP1 cells leads to impaired WNT reception (Lebensohn et al. 2016, Lebensohn and Rohatgi, 2018), we first determined a concentration of WNT3A CM that yielded similar levels of WNT reporter activity in all the cell lines and then titrated RSP03 variants to determine their EC₅o’s. Error bars denote the standard error of the curve fit used to calculate the EC₅o- The statistical significance of differences between the measured EC50’s in LGR4/5/6KO cells and clonal derivatives thereof was determined by a two-tailed, unpaired t-test, and is indicated as ^**** (p<0.0001), ^*** (p<0.001), ^** (p<0.01), ^* (p<0.05) or ns (non-significant).

[0021 ] Figure 5. The interaction with HSPGs potentiates the ability of RSP03 to support the growth of small intestinal organoids. (A) Bright-field microscopy images of B6 mouse small intestinal organoids grown in EN medium supplemented with various purified RSP03 proteins at the indicated concentrations. Green arrows indicate viable, crypt-containing organoids and red asterisk mark dead organoids. The scale bar in the bottom right image represents 10 pm. (B) The viability of organoids grown at various concentrations of the indicated RSP03 variants was quantified from images of the type shown in (A). Each circle represents the quantification of an independent experiment. The inset (right) shows a magnified view of organoid viability at a ligand concentration of 0.1 nM. (C) Confocal microscopy images of mouse small intestinal organoids grown in medium supplemented with 1 nM of the indicated purified RSP03 proteins. DAPI (blue) stains nuclei, phalloidin (red) stains actin filaments at the apical surface of cells, lysozyme (green, top row) stains Paneth cells and Ki67 (green, bottom row) stains proliferating cells. For both images in each row, the scale bars in the right image represent 30 pm.

[0022] Figure 6. A haploid genetic screen for regulators of RSP03 signaling in cells lacking LGRs. (A-B) Circle plots depicting genes enriched for inactivating GT insertions in the screen for regulators of RSP01 -potentiated WNT signaling in HAP1-7TGP cells (A) and in the screen for regulators of RSP03-potentiated WNT signaling in LGR4/5/6^KO cells (B). The y-axis indicates the significance of inactivating GT insertion enrichment in the sorted vs. the control cells (expressed in units of -logioFDR-corrected p-value) and the x-axis indicates genes (in random order) for which inactivating GT insertions were mapped in the sorted cells (see Materials and methods). Genes with FDR-corrected p-value<0.01 are labeled and colored in light blue if they reached this level of significance in both screens, or in orange if they reached this level of significance in only one of the two screens. The diameter of each circle is proportional to the number of unique inactivating GT insertions mapped in the sorted cells, which is also indicated next to the gene name for the most significant hits with FDR-corrected p-valuesdO^-4. See Supplementary file 5 for ranked lists of hits from both screens. (C) Heat map comparing the two screens. Genes enriched for inactivating GT insertions (FDR-corrected p-value<0.01) in at least one of the two screens were clustered based on their IGTIOB score in each screen (see Materials and methods).

[0023] Figure 7. Fusion of the anti-HSPG ABR improves the potency of a soluble WNT inhibitor. (A) Diagrams of fusion proteins tested in the WNT signaling assay shown in (B). The anti-HSPG ABR (HS20) is fused to the DKK1 c (Dickkopf WNT Signaling Pathway Inhibitor 1 ) protein, a known soluble inhibitor of WNT signaling. The inhibitory activity of the DKK1c-HS20 fusion is compared to two controls, HS20 alone and Dkklc alone. (B) WNT signaling assay shows that fusion of the anti-HSPG ABR increases the inhibitory potency of DKK1c by 20-fold.

DETAILED DESCRIPTION OF THE INVENTION

[0024] Before the present methods and compositions are described, it is to be understood that this invention is not limited to particular method or composition described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0025] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0026] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some potential and preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. It is understood that the present disclosure supersedes any disclosure of an incorporated publication to the extent there is a contradiction.

[0027] It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and reference to "the peptide" includes reference to one or more peptides and equivalents thereof, e.g. polypeptides, known to those skilled in the art, and so forth.

[0028] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0029] By “comprising” it is meant that the recited elements are required in the composition/method/kit, but other elements may be included to form the composition/method/kit etc. within the scope of the claim. For example, a composition comprising an engineered RSPO protein is a composition that may comprise other elements in addition to engineered RSPO protein(s), e.g. functional moieties such as polypeptides, small molecules, or nucleic acids bound, e.g. covalently bound, to the engineered RSPO protein; agents that promote the stability of the engineered RSPO protein composition, agents that promote the solubility of the engineered RSPO protein composition, adjuvants, etc. as will be readily understood in the art, with the exception of elements that are encompassed by any negative provisos.

[0030] By “consisting essentially of”, it is meant a limitation of the scope of composition or method described to the specified materials or steps that do not materially affect the basic and novel characteristic(s) of the subject invention. For example, an engineered RSPO protein “consisting essentially of” a disclosed sequence has the amino acid sequence of the disclosed sequence plus or minus about 5 amino acid residues at the boundaries of the sequence based upon the sequence from which it was derived, e.g. about 5 residues, 4 residues, 3 residues, 2 residues or about 1 residue less than the recited bounding amino acid residue, or about 1 residue, 2 residues, 3 residues, 4 residues, or 5 residues more than the recited bounding amino acid residue.

[0031] By “consisting of”, it is meant the exclusion from the composition, method, or kit of any element, step, or ingredient not specified in the claim. For example, an engineered RSPO protein “consisting of” a disclosed sequence consists only of the disclosed amino acid sequence. [0032] The terms "treatment", "treating" and the like are used herein to generally mean obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease. "Treatment" as used herein covers any treatment of a disease in a mammal and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; or (c) relieving the disease, i.e., causing regression of the disease. The therapeutic agent may be administered before, during or after the onset of disease or injury. The treatment of ongoing disease, where the treatment stabilizes or reduces the undesirable clinical symptoms of the patient, is of particular interest. Such treatment is desirably performed prior to complete loss of function in the affected tissues. The subject therapy may be administered during the symptomatic stage of the disease, and in some cases after the symptomatic stage of the disease.

[0033] The terms "individual," "subject," "host," and "patient," are used interchangeably herein and refer to any mammalian subject for whom diagnosis, treatment, or therapy is desired, particularly humans.

[0034] The expression "body fluid" as used herein in intended to include all of those accessible body fluids usable as clinical specimens which may contain a compound being tested for in sufficient concentration in said fluid to be within the limits of detection of the test device or assay being used. Body fluids will thus include whole blood, serum, plasma, urine, cerebrospinal fluid, synovial fluid, and interstitial and other extracellular fluids.

[0035] General methods in molecular and cellular biochemistry can be found in such standard textbooks as Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., CSH Laboratory Press 2001 ); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); Nonviral Vectors for Gene Therapy (Wagner et al. eds., Academic Press 1999); Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998), the disclosures of which are incorporated herein by reference. Reagents, cloning vectors, and kits for genetic manipulation referred to in this disclosure are available from commercial vendors such as BioRad, Thermo Fisher, Sigma-Aldrich, and ClonTech.

[0036] The term "isolated" means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, a naturally-occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated. Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment.

[0037] "Pharmaceutically acceptable excipient” means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for veterinary use as well as for human pharmaceutical use. Such excipients can be solid, liquid, semisolid, or, in the case of an aerosol composition, gaseous.

[0038] "Pharmaceutically acceptable salts and esters" means salts and esters that are pharmaceutically acceptable and have the desired pharmacological properties. Such salts include salts that can be formed where acidic protons present in the compounds are capable of reacting with inorganic or organic bases. Suitable inorganic salts include those formed with the alkali metals, e.g. sodium and potassium, magnesium, calcium, and aluminum. Suitable organic salts include those formed with organic bases such as the amine bases, e.g., ethanolamine, diethanolamine, triethanolamine, tromethamine, N methylglucamine, and the like. Such salts also include acid addition salts formed with inorganic acids {e.g., hydrochloric and hydrobromic acids) and organic acids {e.g., acetic acid, citric acid, maleic acid, and the alkane- and arene-sulfonic acids such as methanesulfonic acid and benzenesulfonic acid). Pharmaceutically acceptable esters include esters formed from carboxy, sulfonyloxy, and phosphonoxy groups present in the compounds, e.g., Ci-₆ alkyl esters. When there are two acidic groups present, a pharmaceutically acceptable salt or ester can be a mono-acid-mono-salt or ester or a di-salt or ester; and similarly, where there are more than two acidic groups present, some or all of such groups can be salified or esterified. Compounds named in this invention can be present in unsalified or unesterified form, or in salified and/or esterified form, and the naming of such compounds is intended to include both the original (unsalified and unesterified) compound and its pharmaceutically acceptable salts and esters. Also, certain compounds named in this invention may be present in more than one stereoisomeric form, and the naming of such compounds is intended to include all single stereoisomers and all mixtures (whether racemic or otherwise) of such stereoisomers.

[0039] The terms "pharmaceutically acceptable", "physiologically tolerable" and grammatical variations thereof, as they refer to compositions, carriers, diluents and reagents, are used interchangeably and represent that the materials are capable of administration to or upon a human without the production of undesirable physiological effects to a degree that would prohibit administration of the composition. [0040] A pharmaceutical formulation is a composition comprising different chemical substances including but not limited to active drugs, excipients, etc. which are combined and formulated to produce a final medicinal product for the treatment of humans or other organisms.

[0041] A sterile formulation is a formulation substantially free of living germs or microorganisms.

[0042] A therapeutically effective amount is that mass of an active drug in a formulation, and the frequency of administration of a formulation, that results in the prevention of the development of symptoms, prevention of development of markers or signs of a disease, prevention of the development of tissue or organ damage, prevention of the progression of a disease, reduction in the severity of a disease, or treatment of disease symptoms as defined above.

[0043] It is within the level of skill of a clinician to determine the preferred route of administration and the corresponding dosage form and amount, as well as the dosing regimen, i.e., the frequency of dosing. Such information may be obtained in a straightforward manner in accordance with the teachings and guidelines contained in the instant specification taken in light of the knowledge and skill of the artisan. The results that are obtained can also be correlated with data from corresponding evaluations of an approved product in the same assays.

[0044] Dose range for an agent is the range of the mass of active drug in, and frequency of administration of, a formulation which results in the prevention of the development of symptoms, prevention of the development of a disease, prevention of development of abnormal markers or signs of a disease, prevention of the development of tissue or organ damage, prevention of the progression of a disease, reduction in the severity of a disease, or treatment of disease symptoms as defined above.

[0045] Regimen means dose, frequency of administration, for example twice-per day, daily, weekly, bi-weekly etc., and duration of treatment, for example one day, several days, one week, several weeks, one month, several months, one year, several years, etc.

[0046] Unit doses (also called dosage forms) are essentially pharmaceutical products in the form in which they are marketed for use, typically involving a mixture of active drug components and nondrug components (excipients), along with other non-reusable material that may not be considered either ingredient or packaging (such as a capsule shell, for example). Depending on the context, multi(ple) unit dose can refer to distinct drug products packaged together, or to a single drug product containing multiple drugs and/or doses. The term dosage form can also sometimes refer only to the chemical formulation of a drug product's constituent drug substance(s) and any blends involved.

[0047] A dose pack is a premeasured amount of drug to be dispensed to a patient in a set or variable dose and in a package including but not limited to a blister pack or other series of container for the purpose of facilitating a dose regimen. A dose pack can be used to facilitate delivery of an initial and/or loading dose to an individual, followed by a maintenance dose.

[0048] An excipient is generally a pharmacologically inactive substance formulated with the active pharmaceutical ingredient ("API") of a medication. Excipients are commonly used to bulk up formulations that contain potent active ingredients (thus often referred to as "bulking agents," "fillers," or "diluents"), to allow convenient and accurate dispensation of a drug substance when producing a dosage form. They also can serve various therapeutic-enhancing purposes, such as facilitating drug absorption or solubility, or other pharmacokinetic considerations.

[0049] “Homology” between two sequences is determined by sequence identity. If two sequences, which are to be compared with each other, differ in length, sequence identity preferably relates to the percentage of the nucleotide residues of the shorter sequence which are identical with the nucleotide residues of the longer sequence. Sequence identity can be determined conventionally with the use of computer programs such as the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive Madison, Wis. 53711). Bestfit utilizes the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2 (1981), 482- 489, in order to find the segment having the highest sequence identity between two sequences. When using Bestfit or another sequence alignment program to determine whether a particular sequence has for instance 95% identity with a reference sequence of the present invention, the parameters are preferably so adjusted that the percentage of identity is calculated over the entire length of the reference sequence and that homology gaps of up to 5% of the total number of the nucleotides in the reference sequence are permitted. When using Bestfit, the so-called optional parameters are preferably left at their preset (“default”) values. The deviations appearing in the comparison between a given sequence and the above-described sequences of the invention may be caused for instance by addition, deletion, substitution, insertion or recombination. Such a sequence comparison can preferably also be carried out with the program “fasta20u66” (version 2.0u66, September 1998 by William R. Pearson and the University of Virginia; see also W. R. Pearson (1990), Methods in Enzymology 183, 63-98, appended examples and http://workbench.sdsc.edu/). For this purpose, the “default” parameter settings may be used.

[0050] "Variant" refers to polypeptides having amino acid sequences that differ to some extent from a native sequence polypeptide. Ordinarily, amino acid sequence variants will possess at least about 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% sequence identity to a corresponding native sequence. The amino acid sequence variants may possess substitutions, deletions, and/or insertions at certain positions within the reference native amino acid sequence.

[0051] The term “vector,” as used herein, is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid”, which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply, “recombinant vectors”). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, “plasmid” and “vector” may be used interchangeably as the plasmid is the most commonly used form of vector.

[0052] The term “host cell” (or “recombinant host cell”), as used herein, is intended to refer to a cell that has been genetically altered, or is capable of being genetically altered by introduction of an exogenous polynucleotide, such as a recombinant plasmid or vector. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein.

[0053] “Binding affinity” generally refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody or other binding molecule) and its binding partner (e.g., an antigen or receptor). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies bind antigen (or receptor) weakly and tend to dissociate readily, whereas high-affinity antibodies bind antigen (or receptor) more tightly and remain bound longer.

[0054] Antigen binding region (ABR). As used herein, the term ABR refers to a combination of variable heavy (VH and variable light (VL) polypeptides to associate to form a variable region domain. An ABR is the minimum antibody fragment that contains a complete antigen-recognition and binding site. This region consists of heavy- and one light-chain variable domain in tight, non- covalent association, as a single polypeptide or as a dimer. It is in this configuration that the three CDRS of each variable domain interact to define an antigen-binding site on the surface of the domain. Collectively, the six CDRs confer antigen-binding specificity to the antibody.

[0055] The term "variable" refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions both in the light-chain and the heavy-chain variable domains. The more highly conserved portions of variable domains are called the framework (FR). The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a b-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the b-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody- dependent cellular toxicity.

[0056] An antibody or ABR “which binds” an antigen of interest, is one that binds the antigen with sufficient affinity such that the antibody or binding molecule is useful as a diagnostic and/or therapeutic agent in targeting the antigen, and does not significantly cross-react with other proteins. In such embodiments, the extent of binding of the antibody or other binding molecule to a non-targeted antigen will usually be no more than 10% as determined by fluorescence activated cell sorting (FACS) analysis or radioimmunoprecipitation (RIA).

[0057] Antibodies, also referred to as immunoglobulins, conventionally comprise at least one heavy chain and one light, where the amino terminal domain of the heavy and light chains is variable in sequence, hence is commonly referred to as a variable region domain, or a variable heavy (VH) or variable light (VH) domain. The two domains conventionally associate to form a specific binding region, although as well be discussed here, a variety of non-natural configurations of antibodies are known and used in the art. [0058] A “functional” or “biologically active” antibody or antigen-binding molecule is one capable of exerting one or more of its natural activities in structural, regulatory, biochemical or biophysical events. For example, a functional antibody or other binding molecule may have the ability to specifically bind an antigen and the binding may in turn elicit or alter a cellular or molecular event such as signaling transduction or enzymatic activity. A functional antibody or other binding molecule may also block ligand activation of a receptor or act as an agonist or antagonist. The capability of an antibody or other binding molecule to exert one or more of its natural activities depends on several factors, including proper folding and assembly of the polypeptide chains.

[0059] The term “antibody” herein is used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, monomers, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), heavy chain only antibodies, three chain antibodies, single chain Fv, nanobodies, etc., and also include antibody fragments, so long as they exhibit the desired biological activity (Miller et al (2003) Jour of Immunology 170:4854-4861). Antibodies may be murine, human, humanized, chimeric, or derived from other species.

[0060] The term antibody may reference a full-length heavy chain, a full-length light chain, an intact immunoglobulin molecule; or an immunologically active portion of any of these polypeptides, i.e., a an scFv polypeptide that comprises an ABR that immunospecifically binds an antigen of interest. The immunoglobulin disclosed herein can be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., lgG1 , lgG2, lgG3, lgG4, lgA1 and lgA2) or subclass of immunoglobulin molecule, including engineered subclasses with altered Fc portions that provide for reduced or enhanced effector cell activity. The immunoglobulins can be derived from any species. In one aspect, the immunoglobulin is of largely human origin.

[0061] The term “variable” refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. Flowever, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called hypervariable regions both in the light chain and the heavy chain variable domains. The more highly conserved portions of variable domains are called the framework regions (FRs). The variable domains of native heavy and light chains each comprise four FRs, largely adopting a beta-sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the beta-sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al (1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md.)· The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody dependent cellular cytotoxicity (ADCC).

[0062] The term “hypervariable region” when used herein refers to the amino acid residues of an antibody which are responsible for antigen-binding. The hypervariable region may comprise amino acid residues from a “complementarity determining region” or “CDR”, and/or those residues from a “hypervariable loop”. “Framework Region” or “FR” residues are those variable domain residues other than the hypervariable region residues as herein defined.

[0063] Variable regions of interest include 3 CDR sequences, which may be obtained from available antibodies with the desired specificity, or may be obtained from antibodies developed for this purpose. One of skill in the art will understand that a number of definitions of the CDRs are commonly in use, including the Kabat definition (see “Zhao et al. A germline knowledge based computational approach for determining antibody complementarity determining regions.” Mol Immunol. 2010;47:694-700), which is based on sequence variability and is the most commonly used. The Chothia definition is based on the location of the structural loop regions (Chothia et al. “Conformations of immunoglobulin hypervariable regions.” Nature. 1989;342:877-883). Alternative CDR definitions of interest include, without limitation, those disclosed by Honegger, “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool.” J Mol Biol. 2001 ;309:657-670; Ofran et al. “Automated identification of complementarity determining regions (CDRs) reveals peculiar characteristics of CDRs and B cell epitopes.” J Immunol. 2008;181 :6230-6235; Almagro “Identification of differences in the specificity-determining residues of antibodies that recognize antigens of different size: implications for the rational design of antibody repertoires.” J Mol Recognit. 2004;17:132-143; and Padlanet al. “Identification of specificity-determining residues in antibodies.” Faseb J. 1995;9:133-139., each of which is herein specifically incorporated by reference.

[0064] The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations, which include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.

[0065] The ABRs utilized herein specifically include “chimeric” antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; and Morrison et al (1984) Proc. Natl. Acad. Sci. USA, 81 :6851- 6855). Chimeric antibodies of interest herein include “primatized” antibodies comprising variable domain antigen-binding sequences derived from a non-human primate (e.g., Old World Monkey, Ape etc) and human constant region sequences.

[0066] An “intact antibody chain” as used herein is one comprising a full-length variable region and a full length constant region. An intact “conventional” antibody comprises an intact light chain and an intact heavy chain, as well as a light chain constant domain (CL) and heavy chain constant domains, CH1 , hinge, CH2 and CH3 for secreted IgG. Other isotypes, such as IgM or IgA may have different CH domains. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof. The intact antibody may have one or more “effector functions” which refer to those biological activities attributable to the Fc constant region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody. Examples of antibody effector functions include C1q binding; complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; and down regulation of cell surface receptors. Constant region variants include those that alter the effector profile, binding to Fc receptors, and the like.

[0067] Depending on the amino acid sequence of the constant domain of their heavy chains, intact antibodies can be assigned to different “classes.” There are five major classes of intact immunoglobulin antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into “subclasses” (isotypes), e.g., lgG1 , lgG2, lgG3, lgG4, IgA, and lgA2. The heavy-chain constant domains that correspond to the different classes of antibodies are called a, d, e, y, and m, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. Ig forms include hinge-modifications or hingeless forms (Roux et al (1998) J. Immunol. 161 :4083-4090; Lund et al (2000) Eur. J. Biochem. 267:7246- 7256; US 2005/0048572; US 2004/0229310). The light chains of antibodies from any vertebrate species can be assigned to one of two clearly distinct types, called k and l, based on the amino acid sequences of their constant domains.

[0068] “Humanized” forms of non-human (e.g., rodent) antibodies, including single chain antibodies, are chimeric antibodies (including single chain antibodies) that contain minimal sequence derived from non-human immunoglobulin. See, for example, Jones et al, (1986) Nature 321 :522-525; Chothia et al (1989) Nature 342:877; Riechmann et al (1992) J. Mol. Biol. 224, 487- 499; Foote and Winter, (1992) J. Mol. Biol. 224:487-499; Presta et al (1993) J. Immunol. 151 , 2623-2632; Werther et al (1996) J. Immunol. Methods 157:4986-4995; and Presta et al (2001) Thromb. Haemost. 85:379-389. For further details, see U.S. Pat. Nos. 5,225,539; 6,548,640; 6,982,321 ; 5,585,089; 5,693,761 ; 6,407,213; Jones et al (1986) Nature, 321 :522-525; and Riechmann et al (1988) Nature 332:323-329.

[0069] Heparan sulfate. Heparan sulfate (HS) is a linear polysaccharide found in all animal tissues. It occurs as a proteoglycan (HSPG) in which two or three HS chains are attached in close proximity to cell surface or extracellular matrix proteins. The major cell membrane HSPGs are the transmembrane syndecans and the glycosylphosphatidylinositol (GPI) anchored glypicans. Heparan sulfate is a member of the glycosaminoglycan family of carbohydrates and is very closely related in structure to heparan. Both consist of a variably sulfated repeating disaccharide unit. The most common disaccharide unit within heparan sulfate is composed of a glucuronic acid (GlcA) linked to N-acetylglucosamine (GlcNAc) typically making up around 50% of the total disaccharide units.

[0070] Antibodies and ABRs specific for HS chains are known in the art, e.g. HS20, a human monoclonal antibody targeting the HS chains on GPC3, a cell surface-associated HSPG involved in Wnt3a-dependent cell proliferation (see Gao et al. (2015) PLoS ONE 10(9): e0137664; and international patent publication WO2012145469A1). HS20 can also bind to HSPG other than GPC3 through the heparan sulphate chains. Other known antibodies for heparan sulfate chains include, without limitation, those described by Christianson et al. PLoS One. 2012; 7(11 ): e49092; van Kuppevelt et al. J Biol Chem. 1998273(21 ):12960-6; van den Born J. Biol. Chem 280, 20516- 20523; and Dennissen et al. (2002) J. Biol. Chem 277, 10982-10986; and Wittrup et al. J Biol Chem. 2009 Nov 20;284(47):32959-67

[0071] In some embodiments, the ABR comprises the set of CDR sequences comprising amino acid residues 26-33, 51-58 and 97-105 of SEG ID NO: 3 and residues 27-32, 50-52 and 89-97 of SEG ID NO: 4. SEG ID NO: 3 is the amino acid sequence of the VH domain of the HS20 antibody and SEQ ID NO:4 is the amino acid sequence of the VI domain of the HS20 antibody in one embodiment the ABR comprises an scFv variant of HS20 antibody, for example as provided in SEQ ID NO:5.

VH (SEQ ID NO: 3):

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSTIQKQGLPTQYAD

SVKGRFT!SRDNSKNTLYLQ NSLRAEDTAVYYCAKNRAKFDYWGQGTLVTVSS

VI {SEQ ID NO: 4):

DIQMTQSPSSLSASVGDRVTITCRASQS!SSYLNWYQQKPGKAPKLLIYNASMLQSGVPSRFSG

SGSGTDFTLTISSLQPEDFATYYCQQNRGFPLTFGQGTKVEIK

HS20 scFv Protein Sequence (SEQ ID NO: 5):

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSTIQKQGLPTEYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKNRAKFDYWGQGTLVTVSSGGGGSGG GGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYNASML QSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNR GFPLTFGQGTKVEIK

[0072] R-spondins (RSPOs) are secreted proteins that regulate beta-catenin through the control of the internalization of Frizzled and LRP5/6 receptors. RSPO proteins contains an N-terminal signal peptide, 2 furin-like domains, a thrombospondin type-1 domain, and a C-terminal low- complexity region enriched with positively charged amino acids. The term “R-spondin” specifically includes proteins R-spondin 1 , R-spondin 2, R-spondin 3 and R-spondin 4, e.g., the native human proteins, or protein derived from a mammal of interest, such as by truncation of the proteins, fusion or chimeras of the native proteins, e.g., fusion products with a portion of an immunoglobulin; pegylated version of the native protein, etc.

[0073] Included in the R-spondin molecules of interest are “chimeric” polypeptides comprising an R-spondin polypeptide or portion thereof, e.g., one or more domains, fused or bonded to a heterologous polypeptide. The chimeric polypeptide will generally share at least one biological property in common with a native R-spondin polypeptide. Examples of chimeric polypeptides include immunoadhesins, which combine a portion of the native polypeptide with an immunoglobulin sequence. For example, a commercially available product is the full-length human R-spondin1 fused at its C-terminus to the Fc domain of human lgG1 . This fusion increases the stability of the protein in vitro and in vivo without compromising its biological activity. See de Lau, et al (2011 ) Nature 476: 293; Carmon et al. (2011 ) PNAS 108: 11452, each herein specifically incorporated by reference.

[0074] Relative to the reference sequence for human RSP03 precursor, NP 116173, provided here as SEQ ID NO:1 , RSPQ3 has the following features: aa 1-21 signal protein; aa 22-272 mature protein; aa 35-86 Furin-like 1 region; aa 36 glycosylation site; aa 42-144 Furin-like repeat region; aa 150-202 TSP1 , Thrombospondin type 1 repeats.

(SEQ ID NO:1)

MHLRLISWLFIILNFMEYIGSQNASRGRRQRRMHPNVSQGCQGGCATCSDYNGCLSCKPRLFF

ALERIGMKQIGVCLSSCPSGYYGTRYPDINKCTKCKADCDTCFNKNFCTKCKSGFYLHLGKCL

DNCPEGLEANNHTMECVSIVHCEVSEWNPWSPCTKKGKTCGFKRGTETRVREIIQHPSAKGN

LCPPTNETRKCTVQRKKCQKGERGKKGRERKRKKPNKGESKEAIPDSKSLESSKEIPEQRENK

QQQKKRKVQDKQKSVSVSTVH

[0075] Relative to the reference sequence for human RSP02 precursor, NP 848660, provided here as SEQ ID NO:2. RSP02 has the following features: aa 1-21 signal peptide; aa 22-243 mature protein; aa 40-144 Furin-like 2 region; aa 160 glycosylation;

[0076] (SEQ ID NO:2) MQFRLFSFAL IILNCMDYSH CQGNRWRRSK RASYVSNPIC KGCLSCSKDN GCSRCQQKLF FFLRREGMRQ YGECLHSCPS GYYGHRAPDM NRCARCRIEN CDSCFSKDFC TKCKVGFYLH RGRCFDECPD GFAPLEETME CVEGCEVGHW SEWGTCSRNN RTCGFKWGLE TRTRQIVKKP VKDTILCPTI AESRRCKMTM RHCPGGKRTP KAKEKRNKKK KRKLIERAQE QHSVFLATDR ANQ

[0077] Suitable inhibitor (antagonist) proteins include without limitation, DKK1 , DKK2, DKK3, DKK4, WIF; fusions proteins comprising any of the above; derivatives of any of the above; variants of any of the above; and biologically active fragments of any of the above, and the like. Members of the Dickkopf (Dkk) gene family (see Krupnik et al. (1999) Gene 238(2):301 -13) include Dkk-1 , Dkk-2, Dkk-3, and Dkk-4, and the Dkk-3 related protein Soggy (Sgy). hDkks 1-4 contain two distinct cysteine-rich domains in which the positions of 10 cysteine residues are highly conserved between family members. Exemplary sequences of human Dkk genes and proteins are publicly available, e.g. Genbank accession number NM 014419 (soggy-1); NM 014420 (DKK4); AF177394 (DKK-1); AF177395 (DKK-2); NM_015881 (DKK3); and NM_014421 (DKK2). In some embodiments of the invention, the Lrp6 binding moiety is a DKK1 peptide, including without limitation the C-terminal domain of human DKK1. The C-terminal domain may comprise the sequence (SEQ ID NO:22)

KMYHTKGQEGSVCLRSSDCASGLCCARHFWSKICKPVLKEGQVCTKHRRKGSHGLEIFQRCY CGEGLSCRIQKDHHQASNSSRLHTCQRH (see Genbank accession number NP_036374) or a biologically active fragment thereof. The corresponding region of human DKK2 (Genbank reference NP 055236) may comprise the sequence (SEQ ID NO:23) KMSHIKGHEGDPCLRSSDCIEGFCCARHFWTKICKPVLHQGEVCTKQRKKGSHGLEIFQRCDC AKGLSCKVWKDATYSSKARLHVCQK or a biologically active fragment thereof.

A "native sequence" polypeptide is one that has the same amino acid sequence as a polypeptide derived from nature. Such native sequence polypeptides can be isolated from cells producing endogenous protein or can be produced by recombinant or synthetic means. Thus, a native sequence polypeptide can have the amino acid sequence of, e.g., naturally occurring human polypeptide, murine polypeptide, or polypeptide from any other mammalian species, or from non mammalian species, e.g. Drosophila, C. elegans, or the like. In some instances, the “native sequence” polypeptide also includes the N-terminal methionine. In some instances, the “native sequence” polypeptide does not include the N-terminal methionine.

[0078] Engineered WNT signaling agonist or antagonist proteins may be modified using ordinary molecular biological techniques and synthetic chemistry so as to improve their resistance to proteolytic degradation or to optimize solubility properties or to render them more suitable as a therapeutic agent. Analogs of such polypeptides include those containing residues other than naturally occurring L-amino acids, e.g. D-amino acids or non-naturally occurring synthetic amino acids. D-amino acids may be substituted for some or all of the amino acid residues.

[0079] The engineered WNT signaling agonist or antagonist proteins may be prepared by in vitro synthesis, using conventional methods as known in the art. Various commercial synthetic apparatuses are available, for example, automated synthesizers by Applied Biosystems, Inc., Beckman, etc. By using synthesizers, naturally occurring amino acids may be substituted with unnatural amino acids. The particular sequence and the manner of preparation will be determined by convenience, economics, purity required, and the like. If desired, various groups may be introduced into the peptide during synthesis or during expression, which allow for linking to other molecules or to a surface. Thus cysteines can be used to make thioethers, histidines for linking to a metal ion complex, carboxyl groups for forming amides or esters, amino groups for forming amides, and the like.

[0080] The effective dose of an engineered WNT signaling agonist or antagonist protein may be at least about 0.01 mg/dose, at least about 0.05 mg/dose, at least about 0.1 mg/dose, at least about

0.25 mg/dose, at least about 0.5 mg/dose, at least about 1 mg/dose, at least about 2 mg/dose, at least about 2.5 mg/dose, at least about 5 mg/dose, at least about 10 mg/dose, at least about 50 mg/dose, at least about 100 mg/dose, at least about 250 mg/dose, at least about 500 mg/dose, at least about 750 mg/dose, at least about 1 mg/dose, at least about 10 mg/dose, at least about 50 mg/dose, or at least about 100 mg/dose. In certain embodiments, the dose of engineered WNT signaling agonist or antagonist may be administered one time per day, two times per day, three times per day, four times per day or even five times per day. In certain embodiments, the dose of engineered WNT signaling agonist or antagonist is administered at least one time every other day, at least one time every third day, at least one time every fourth day, at least one time every fifth day, at least one time every sixth day or at least one time every seventh day.

[0081] The term "wnt agonist or antagonist activity" refers to the ability of an agonist to modulate the effect or activity of a wnt protein binding to a frizzled protein. The ability of wnt agonists, antagonists or Wnt enhancing agents, such as the RPSPO proteins described herein to modulate activity of wnt can be confirmed by a number of assays. In particular, the proteins of the invention can enhance the canonical Wnt^-catenin signaling pathway. As used herein, the term "enhances" refers to a measurable increase in the level of Wnt^-catenin signaling compared with the level in the absence of a protein of the invention. The term “inhibits” refers to a measurable decrease in the level of Wnt^-catenin signaling compared with the level in the absence of a protein of the invention

[0082] The engineered RSPO proteins described herein will amplify WNT responses generated by endogenous WNTs or exogenous WNTS or synthetic WNT surrogates, for example as described in WO2015023851 A1 ; WO2019159084A1 ; US20190040144A1 ; Tao et al. eLife 2019;8:e46134, each herein specifically incorporated by reference/

[0083] Various methods are known in the art for measuring the level of canonical Wnt^-catenin signaling. These include, but are not limited to assays that measure: Wnt^-catenin target gene expression; TCF reporter gene expression; beta-catenin stabilization; LRP phosphorylation; Axin translocation from cytoplasm to cell membrane and binding to LRP. The canonical Wnt^-catenin signaling pathway ultimately leads to changes in gene expression through the transcription factors TCF7, TCF7L1 , TCF7L2 and LEF. The transcriptional response to Wnt activation has been characterized in a number of cells and tissues. As such, global transcriptional profiling by methods well known in the art can be used to assess Wnt^-catenin signaling activation.

[0084] Changes in wnt-responsive gene expression are generally mediated by TCF and LEF transcription factors. A TCF reporter assay assesses changes in the transcription of TCF/LEF controlled genes to determine the level of Wnt/.beta.-catenin signaling. A TCF reporter assay was first described by Korinek, V. et al., 1997. Also known as TOP/FOP this method involves the use of three copies of the optimal TCF motif CCTTTGATC, or three copies of the mutant motif CCTTTGGCC, upstream of a minimal c-Fos promoter driving luciferase expression (pTOPFLASH and pFOPFLASH, respectively) to determine the transactivational activity of endogenous b- catenin/TCF4. A higher ratio of these two reporter activities (TOP/FOP) indicates higher b- catenin/TCF4 activity.

[0085] Various other reporter transgenes that respond to Wnt signals exist intact in animals and therefore, effectively reflect endogenous Wnt signaling. These reporters are based on a multimerized TCF binding site, which drives expression of LacZ or GFP, which are readily detectable by methods known in the art. These reporter genes include: TOP-GAL, BAT-GAL, ins- TOPEGFP, ins-TOPGAL, LEF-EGFP, Axin2-LacZ, Axin2-d2EGFP, Lgr5tm1(cre/ERT2), TOPdGFP.

[0086] The recruitment of dephosphorylated b-catenin to the membrane, stabilization and phosphorylation status of b-catenin and translocation of b-catenin to the nucleus (Klapholz-Brown Z et al., PLoS One. 2(9) e945, 2007) in some cases mediated by complex formation with TCF transcription factors and TNIK are key steps in the Wnt signaling pathway. Stabilisation is mediated by Disheveled family proteins that inhibit the "destruction" complex so that degradation of intracellular b-catenin is reduced, and translocation of b-catenin to the nucleus follows thereafter. Therefore, measuring the level and location of b-catenin in a cell is a good reflection of the level of Wnt^-catenin signaling. A non-limiting example of such an assay is the "Biolmage b-Catenin Redistribution Assay" (Thermo Scientific) which provides recombinant U20S cells that stably express human b-catenin fused to the C-terminus of enhanced green fluorescent protein (EGFP). Imaging and analysis is performed with a fluorescence microscope or HCS platform allowing the levels and distribution of EGFP^-catenin to be visualized.

[0087] Another way, in which the destruction complex is inhibited, is by removal of Axin by recruitment of Axin to the cytoplasmic tail of the Wnt co-receptor LRP. Axin has been shown to bind preferentially to a phosphorylated form of the LRP tail. Visualisation of Axin translocation, for example with a GFP-Axin fusion protein, is therefore another method for assessing levels of Wnt^-catenin signaling.

[0088] In certain embodiments, the proteins of the invention may enhance b-catenin signaling by at least 30%, 35%, 40%, 45%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 150%, 200%, 250%, 300%, 400% or 500% compared to the b-catenin signaling induced in the absence of the protein as measured in an assay described above, for example as measured in the TOPFIash assay. A negative control may be included in these assays. In particular embodiments, the proteins of the invention may enhance b-catenin signaling by a factor of 2x, 5x, 10x, 100x, 1000x, 10000X or more as compared to the activity in the absence of the protein when measured in an assay described above, for example when measured in the TOPFIash assay, or any of the other assays mentioned herein.

[0089] In certain embodiments, the proteins of the invention may inhibit b-catenin signaling by at least 30%, 35%, 40%, 45%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 150%, 200%, 250%, 300%, 400% or 500% compared to the b-catenin signaling induced in the absence of the protein as measured in an assay described above, for example as measured in the TOPFIash assay. A negative control may be included in these assays. In particular embodiments, the proteins of the invention may inhibit b-catenin signaling by a factor of 2x, 5x, 10x, 10Ox, 10OOx, 10000X or more as compared to the activity in the absence of the protein when measured in an assay described above, for example when measured in the TOPFIash assay, or any of the other assays mentioned herein.

[0090] "Wnt gene product" or "Wnt polypeptide" when used herein encompass native sequence Wnt polypeptides, Wnt polypeptide variants, Wnt polypeptide fragments and chimeric Wnt polypeptides. In particular embodiments, a Wnt polypeptide is a native human full length mature Wnt protein.

[0091] For example, human native sequence Wnt proteins of interest in the present application include the following: Wnt-1 (GenBank Accession No. NM 005430); Wnt-2 (GenBank Accession No. NM 003391); Wnt-2B (Wnt-13) (GenBank Accession No. NM_004185 (isoform 1), NM 024494.2 (isoform 2)), Wnt-3 (RefSeq.: NM 030753), Wnt3a (GenBank Accession No. NM 033131), Wnt-4 (GenBank Accession No. NM 030761), Wnt-5A (GenBank Accession No. NM 003392), Wnt-5B (GenBank Accession No. NM 032642), Wnt-6 (GenBank Accession No. NM 006522), Wnt-7A (GenBank Accession No. NM 004625), Wnt-7B (GenBank Accession No. NM 058238), Wnt-8A (GenBank Accession No. NM 058244), Wnt-8B (GenBank Accession No. NM 003393), Wnt-9A (Wnt-14) (GenBank Accession No. NM_003395), Wnt-9B (Wnt-15) (GenBank Accession No. NM 003396), Wnt-10A (GenBank Accession No. NM 025216), Wnt- 10B (GenBank Accession No. NM 003394), Wnt-11 (GenBank Accession No. NM 004626), Wnt-16 (GenBank Accession No. NM 016087)). Although each member has varying degrees of sequence identity with the family, all encode small (i.e., 39-46 kD), acylated, palmitoylated, secreted glycoproteins that contain 23-24 conserved cysteine residues whose spacing is highly conserved (McMahon, A P et al., Trends Genet. 1992; 8: 236-242; Miller, J R. Genome Biol. 2002; 3(1): 3001.1-3001.15). Other native sequence Wnt polypeptides of interest include orthologs of the above from any mammal, including domestic and farm animals, and zoo, laboratory or pet animals, such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, rats, mice, frogs, zebra fish, fruit fly, worm, etc.

[0092] “Wnt protein signaling” or “Wnt signaling” is used herein to refer to the mechanism by which a biologically active Wnt exerts its effects upon a cell to modulate a cell’s activity. Wnt proteins modulate cell activity by binding to Wnt receptors, including proteins from the Frizzled (Fz) family of proteins, proteins from the ROR family of proteins, the proteins LRP5, LRP6 from the LRP family of proteins, the protein FRL1 /crypto, and the protein Derailed/Ryk. Once activated by Wnt binding, the Wnt receptor(s) will activate one or more intracellular signaling cascades. These include the canonical Wnt signaling pathway; the Wnt/planar cell polarity (Wnt/PCP) pathway; the Wnt-calcium (Wnt/Ca²⁺) pathway (Giles, RH et al. (2003) Biochim Biophys Acta 1653, 1-24; Peifer, M. et al. (1994) Development 120: 369-380; Papkoff, J. et al (1996) Mol. Cell Biol. 16: 2128-2134; Veeman, M. T. et al. (2003) Dev. Cell 5: 367-377); and other Wnt signaling pathways as is well known in the art.

[0093] For example, activation of the canonical Wnt signaling pathway results in the inhibition of phosphorylation of the intracellular protein 8-catenin, leading to an accumulation of 8-catenin in the cytosol and its subsequent translocation to the nucleus where it interacts with transcription factors, e.g. TCF/LEF, to activate target genes. Activation of the Wnt/PCP pathway activates RhoA, c-Jun N-terminal kinase (JNK), and nemo-like kinase (NLK) signaling cascades to control such biological processes as tissue polarity and cell movement. Activation of the Wnt/Ca²⁺ by, for example, binding of Wnt-4, Wnt-5A or Wnt-11 , elicits an intracellular release of calcium ions, which activates calcium sensitive enzymes like protein kinase C (PKC), calcium-calmodulin dependent kinase II (CamKII) or calcineurin (CaCN). By assaying for activity of the above signaling pathways, the biological activity of a Wnt composition can be readily determined. A “biologically active engineered RSPO protein” is an engineered RSPO protein composition that is able to enhance Wnt signaling when provided to a cell in vitro or in vivo, that is, when administered to an animal, e.g. a mammal.

[0094] The term "specific binding" refers to that binding which occurs between such paired species as enzyme/substrate, receptor/ligand, antibody/antigen, and lectin/carbohydrate which may be mediated by covalent or non-covalent interactions or a combination of covalent and non-covalent interactions. When the interaction of the two species produces a non-covalently bound complex, the binding which occurs is typically electrostatic, hydrogen-bonding, or the result of lipophilic interactions. Accordingly, "specific binding" occurs between a paired species where there is interaction between the two which produces a bound complex having the characteristics of an antibody/antigen or ligand/receptor interaction. One may determine the biological activity of an engineered WNT signaling agonist or antagonist, e.g. engineered RSPO protein in a composition by determining the level of activity in a functional assay after in vivo administration, e.g. accelerating bone regeneration, enhancing stem cell proliferation, etc., nuclear localization of b- catenin, increased transcription of wnt-responsive genes; etc.

[0095] An engineered WNT signaling agonist or antagonist, e.g. engineered RSPO protein may be fused or bonded to an additional polypeptide sequence. Examples include immunoadhesins, which combine an engineered RSPO protein with an immunoglobulin sequence particularly an Fc sequence, and epitope tagged polypeptides, which comprise a native inhibitors polypeptide or portion thereof fused to a "tag polypeptide". The tag polypeptide has enough residues to provide an epitope against which an antibody can be made, yet is short enough such that it does not interfere with biological activity of the native inhibitors polypeptide. Suitable tag polypeptides generally have at least six amino acid residues and usually between about 6-60 amino acid residues. The engineered WNT signaling agonist or antagonist, e.g. engineered RSPO protein may also be fused or combined in a formulation, or co-administered with an agent that enhances wnt activity, e.g. wnt proteins, wnt surrogates, anti-wnt antibodies, etc.

[0096] Linker. The amino acid linkers that join domains can play an important role in the structure and function of multi-domain proteins, for example between an RSPO; DKK, etc. domain and an ABR domain. In general, altering the length of linkers connecting domains has been shown to affect protein stability, folding rates and domain-domain orientation (see George and Hering (2003) Prot. Eng. 15:871-879). The length of the linker in the engineered WNT signaling agonist or antagonist, e.g. engineered RSPO protein, and therefore the spacing between the binding domains, can be used to modulate the signal strength of the engineered WNT signaling agonist or antagonist, e.g. engineered RSPO protein, and can be selected depending on the desired use of the engineered WNT signaling agonist or antagonist, e.g. engineered RSPO protein.

[0097] In some embodiments the linker is a rigid linker, in other embodiments the linker is a flexible linker. In some embodiments, the linker moiety is a peptide linker. In some embodiments, the peptide linker comprises 2 to 100 amino acids. In some embodiments, the peptide linker comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26,

27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78,

79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 but no greater than 100 amino acids. In some embodiments, the peptide linker is between 5 to 75, 5 to 50, 5 to 25, 5 to 20, 5 to 15, 5 to 10 or 5 to 9 amino acids in length. Exemplary linkers include linear peptides having at least two amino acid residues such as Gly-Gly, Gly-Ala-Gly, Gly-Pro-Ala, Gly-Gly-Gly- Gly-Ser. Suitable linear peptides include poly glycine, polyserine, polyproline, polyalanine and oligopeptides consisting of alanyl and/or serinyl and/or prolinyl and/or glycyl amino acid residues. In some embodiments, the peptide linker comprises the amino acid sequence selected from the group consisting of Gly₉, Glu₉, Ser₉, Gly₅-Cys-Pro2-Cys, (Gly -Ser)₃, Ser-Cys-Val-Pro-Leu-Met- Arg-Cys-Gly-Gly-Cys-Cys-Asn, Pro-Ser-Cys-Val-Pro-Leu-Met-Arg-Cys-Gly-Gly-Cys-Cys-Asn, Gly-Asp-Leu-lle-Tyr-Arg-Asn-Gln-Lys, and Gly₉-Pro-Ser-Cys-Val-Pro-Leu-Met-Arg-Cys-Gly-Gly- Cys-Cys-Asn. In one embodiment a linker comprises the amino acid sequence GSTSGSGKSSEGKG, or (GGGGS)n, where n is 1 , 2, 3, 4, 5, etc.; however many such linkers are known and used in the art and may serve this purpose.

[0098] The engineered RPO protein can be provided in single-chain form, which means that the ABR is linked by peptide bonds through a linker peptide to the RSPO moiety. In other embodiments, the binding domains are individual peptides and can be joined through a non- peptidic linker.

[0099] Chemical groups that find use in linking binding domains include carbamate; amide (amine plus carboxylic acid); ester (alcohol plus carboxylic acid), thioether (haloalkane plus sulfhydryl; maleimide plus sulfhydryl), Schiff's base (amine plus aldehyde), urea (amine plus isocyanate), thiourea (amine plus isothiocyanate), sulfonamide (amine plus sulfonyl chloride), disulfide; hyrodrazone, lipids, and the like, as known in the art.

[00100] The linkage between binding domains may comprise spacers, e.g. alkyl spacers, which may be linear or branched, usually linear, and may include one or more unsaturated bonds; usually having from one to about 300 carbon atoms; more usually from about one to 25 carbon atoms; and may be from about three to 12 carbon atoms. Spacers of this type may also comprise heteroatoms or functional groups, including amines, ethers, phosphodiesters, and the like. Specific structures of interest include: (CH₂CH₂0)n where n is from 1 to about 12; (CH₂CH₂NH)n, where n is from 1 to about 12; [(CH₂)n(C=0)NH(CH₂)_m]z, where n and m are from 1 to about 6, and z is from 1 to about 10; [(CH₂)n0P0₃(CH₂)_m]_z where n and m are from 1 to about 6, and z is from 1 to about 10. Such linkers may include polyethylene glycol, which may be linear or branched.

[00101] The binding domains may be joined through a homo- or heterobifunctional linker having a group at one end capable of forming a stable linkage to the hydrophilic head group, and a group at the opposite end capable of forming a stable linkage to the targeting moiety. Illustrative entities include: azidobenzoyl hydrazide, N-[4-(p-azidosalicylamino)butyl]-3'-[2'- pyridyldithio]propionamide), bis-sulfosuccinimidyl suberate, dimethyladipimidate, disuccinimidyltartrate, N-g-maleimidobutyryloxysuccinimide ester, N-hydroxy sulfosuccinimidyl-4- azidobenzoate, N-succinimidyl [4-azidophenyl]-1 ,3'-dithiopropionate, N-succinimidyl [4- iodoacetyl]aminobenzoate, glutaraldehyde, NHS-PEG-MAL; succinimidyl 4-[N- maleimidomethyl]cyclohexane-1-carboxylate; 3-(2-pyridyldithio)propionic acid N- hydroxysuccinimide ester (SPDP); N, N'-(1 ,3-phenylene) bismaleimide; N, N'-ethylene-bis- (iodoacetamide); or 4-(N-maleimidomethyl)-cyclohexane-1 -carboxylic acid N-hydroxysuccinimide ester (SMCC); m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), and succinimide 4-(p- maleimidophenyl)butyrate (SMPB), an extended chain analog of MBS. The succinimidyl group of these cross-linkers reacts with a primary amine, and the thiol-reactive maleimide forms a covalent bond with the thiol of a cysteine residue.

[00102] Other reagents useful for this purpose include: p,p'-difluoro-m,m'-dinitrodiphenylsulfone (which forms irreversible cross-linkages with amino and phenolic groups); dimethyl adipimidate (which is specific for amino groups); phenol-1 ,4-disulfonylchloride (which reacts principally with amino groups); hexamethylenediisocyanate or diisothiocyanate, or azophenyl-p-diisocyanate (which reacts principally with amino groups); disdiazobenzidine (which reacts primarily with tyrosine and histidine); O-benzotriazolyloxy tetramethuluronium hexafluorophosphate (HATU), dicyclohexyl carbodiimde, bromo-tris (pyrrolidino) phosphonium bromide (PyBroP); N,N- dimethylamino pyridine (DMAP); 4-pyrrolidino pyridine; N-hydroxy benzotriazole; and the like. Homobifunctional cross-linking reagents include bismaleimidohexane ("BMH").

[00103] Antibody : As used herein, the term “antibody” refers to a polypeptide that includes canonical immunoglobulin sequence elements sufficient to confer specific binding to a particular target antigen. As is known in the art, intact antibodies as produced in nature are approximately 150 kD tetrameric agents comprised of two identical heavy chain polypeptides (about 50 kD each) and two identical light chain polypeptides (about 25 kD each) that associate with each other into what is commonly referred to as a Ύ-shaped” structure. Each heavy chain is comprised of at least four domains (each about 110 amino acids long)- an amino-terminal variable (VH) domain (located at the tips of the Y structure), followed by three constant domains: CH1 , CH2, and the carboxy-terminal CH3 (located at the base of the Y’s stem). A short region, known as the “switch”, connects the heavy chain variable and constant regions. The “hinge” connects CH2 and CH3 domains to the rest of the antibody. Two disulfide bonds in this hinge region connect the two heavy chain polypeptides to one another in an intact antibody. Each light chain is comprised of two domains - an amino-terminal variable (VL) domain, followed by a carboxy-terminal constant (CL) domain, separated from one another by another “switch”. Intact antibody tetramers are comprised of two heavy chain-light chain dimers in which the heavy and light chains are linked to one another by a single disulfide bond; two other disulfide bonds connect the heavy chain hinge regions to one another, so that the dimers are connected to one another and the tetramer is formed. Naturally-produced antibodies are also glycosylated, typically on the CH2 domain. Each domain in a natural antibody has a structure characterized by an “immunoglobulin fold” formed from two beta sheets (e.g., 3-, 4-, or 5-stranded sheets) packed against each other in a compressed antiparallel beta barrel. Each variable domain contains three hypervariable loops known as “complement determining regions” (CDR1 , CDR2, and CDR3) and four somewhat invariant “framework” regions (FR1 , FR2, FR3, and FR4). When natural antibodies fold, the FR regions form the beta sheets that provide the structural framework for the domains, and the CDR loop regions from both the heavy and light chains are brought together in three-dimensional space so that they create a single hypervariable antigen binding site located at the tip of the Y structure.

[00104] The Fc region of naturally-occurring antibodies binds to elements of the complement system, and also to receptors on effector cells, including for example effector cells that mediate cytotoxicity. As is known in the art, affinity and/or other binding attributes of Fc regions for Fc receptors can be modulated through glycosylation or other modification. In some embodiments, antibodies produced and/or utilized in accordance with the present invention include glycosylated Fc domains, including Fc domains with modified or engineered such glycosylation.

[00105] Any polypeptide or complex of polypeptides that includes sufficient immunoglobulin domain sequences as found in natural antibodies can be referred to and/or used as an “antibody”, whether such polypeptide is naturally produced (e.g., generated by an organism reacting to an antigen), or produced by recombinant engineering, chemical synthesis, or other artificial system or methodology. In some embodiments, antibody sequence elements are humanized, primatized, chimeric, etc, as is known in the art.

[00106] Moreover, the term “antibody” as used herein, can refer in appropriate embodiments (unless otherwise stated or clear from context) to any of the art-known or developed constructs or formats for utilizing antibody structural and functional features in alternative presentation. For example, embodiments, an antibody utilized in accordance with the present invention is in a format selected from, but not limited to, intact IgG, IgE and IgM, bi- or multi- specific antibodies (e.g., Zybodies®, etc), single chain Fvs, Fabs, Small Modular ImmunoPharmaceuticals (“SMIPs™^”), single chain or Tandem diabodies (TandAb®), VHHs, Anticalins®, Nanobodies®, minibodies, BiTE®s, ankyrin repeat proteins or DARPINs®, Avimers®, a DART, a TCR-like antibody, Adnectins®, Affilins®, Trans-bodies®, Affibodies®, a TrimerX®, MicroProteins, Fynomers®, Centyrins®, and a KALBITOR®. In some embodiments, an antibody may lack a covalent modification (e.g., attachment of a glycan) that it would have if produced naturally. In some embodiments, an antibody may contain a covalent modification (e.g., attachment of a glycan, a payload [e.g., a detectable moiety, a therapeutic moiety, a catalytic moiety, etc], or other pendant group [e.g., poly-ethylene glycol, etc.]

[00107] In many embodiments, an antibody agent is or comprises a polypeptide whose amino acid sequence includes one or more structural elements recognized by those skilled in the art as a complementarity determining region (CDR); in some embodiments an antibody agent is or comprises a polypeptide whose amino acid sequence includes at least one CDR (e.g., at least one heavy chain CDR and/or at least one light chain CDR) that is substantially identical to one found in a reference antibody. In some embodiments an included CDR is substantially identical to a reference CDR in that it is either identical in sequence or contains between 1-5 amino acid substitutions as compared with the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that it shows at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that it shows at least 96%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that at least one amino acid within the included CDR is deleted, added, or substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical with that of the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that 1-5 amino acids within the included CDR are deleted, added, or substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical to the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that at least one amino acid within the included CDR is substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical with that of the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that 1-5 amino acids within the included CDR are deleted, added, or substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical to the reference CDR. In some embodiments, an antibody agent is or comprises a polypeptide whose amino acid sequence includes structural elements recognized by those skilled in the art as an immunoglobulin variable domain. In some embodiments, an antibody agent is a polypeptide protein having a binding domain which is homologous or largely homologous to an immunoglobulin-binding domain.

[00108] Expression construct In the present methods, a engineered WNT signaling agonist or antagonist, e.g. engineered RSPO protein, if a polypeptide, may be produced by recombinant methods. Amino acid sequence variants of are prepared by introducing appropriate nucleotide changes into the DNA coding sequence. Such variants represent insertions, substitutions, and/or specified deletions of, residues within or at one or both of the ends of the amino acid sequence. Any combination of insertion, substitution, and/or specified deletion is made to arrive at the final construct, provided that the final construct possesses the desired biological activity as defined herein. The amino acid changes also may alter post-translational processes of the polypeptide, such as changing the number or position of glycosylation sites, altering the membrane anchoring characteristics, and/or altering the cellular location by inserting, deleting, or otherwise affecting the leader sequence of a polypeptide.

[00109] The nucleic acid encoding the engineered WNT signaling agonist or antagonist, e.g. engineered RSPO protein can be inserted into a replicable vector for expression. Many such vectors are available. The vector components generally include, but are not limited to, one or more of the following: an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

[00110] Expression vectors will contain a promoter that is recognized by the host organism and is operably linked to the engineered WNT signaling agonist or antagonist, e.g. engineered RSPO protein coding sequence. Promoters are untranslated sequences located upstream (5') to the start codon of a structural gene (generally within about 100 to 1000 bp) that control the transcription and translation of particular nucleic acid sequence to which they are operably linked. Such promoters typically fall into two classes, inducible and constitutive. Inducible promoters are promoters that initiate increased levels of transcription from DNA under their control in response to some change in culture conditions, e.g., the presence or absence of a nutrient or a change in temperature.

[00111] Promoters suitable for use with prokaryotic hosts include the b-lactamase and lactose promoter systems, alkaline phosphatase, a tryptophan (trp) promoter system, and hybrid promoters such as the tac promoter. However, other known bacterial promoters are also suitable. Such nucleotide sequences have been published, thereby enabling a skilled worker operably to ligate them to a DNA coding sequence. Promoters for use in bacterial systems also will contain a Shine-Dalgarno (S.D.) sequence operably linked to the coding sequence. [00112] Promoter sequences are known for eukaryotes. Examples of suitable promoting sequences for use with yeast hosts include the promoters for 3-phosphoglyceratekinase or other glycolytic enzymes, such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3- phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. Other yeast promoters, which are inducible promoters having the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Suitable vectors and promoters for use in yeast expression are further described in EP 73,657. Yeast enhancers also are advantageously used with yeast promoters.

[00113] Transcription from vectors in mammalian host cells may be controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and most preferably Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g., the actin promoter, PGK (phosphoglycerate kinase), or an immunoglobulin promoter, from heat-shock promoters, provided such promoters are compatible with the host cell systems. The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment that also contains the SV40 viral origin of replication. The immediate early promoter of the human cytomegalovirus is conveniently obtained as a Hindll I E restriction fragment.

[00114] Transcription by higher eukaryotes is often increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp, which act on a promoter to increase its transcription. Enhancers are relatively orientation and position independent, having been found 5' and 3' to the transcription unit, within an intron, as well as within the coding sequence itself. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, D-fetoprotein, and insulin). Typically, however, one will use an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on the late side of the replication origin, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. The enhancer may be spliced into the expression vector at a position 5' or 3' to the coding sequence, but is preferably located at a site 5' from the promoter. [00115] Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal, human, or nucleated cells from other multicellular organisms) may also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5' and, occasionally 3', untranslated regions of eukaryotic or viral DNAs or cDNAs.

[00116] Construction of suitable vectors containing one or more of the above-listed components employs standard techniques. Isolated plasmids or DNA fragments can be cleaved, tailored, and re-ligated in the form desired to generate the plasmids required. For analysis to confirm correct sequences in plasmids constructed, the ligation mixtures are used to transform host cells, and successful transformants selected by ampicillin or tetracycline resistance where appropriate. Plasmids from the transformants are prepared, analyzed by restriction endonuclease digestion, and/or sequenced.

[00117] Suitable host cells for cloning or expressing the DNA in the vectors herein are the prokaryote, yeast, or higher eukaryote cells described above. Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis, Pseudomonas such as P. aeruginosa, and Streptomyces. These examples are illustrative rather than limiting.

[00118] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable expression hosts. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms. However, a number of other genera, species, and strains are commonly available and useful herein, such as Schizosaccharomyces pombe; Kluyveromyces hosts such as K. lactis, K. fragilis, etc.; Pichia pastoris; Candida; Neurospora crassa; Schwanniomyces such as Schwanniomyces occidentalis; and filamentous fungi such as Penicillium, Tolypocladium, and Aspergillus hosts such as A. nidulan, and A. niger.

[00119] Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can be utilized as hosts. Typically, plant cells are transfected by incubation with certain strains of the bacterium Agrobacterium tumefaciens. During such incubation of the plant cell culture, the DNA coding sequence is transferred to the plant cell host such that it is transfected, and will, under appropriate conditions, express the DNA. In addition, regulatory and signal sequences compatible with plant cells are available, such as the nopaline synthase promoter and polyadenylation signal sequences. [00120] Examples of useful mammalian host cell lines are mouse L cells (L-M[TK-], ATCC#CRL- 2648), monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture; baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR (CHO); mouse sertoli cells (TM4); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO- 76, ATCC CRL-1 587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51 ); TRI cells; MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).

[00121] Host cells are transfected with the above-described expression vectors for engineered RSPO protein production, and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. Mammalian host cells may be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), Sigma), RPMI 1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing the host cells. Any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleosides (such as adenosine and thymidine), antibiotics, trace elements, and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art. The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.

Pharmaceutical Compositions

[00122] For therapeutic applications, the engineered WNT signaling agonist or antagonist, e.g. engineered RSPO protein is administered to a mammal, preferably a human, in a physiologically acceptable dosage form, including those that may be administered to a human intravenously as a bolus or by continuous infusion over a period of time. Alternative routes of administration include topical, intramuscular, intraperitoneal, intra-cerobrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes. The engineered RSPO proteins also are suitably administered by intratumoral, peritumoral, intralesional, or perilesional routes or to the lymph, to exert local as well as systemic therapeutic effects. [00123] Pharmaceutical compositions may also comprise combinations of the molecules of the invention with cells, including stem cells, progenitor cells, and the like. In some embodiments, the compositions comprise the molecules of the invention in combination with regenerative somatic stem cells, e.g. epithelial stem cells, neural stem cells, liver stem cells, hematopoietic stem cells, osteoblasts, muscle stem cells, mesenchymal stem cells, pancreatic stem cells, etc. In such combinations, cells can be pre-treated with a molecule of the invention prior to use, e.g. ex vivo treatment of cells with the engineered WNT signaling agonist or antagonist, e.g. engineered RSPO protein; cells can be administered concomitantly with a molecule of the invention in a separate or combined formulation; cells can be provided to an individual prior to treatment with a molecule of the invention, and the like.

[00124] The terms “stem cell” as used herein, refer to a cell that has the property of self-renewal and has the developmental potential to differentiate into multiple cell types. A stem cell is capable of proliferation and giving rise to more such stem cells while maintaining its developmental potential. Stem cells can divide asymmetrically, with one daughter cell retaining the developmental potential of the parent stem cell and the other daughter cell expressing some distinct other specific function, phenotype and/or developmental potential from the parent cell. The daughter cells themselves can be induced to proliferate and produce progeny that subsequently differentiate into one or more mature cell types, while also retaining one or more cells with parental developmental potential. A differentiated cell may derive from a multipotent cell, which itself is derived from a multipotent cell, and so on. While each of these multipotent cells may be considered stem cells, the range of cell types each such stem cell can give rise to, i.e., their developmental potential, can vary considerably. Alternatively, some of the stem cells in a population can divide symmetrically into two stem cells, known as stochastic differentiation, thus maintaining some stem cells in the population as a whole, while other cells in the population give rise to differentiated progeny only. Accordingly, the term “stem cell” refers to any subset of cells that have the developmental potential, under particular circumstances, to differentiate to a more specialized or differentiated phenotype, and which retain the capacity, under certain circumstances, to proliferate without substantially differentiating.

[00125] The term “somatic stem cell” is used herein to refer to any pluripotent or multipotent stem cell derived from non-embryonic tissue, including fetal, juvenile, and adult tissue. Natural somatic stem cells have been isolated from a wide variety of adult tissues including blood, bone marrow, brain, olfactory epithelium, skin, pancreas, skeletal muscle, and cardiac muscle. The term “progenitor cell” is used herein to refer to cells that are at an earlier stage along a developmental pathway or progression, relative to a cell which it can give rise to by differentiation. Often, progenitor cells have significant or very high proliferative potential. Progenitor cells can give rise to multiple distinct differentiated cell types, or to a single differentiated cell type, depending on the developmental pathway and on the environment in which the cells develop and differentiate.

[00126] Pharmaceutical compositions can include, depending on the formulation desired, pharmaceutically-acceptable, non-toxic carriers of diluents, which are defined as vehicles commonly used to formulate pharmaceutical compositions for animal or human administration. The diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, buffered water, physiological saline, PBS, Ringer's solution, dextrose solution, and Hank's solution. In addition, the pharmaceutical composition or formulation can include other carriers, adjuvants, or non-toxic, nontherapeutic, nonimmunogenic stabilizers, excipients and the like. The compositions can also include additional substances to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, wetting agents and detergents.

[00127] The composition can also include any of a variety of stabilizing agents, such as an antioxidant for example. When the pharmaceutical composition includes a polypeptide, the polypeptide can be complexed with various well-known compounds that enhance the in vivo stability of the polypeptide, or otherwise enhance its pharmacological properties (e.g., increase the half-life of the polypeptide, reduce its toxicity, enhance solubility or uptake). Examples of such modifications or complexing agents include sulfate, gluconate, citrate and phosphate. The polypeptides of a composition can also be complexed with molecules that enhance their in vivo attributes. Such molecules include, for example, carbohydrates, polyamines, amino acids, other peptides, ions (e.g., sodium, potassium, calcium, magnesium, manganese), and lipids.

[00128] Further guidance regarding formulations that are suitable for various types of administration can be found in Remington's Pharmaceutical Sciences, Mace Publishing Company, Philadelphia, Pa., 17th ed. (1985). For a brief review of methods for drug delivery, see, Langer, Science 249:1527-1533 (1990).

[00129] The pharmaceutical compositions can be administered for prophylactic and/or therapeutic treatments. Toxicity and therapeutic efficacy of the active ingredient can be determined according to standard pharmaceutical procedures in cell cultures and/or experimental animals, including, for example, determining the LD₅o (the dose lethal to 50% of the population) and the ED₅o (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD₅o/ED₅o Compounds that exhibit large therapeutic indices are preferred. [00130] The data obtained from cell culture and/or animal studies can be used in formulating a range of dosages for humans. The dosage of the active ingredient typically lines within a range of circulating concentrations that include the ED₅o with low toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized.

[00131] For oral administration, the active ingredient can be administered in solid dosage forms, such as capsules, tablets, and powders, or in liquid dosage forms, such as elixirs, syrups, and suspensions. The active component(s) can be encapsulated in gelatin capsules together with inactive ingredients and powdered carriers, such as glucose, lactose, sucrose, mannitol, starch, cellulose or cellulose derivatives, magnesium stearate, stearic acid, sodium saccharin, talcum, magnesium carbonate. Examples of additional inactive ingredients that may be added to provide desirable color, taste, stability, buffering capacity, dispersion or other known desirable features are red iron oxide, silica gel, sodium lauryl sulfate, titanium dioxide, and edible white ink. Similar diluents can be used to make compressed tablets. Both tablets and capsules can be manufactured as sustained release products to provide for continuous release of medication over a period of hours. Compressed tablets can be sugar coated or film coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric-coated for selective disintegration in the gastrointestinal tract. Liquid dosage forms for oral administration can contain coloring and flavoring to increase patient acceptance.

[00132] The active ingredient, alone or in combination with other suitable components, can be made into aerosol formulations (i.e., they can be "nebulized") to be administered via inhalation. Aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen.

[00133] Formulations suitable for parenteral administration, such as, for example, by intraarticular (in the joints), intravenous, intramuscular, intradermal, intraperitoneal, and subcutaneous routes, include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacterio stats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.

[00134] The components used to formulate the pharmaceutical compositions are preferably of high purity and are substantially free of potentially harmful contaminants (e.g., at least National Food (NF) grade, generally at least analytical grade, and more typically at least pharmaceutical grade). Moreover, compositions intended for in vivo use are usually sterile. To the extent that a given compound must be synthesized prior to use, the resulting product is typically substantially free of any potentially toxic agents, particularly any endotoxins, which may be present during the synthesis or purification process. Compositions for parental administration are also sterile, substantially isotonic and made under GMP conditions.

[00135] The effective amount of a therapeutic composition to be given to a particular patient will depend on a variety of factors, several of which will be different from patient to patient. A formulation may be provided, for example, in a unit dose. A competent clinician will be able to determine an effective amount of a therapeutic agent to administer to a patient. Dosage of the protein will depend on the treatment, route of administration, the nature of the therapeutics, sensitivity of the disease to the therapeutics, etc. Utilizing LD₅o animal data, and other information available, a clinician can determine the maximum safe dose for an individual, depending on the route of administration. Compositions which are rapidly cleared from the body may be administered at higher doses, or in repeated doses, in order to maintain a therapeutic concentration. Utilizing ordinary skill, the competent clinician will be able to optimize the dosage of a particular therapeutic or imaging composition in the course of routine clinical trials. Typically the dosage will be 0.001 to 100 milligrams of agent per kilogram subject body weight.

[00136] The compositions can be administered to the subject in a series of more than one administration. For therapeutic compositions, regular periodic administration (e.g., every 2-3 days) will sometimes be required, or may be desirable to reduce toxicity. For therapeutic compositions which will be utilized in repeated-dose regimens, moieties which do not provoke immune responses are preferred.

[00137] In another embodiment of the invention, an article of manufacture containing materials useful for the treatment of the conditions described herein is provided. The article of manufacture comprises a container and a label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition that is effective for treating the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The active agent in the composition is the engineered WNT signaling agonist or antagonist, e.g. engineered RSPO protein. The label on, or associated with, the container indicates that the composition is used for treating the condition of choice. Further container(s) may be provided with the article of manufacture which may hold, for example, a pharmaceutically-acceptable buffer, such as phosphate-buffered saline, Ringer's solution or dextrose solution. The article of manufacture may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. [00138] As used herein, the term “therapeutically effective amount” means an amount that is sufficient, when administered to a population suffering from or susceptible to a disease, disorder, and/or condition in accordance with a therapeutic dosing regimen, to treat the disease, disorder, and/or condition. In some embodiments, a therapeutically effective amount is one that reduces the incidence and/or severity of, stabilizes one or more characteristics of, and/or delays onset of, one or more symptoms of the disease, disorder, and/or condition. Those of ordinary skill in the art will appreciate that the term “therapeutically effective amount” does not in fact require successful treatment be achieved in a particular individual. Rather, a therapeutically effective amount may be that amount that provides a particular desired pharmacological response in a significant number of subjects when administered to patients in need of such treatment.

[00139] For example, in some embodiments, term "therapeutically effective amount", refers to an amount which, when administered to an individual in need thereof in the context of inventive therapy, will block, stabilize, attenuate, or reverse a disease process occurring in said individual.

Methods of Use

[00140] The engineered WNT signaling agonist or antagonist, e.g. engineered RSPO proteins are useful for both prophylactic and therapeutic purposes. Thus, as used herein, the term “treating” is used to refer to both prevention of disease, and treatment of a pre-existing condition. In certain instances, prevention indicates inhibiting or delaying the onset of a disease or condition, in a patient identified as being at risk of developing the disease or condition. The treatment of ongoing disease, to stabilize or improve the clinical symptoms of the patient, is a particularly important benefit provided by the present invention. Such treatment is desirably performed prior to loss of function in the affected tissues; consequently, the prophylactic therapeutic benefits provided by the invention are also important. Evidence of therapeutic effect may be any diminution in the severity of disease. The therapeutic effect can be measured in terms of clinical outcome or can be determined by immunological or biochemical tests. Patients for treatment may be mammals, e.g. primates, including humans, may be laboratory animals, e.g. rabbits, rats, mice, etc., particularly for evaluation of therapies, horses, dogs, cats, farm animals, etc.

[00141] The dosage of the therapeutic formulation, e.g., pharmaceutical composition, will vary widely, depending upon the nature of the condition, the frequency of administration, the manner of administration, the clearance of the agent from the host, and the like. In particular embodiments, the initial dose can be larger, followed by smaller maintenance doses. In certain embodiments, the dose can be administered as infrequently as weekly or biweekly, or more often fractionated into smaller doses and administered daily, semi-weekly, or otherwise as needed to maintain an effective dosage level.

[00142] In some embodiments of the invention, administration of the composition or formulation comprising the engineered WNT signaling agonist or antagonist, e.g. engineered RSPO protein is performed by local administration. Local administration, as used herein, may refer to topical administration, but also refers to injection or other introduction into the body at a site of treatment. Examples of such administration include intramuscular injection, subcutaneous injection, intraperitoneal injection, and the like. In other embodiments, the composition or formulation comprising the engineered WNT signaling agonist or antagonist, e.g. engineered RSPO protein is administered systemically, e.g., orally or intravenously. In one embodiment, the composition of formulation comprising the engineered RSPO protein is administered by infusion, e.g., continuous infusion over a period of time, e.g., 10 min, 20 min, 3 min, one hour, two hours, three hours, four hours, or greater.

[00143] In some embodiments of the invention, the compositions or formulations are administered on a short term basis, for example a single administration, or a series of administrations performed over, e.g. 1 , 2, 3 or more days, up to 1 or 2 weeks, in order to obtain a rapid, significant increase in activity. The size of the dose administered must be determined by a physician and will depend on a number of factors, such as the nature and gravity of the disease, the age and state of health of the patient and the patient's tolerance to the drug itself.

[00144] In certain methods of the present invention, an effective amount of a composition comprising a engineered WNT signaling agonist or antagonist, e.g. engineered RSPO protein is provided to cells, e.g. by contacting the cell with an effective amount of that composition to achieve a desired effect, e.g. to enhance Wnt signaling, proliferation, etc. In particular embodiments, the contacting occurs in vitro, ex vivo or in vivo. In particular embodiments, the cells are derived from or present within a subject in need or increased Wnt signaling.

[00145] In some methods of the invention, an effective amount of the subject composition is provided to enhance Wnt signaling in a cell. Biochemically speaking, an effective amount or effective dose of an engineered WNT signaling agonist or antagonist, e.g. engineered RSPO protein is an amount to increase or decrease Wnt signaling in a cell by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or by 100% relative to the signaling in the absence of the engineered WNT signaling agonist or antagonist, e.g. engineered RSPO protein. The amount of modulation of a cell’s activity can be determined by a number of ways known to one of ordinary skill in the art of wnt biology. [00146] In a clinical sense, an effective dose of a engineered WNT signaling agonist or antagonist, e.g. engineered RSPO protein composition is the dose that, when administered to a subject for a suitable period of time, e.g., at least about one week, and maybe about two weeks, or more, up to a period of about 4 weeks, 8 weeks, or longer, will evidence an alteration in the symptoms associated with lack of wnt signaling. In some embodiments, an effective dose may not only slow or halt the progression of the disease condition but may also induce the reversal of the condition. It will be understood by those of skill in the art that an initial dose may be administered for such periods of time, followed by maintenance doses, which, in some cases, will be at a reduced dosage.

[00147] The calculation of the effective amount or effective dose of engineered WNT signaling agonist or antagonist, e.g. engineered RSPO protein composition to be administered is within the skill of one of ordinary skill in the art, and will be routine to those persons skilled in the art. Needless to say, the final amount to be administered will be dependent upon the route of administration and upon the nature of the disorder or condition that is to be treated.

[00148] Cells suitable for use in the subject methods are cells that comprise one or more Fzd receptors. The cells may further express one of the two receptors for the RSPO proteins — ZNRF3 and RNF43. The cells to be contacted may be in vitro, that is, in culture, or they may be in vivo, that is, in a subject. Cells may be from/in any organism, but are preferably from a mammal, including humans, domestic and farm animals, and zoo, laboratory or pet animals, such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, rats, mice, frogs, zebrafish, fruit fly, worm, etc. Preferably, the mammal is human. Cells may be from any tissue. Cells may be frozen, or they may be fresh. They may be primary cells, or they may be cell lines. Often cells are primary cells used in vivo, or treated ex vivo prior to introduction into a recipient.

[00149] Cells in vitro may be contacted with a composition comprising a engineered WNT signaling agonist or antagonist, e.g. engineered RSPO protein by any of a number of well-known methods in the art. For example, the composition may be provided to the cells in the media in which the subject cells are being cultured. Nucleic acids encoding the engineered WNT signaling agonist or antagonist, e.g. engineered RSPO protein may be provided to the subject cells or to cells co cultured with the subject cells on vectors under conditions that are well known in the art for promoting their uptake, for example electroporation, calcium chloride transfection, and lipofection. Alternatively, nucleic acids encoding the engineered WNT signaling agonist or antagonist, e.g. engineered RSPO protein may be provided to the subject cells or to cells cocultured with the subject cells via a virus, i.e. the cells are contacted with viral particles comprising nucleic acids encoding the engineered WNT signaling agonist or antagonist, e.g. engineered RSPO protein. Retroviruses, for example, lentiviruses, are particularly suitable to the method of the invention, as they can be used to transfect non-dividing cells (see, for example, Uchida et al. (1998) P.N.A.S. 95(20):11939-44). Commonly used retroviral vectors are “defective”, i.e. unable to produce viral proteins required for productive infection. Rather, replication of the vector requires growth in a packaging cell line.

[00150] Likewise, cells in vivo may be contacted with the subject engineered WNT signaling agonist or antagonist, e.g. engineered RSPO protein compositions by any of a number of well-known methods in the art for the administration of peptides, small molecules, or nucleic acids to a subject. The engineered WNT signaling agonist or antagonist, e.g. engineered RSPO protein composition can be incorporated into a variety of formulations or pharmaceutical compositions, which in some embodiments will be formulated in the absence of detergents, liposomes, etc., as have been described for the formulation of full-length Wnt proteins.

[00151] WNT signaling is required for the healing of almost every tissue in the human body. For example, WNTs have been shown to activate adult, tissue-resident stem cells. These stem cells self-renew and divide, and in doing so give rise to progeny cells that mature into the tissue of interest. The molecules of the present invention provide WNT activity in a pharmacologically acceptable format, which can be tailored to the Fzd receptors present in the tissue of interest.

[00152] In some embodiments, the compounds of the invention are administered for use in treating diseased or damaged tissue, for use in tissue regeneration and for use in cell growth and proliferation, and/or for use in tissue engineering. In particular, the present invention provides a engineered WNT signaling agonist or antagonist, e.g. engineered RSPO protein, or a composition comprising one or more engineered WNT signaling agonist or antagonist, e.g. engineered RSPO proteins according to the invention for use in treating tissue loss or damage due to aging, trauma, infection, or other pathological conditions.

[00153] Conditions of interest for treatment with the compositions of the invention include, without limitation, a number of conditions in which regenerative cell growth is desired. Such conditions can include, for example, enhanced bone growth or regeneration, e.g. on bone regeneration, bone grafts, healing of bone fractures, etc.; treatment of alopecia; enhanced regeneration of sensory organs, e.g. treatment of hearing loss, treatment of macular degeneration, etc.; tooth growth, tooth regeneration, treatment of stroke, traumatic brain injury, Alzheimer’s disease, multiple sclerosis and other conditions affecting the blood brain barrier; treatment of oral mucositis, conditions where enhanced epidermal regeneration is desired, e.g. epidermal wound healing, treatment of diabetic foot ulcers, etc., enhanced growth of hematopoietic cells, e.g. enhancement of hematopoietic stem cell transplants from bone marrow, mobilized peripheral blood, treatment of immunodeficiencies, etc.; enhanced regeneration of liver cells, e.g. liver regeneration, treatment of cirrhosis, enhancement of liver transplantations, and the like.

[00154] Conditions in which enhanced bone growth is desired may include, without limitation, fractures, grafts, ingrowth around prosthetic devices, and the like. WNT proteins are critical regulators of bone turnover, and abundant scientific data supports a role for these proteins in promoting bone regeneration. In some embodiments, bone marrow cells are exposed to molecules of the invention, such that stem cells within that marrow become activated. These activated cells can remain in situ for the benefit of the individual, or can be used in bone grafting procedures.

[00155] In some embodiments, bone regeneration is enhanced by contacting a responsive cell population, e.g. bone marrow, bone progenitor cells, bone stem cells, etc. with an effective dose of a molecule of the invention. In some such embodiments, the contacting is performed in vivo. In other such embodiments, the contacting is performed ex vivo. The molecule may be localized to the site of action, e.g. by loading onto a matrix, which is optionally biodegradable, and optionally provides for a sustained release of the active agent. Matrix carriers include, without limitation, absorbable collagen sponges, ceramics, hydrogels, bone cements, and the like.

[00156] Compositions comprising one or more of the molecules of the invention can be used in the in vitro generation of skeletal tissue, such as from skeletogenic stem cells, as well as the in vivo treatment of skeletal tissue deficiencies. The subject compounds may be used to regulate the rate of chondrogenesis and/or osteogenesis. By “skeletal tissue deficiency”, it is meant a deficiency in bone or other skeletal connective tissue at any site where it is desired to restore the bone or connective tissue, no matter how the deficiency originated, e.g. whether as a result of surgical intervention, removal of tumor, ulceration, implant, fracture, or other traumatic or degenerative conditions. For example, the compositions of the present invention can be used as part of a regimen for restoring cartilage function to a connective tissue. Such methods are useful in, for example, the repair of defects or lesions in cartilage tissue which is the result of degenerative wear such as that which results in arthritis, as well as other mechanical derangements which may be caused by trauma to the tissue, such as a displacement of torn meniscus tissue, meniscectomy, a laxation of a joint by a torn ligament, malignment of joints, bone fracture, or by hereditary disease.

[00157] The compositions of the invention also find use in regeneration of tissues in the eye. Age- related macular degeneration (AMD) is characterized by progressively decreased central vision and visual acuity and remains a leading cause of vision loss and blindness in aged Americans. Currently, the standard of care for AMD is intravitreal vascular endothelial growth factor (VEGF) inhibitors. AMD is a multi-factorial disease involving numerous pathogenic factors, such as VEGF, platelet-derived growth factor (PDGF), intercellular adhesion molecule-1 (ICAM-1), tumor necrosis factor-alpha (TNF-oc), cyclooxygenase-2 (Cox-2), connective tissue growth factor (CTGF), and fibronectin (FN), that contribute to angiogenesis, inflammation, fibrosis and oxidative stress in AMD. Compositions of the present invention can be used, for example, in an infusion; in a matrix or other depot system; or other topical application to the eye for treatment of macular degeneration.

[00158] In other embodiments, the compositions of the invention are used in the regeneration of retinal tissue. In the adult mammalian retina, Muller glia dedifferentiate and produce retinal cells, including photoreceptors, for example after neurotoxic injury in vivo. However, the number of newly generated retinal neurons is very limited. However wnt signaling can promote proliferation of Muller glia-derived retinal progenitors and neural regeneration after damage or during degeneration. Compositions of the present invention can be used, for example, in an infusion; in a matrix or other depot system; or other topical application to the eye for enhancement of retinal regeneration.

[00159] Other sensory organs, such as the cells involved in hearing loss, also benefit from the compositions of the invention. In the inner ear, the auditory organ houses mechanosensitive hair cells required for translating sound vibration to electric impulses. The vestibular organs, comprised of the semicircular canals (SSCs), the utricle, and the saccule, also contain sensory hair cells in order to detect head position and motion. Both auditory and vestibular signals are in turn relayed centrally via the spiral and vestibular ganglion neurons, allowing for sound and balance perception. Numerous studies have characterized the multiple roles of the Wnt signaling during cochlear development and in promoting hair cell regeneration. Mature mammalian auditory and vestibular organs do not spontaneously mount a proliferative response after hair cell degeneration. However, active Wnt^-catenin signaling can promote proliferation of hair cells, where Lgr5-positive supporting cells can behave as hair cell progenitors. Lgr5-positive supporting cells can mitotically regenerate hair cells, where Wnt signaling augments both the mitotic response and the extent of hair cell regeneration. Wnt signaling can also induce ectopic hair cell formation. Compositions of the present invention can be used, for example, in an infusion; in a matrix or other depot system; or other topical application to the ear for enhancement of auditory regeneration.

[00160] Periodontal diseases are a leading cause of tooth loss and are linked to multiple systemic conditions. Reconstruction of the support and function of affected tooth-supporting tissues represents an important therapeutic endpoint for periodontal regenerative medicine. An improved understanding of periodontal biology coupled with current advances in scaffolding matrices provides treatments that provide the compositions of the invention, optionally in combination with delivery of regenerative cells for the predictable tissue regeneration of supporting alveolar bone, periodontal ligament, and cementum. In some embodiments, tooth or underlying bone regeneration is enhanced by contacting a responsive cell population with an effective dose of a molecule of the invention. In some such embodiments, the contacting is performed in vivo. In other such embodiments, the contacting is performed ex vivo, with subsequent implantation of the activated stem or progenitor cells. The molecule may be localized to the site of action, e.g. by loading onto a matrix, which is optionally biodegradable, and optionally provides for a sustained release of the active agent. Matrix carriers include, without limitation, absorbable collagen sponges, ceramics, hydrogels, bone cements, and the like.

[00161 ] Hair loss is a common problem with multiple causes that range from hormone sensitivity to autoimmunity. Androgenetic alopecia, often called male pattern baldness, is the most common form of hair loss in men, which affects as many as 50% of men as they age. In androgenetic alopecia, hair loss is caused by a sensitivity of hair follicles in the top of the scalp to the androgen 5a-dihydrotestosterone (DHT). DHT causes those follicles to undergo a progressive miniaturization to the point where they no longer produce a clinically apparent hair shaft. The cells affected by DHT are the dermal papilla cells, which cease growing and lose their ability to direct hair growth. Epidermal Wnt signaling is critical for adult hair follicle regeneration. In some embodiments, hair follicle regeneration is enhanced by contacting a responsive cell population with an effective dose of a molecule of the invention. In some such embodiments, the contacting is performed in vivo. In other such embodiments, the contacting is performed ex vivo, with subsequent implantation of the activated stem or progenitor cells, e.g. follicular cells. The molecule may be localized to the site of action, e.g. topical lotions, gels, creams and the like.

[00162] Various epidermal conditions benefit from treatment with the compounds of the invention. Mucositis occurs when there is a break down of the rapidly divided epithelial cells lining the gastro intestinal tract, leaving the mucosal tissue open to ulceration and infection. Mucosal tissue, also known as mucosa or the mucous membrane, lines all body passages that communicate with the air, such as the respiratory and alimentary tracts, and have cells and associated glands that secrete mucus. The part of this lining that covers the mouth, called the oral mucosa, is one of the most sensitive parts of the body and is particularly vulnerable to chemotherapy and radiation. The oral cavity is the most common location for mucositis. Oral mucositis is probably the most common, debilitating complication of cancer treatments, particularly chemotherapy and radiation. It can lead to several problems, including pain, nutritional problems as a result of inability to eat, and increased risk of infection due to open sores in the mucosa. It has a significant effect on the patient’s quality of life and can be dose-limiting (i.e., requiring a reduction in subsequent chemotherapy doses). Other epidermal conditions include epidermal wound healing, diabetic foot ulcers, and the like. Molecules of the invention can find use in such conditions, where regenerative cells are contacted with compounds of the invention. Contacting can be, for example, topical, including intradermal, subdermal, in a gel, lotion, cream etc. applied at targeted site, etc.

[00163] The liver has a capacity for regeneration, which can be enhanced by wnt signaling. Adult hepatic progenitor (oval) cells are facultative stem cells in liver. Active Wnt^-catenin signaling occurs preferentially within the oval cell population, and wnt signaling promotes expansion of the oval cell population in a regenerated liver. Methods for regeneration of liver tissue benefits from administration of the compounds of the invention, which can be systemic or localized, e.g. by injection into the liver tissue, by injection into veins leading into the liver, by implantation of a sustained release formulation, and the like. Liver damage can be associated with infection, alcohol abuse, etc.

[00164] Stroke, traumatic brain injury, Alzheimer's, multiple sclerosis and other conditions affecting the blood-brain barrier. Angiogenesis is critical to ensure the supply of oxygen and nutrients to many tissues throughout the body, and is especially important for the CNS as the neural tissue is extremely sensitive to hypoxia and ischemia. The blood vessels in the brain form a specialized structure, termed the blood brain barrier (BBB), which limits the flow of molecules and ions from the blood to the brain. This BBB is critical to maintain brain homeostasis and protect the CNS from toxins and pathogens. CNS endothelial cells which form the BBB differ from endothelial cells in non-neural tissue, in that they are highly polarized cells held together by tight junctions that limit the paracellular flow of molecules and ions. In addition, CNS endothelial cells also express specific transporters, both to provide selective transport of essential nutrients across the BBB into the brain and to efflux potential toxins from the brain. Wnt signaling specifically regulates CNS vessel formation and/or function. Conditions in which the BBB is compromised can benefit from administration of the compounds of the invention, e.g. by direct injection, intrathecal administration, implantation of sustained release formulations, and the like.

[00165] The patient may be any animal (e.g., a mammal), including, but not limited to, humans, non-human primates, rodents, and the like. Typically, the patient is human. The methods of treatment and medical uses of the engineered RSPO proteins of the invention or compounds or compositions comprising engineered RSPO proteins of the invention promote tissue regeneration. The term "tissue" refers to part of an organism consisting of a cell or an aggregate of cells, optionally having a similar structure, function and/or origin. Examples of tissues include but are not limited to: epithelial tissues, such as skin tissue, stomach lining, pancreatic lining, liver; connective tissues, such as inner layers of skin, tendons, ligaments, cartilage, bone, fat, hair, blood; muscle tissues; and nerve tissues, such as glial cells and neurons. The loss or damage can be anything which causes the cell number to diminish. For example, an accident, an autoimmune disorder, a therapeutic side-effect or a disease state could constitute trauma. Specific examples of conditions which may cause cell number to diminish include, but are not limited to: radiation/chemotherapy, mucositis, IBD, short bowel syndrome, hereditary bowel disorders, celiac disease, metabolic diseases, hereditary syndromes, (viral) infections (hepB/C), toxic states, alcoholic liver, fatty liver, cirrhosis, infections, pernicious anemia, ulceration, diabetes, diabetic foot ulcers (e.g., refractory diabetic foot ulcers), destruction of islet cells, loss of bone mass (osteoporosis), loss of functional skin, loss of hair, loss of functional lung tissue, loss of kidney tissue (for instance acute tubulus necrosis), loss of sensory cells in the inner ear. Tissue regeneration increases the cell number within the tissue and preferably enables connections between cells of the tissue to be re-established, and more preferably the functionality of the tissue to be regained.

[00166] Other conditions that may be treated with the engineered WNT signaling agonist or antagonist, e.g. engineered RSPO proteins or compositions comprising one or more engineered WNT signaling agonist or antagonist, e.g. engineered RSPO proteins of the invention include but are not limited to: joint disorders, osteoporosis and related bone diseases, baldness, graft-versus- host disease.

[00167] Engineered WNT signaling agonist or antagonist, e.g. engineered RSPO proteins or compositions comprising one or more engineered WNT signaling agonist or antagonist, e.g. engineered RSPO proteins of the invention may also be used for wound healing and generation of smooth muscle tissues in many organs (e.g. airways, large arteries, uterus).

[00168] In some embodiments, the invention provides methods of treatment and medical uses, as described previously, wherein two or more engineered WNT signaling agonist or antagonist, e.g. engineered RSPO proteins of the invention or compounds or compositions comprising engineered WNT signaling agonist or antagonist, e.g. engineered RSPO proteins of the invention, are administered to an animal or patient simultaneously, sequentially, or separately. The engineered WNT signaling agonist or antagonist, e.g. engineered RSPO protein (s) may also be administered simultaneously, sequentially, or separately with a wnt protein or wnt surrogate, etc. [00169] In some embodiments, the invention provides methods of treatment and medical uses, as described previously, wherein one or more engineered WNT signaling agonist or antagonist, e.g. engineered RSPO proteins of the invention or compounds or compositions comprising engineered WNT signaling agonist or antagonist, e.g. engineered RSPO proteins of the invention, is administered to an animal or patient in combination with one or more further compound or drug, and wherein said engineered WNT signaling agonist or antagonist, e.g. engineered RSPO proteins of the invention or compounds or compositions comprising engineered WNT signaling agonist or antagonist, e.g. engineered RSPO proteins of the invention and said further compound or drug are administered simultaneously, sequentially, or separately.

[00170] The engineered WNT signaling agonist or antagonist, e.g. engineered RSPO proteins of the invention also have widespread applications in non-therapeutic methods, for example in vitro research methods.

[00171] The invention provides a method for tissue regeneration of damaged tissue, such as the tissues discussed in the section of medical uses above, comprising administering a protein of the invention. The protein may be administered directly to the cells in vivo, administered to the patient orally, intravenously, or by other methods known in the art, or administered to ex vivo cells. In some embodiments where the protein of the invention is administered to ex vivo cells, these cells may be transplanted into a patient before, after or during administration of the agonist of the invention.

[00172] The invention also provides a method for enhancing the proliferation of cells comprising supplying the cells with a protein of the invention. These methods may be carried out in vivo, ex vivo or in vitro Wnt signaling is a key component of stem cell culture. For example, the stem cell culture media as described in WO2010/090513, WO2012/014076, Sato et al., 2011 (GASTROENTEROLOGY 2011 ; 141 :1762-1772) and Sato et al., 2009 (Nature 459, 262-5). The proteins of the invention are suitable alternatives to natuve Rspondin for use in these stem cell culture media.

[00173] Accordingly, in one embodiment, the invention provides a method for enhancing the proliferation of stem cells comprising supplying stem cells with engineered RSPO proteins of the invention. In one embodiment, the invention provides a cell culture medium comprising one or more proteins of the invention. In some embodiments, the cell culture medium may be any cell culture medium already known in the art that normally comprises Wnt or Rspondin, but wherein the Wnt or Rspondin is replaced (wholly or partially) or supplemented by the proteins of the invention. For example, the culture medium may be as described in as described in WO2010/090513, WO2012/014076, Sato et al., 2011 (GASTROENTEROLOGY 2011 ; 141 :1762- 1772) and Sato et al., 2009 (Nature 459, 262-5), which are hereby incorporated by reference in their entirety.

[00174] Stem cell culture media often comprise additional growth factors. This method may thus additionally comprise supplying the stem cells with a growth factor. Growth factors commonly used in cell culture medium include epidermal growth factor (EGF, (Peprotech), Transforming Growth Factor-alpha (TGF-alpha, Peprotech), basic Fibroblast Growth Factor (bFGF, Peprotech), brain-derived neurotrophic factor (BDNF, R&D Systems), Human Growth Factor (HGF) and Keratinocyte Growth Factor (KGF, Peprotech, also known as FGF7). EGF is a potent mitogenic factor for a variety of cultured ectodermal and mesodermal cells and has a profound effect on the differentiation of specific cells in vivo and in vitro and of some fibroblasts in cell culture. The EGF precursor exists as a membrane-bound molecule which is proteolytically cleaved to generate the 53-amino acid peptide hormone that stimulates cells. EGF or other mitogenic growth factors may thus be supplied to the stem cells. During culturing of stem cells, the mitogenic growth factor may be added to the culture medium every second day, while the culture medium is refreshed preferably every fourth day. In general, a mitogenic factor is selected from the groups consisting of: i) EGF, TGF-.alpha. and KGF, ii) EGF, TGF-.alpha. and FGF7; iii) EGF, TGF-.alpha. and FGF; iv) EGF and KGF; v) EGF and FGF7; vi) EGF and a FGF; vii) TGF-oc and KGF; viii) TGF-.alpha. and FGF7; ix) or from TGF a and a FGF.

[00175] These methods of enhancing proliferation of stem cells can be used to grow new organoids and tissues from stem cells, as for example described in WO2010/090513 WO2012/014076, Sato et al., 2011 (GASTROENTEROLOGY 2011 ; 141 :1762-1772) and Sato et al., 2009 (Nature 459, 262-5).

[00176] A number of clinically relevant conditions are characterized by an inability to regenerate tissues, where upregulation of wnt signaling is desirable.

[00177] In some embodiments the engineered RSPO protein is used to enhance stem cell regeneration. Stem cells of interest include muscle satellite cells; hematopoietic stem cells and progenitor cells derived therefrom (U.S. Pat. No. 5,061 ,620); neural stem cells (see Morrison et al. (1999) Cell 96: 737-749); embryonic stem cells; mesenchymal stem cells; mesodermal stem cells; liver stem cells, etc.

[00178] The engineered RSPO proteins find use in enhancing bone healing. In many clinical situations, the bone healing condition are less ideal due to decreased activity of bone forming cells, e.g. within aged people, following injury, in osteogenesis imperfecta, etc. A variety of bone and cartilage disorders affect aged individuals. Such tissues are normally regenerated by mesenchymal stem cells. Included in such conditions is osteoarthritis. Osteoarthritis occurs in the joints of the body as an expression of “wear-and-tear”. Thus athletes or overweight individuals develop osteoarthritis in large joints (knees, shoulders, hips) due to loss or damage of cartilage. This hard, smooth cushion that covers the bony joint surfaces is composed primarily of collagen, the structural protein in the body, which forms a mesh to give support and flexibility to the joint. When cartilage is damaged and lost, the bone surfaces undergo abnormal changes. There is some inflammation, but not as much as is seen with other types of arthritis. Nevertheless, osteoarthritis is responsible for considerable pain and disability in older persons.

[00179] In methods of accelerating bone repair, a pharmaceutical wnt composition of the present invention is administered to a patient suffering from damage to a bone, e.g. following an injury. The formulation is preferably administered at or near the site of injury, following damage requiring bone regeneration. The wnt formulation is preferably administered for a short period of time, and in a dose that is effective to increase the number of bone progenitor cells present at the site of injury. In some embodiments the wnt is administered within about two days, usually within about 1 day of injury, and is provided for not more than about two weeks, not more than about one week, not more than about 5 days, not more than about 3 days, etc.

[00180] In an alternative method, patient suffering from damage to a bone is provided with a composition comprising bone marrow cells, e.g. a composition including mesenchymal stem cells, bone marrow cells capable of differentiating into osteoblasts; etc. The bone marrow cells may be treated ex vivo with a pharmaceutical composition comprising a wnt protein or proteins in a dose sufficient to enhance regeneration; or the cell composition may be administered to a patient in conjunction with a wnt formulation of the invention.

[00181] In yet other embodiments, methods of the present invention include administering to a subject in need of treatment a therapeutically effective amount or an effective dose of a therapeutic entity {e.g., inhibitor agent) of the present invention. In some embodiments, effective doses of the therapeutic entity of the present invention, e.g. for the treatment of cancer vary depending upon many different factors, including means of administration, target site, physiological state of the patient, whether the patient is human or an animal, other medications administered, and whether treatment is prophylactic or therapeutic. Usually, the patient is a human but nonhuman mammals including transgenic mammals can also be treated. Treatment dosages need to be titrated to optimize safety and efficacy. [00182] The types of cancer that can be treated using the subject methods of the present invention include but are not limited to adrenal cortical cancer, anal cancer, aplastic anemia, bile duct cancer, bladder cancer, bone cancer, bone metastasis, brain cancers, central nervous system (CNS) cancers, peripheral nervous system (PNS) cancers, breast cancer, cervical cancer, childhood Non-Hodgkin's lymphoma, colon and rectum cancer, endometrial cancer, esophagus cancer, Ewing's family of tumors (e.g. Ewing's sarcoma), eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, gestational trophoblastic disease, hairy cell leukemia, Hodgkin's lymphoma, Kaposi's sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, acute lymphocytic leukemia, acute myeloid leukemia, children's leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, liver cancer, lung cancer, lung carcinoid tumors, Non-Hodgkin's lymphoma, male breast cancer, malignant mesothelioma, multiple myeloma, myelodysplastic syndrome, myeloproliferative disorders, nasal cavity and paranasal cancer, nasopharyngeal cancer, neuroblastoma, oral cavity and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumor, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcomas, melanoma skin cancer, non-melanoma skin cancers, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, uterine cancer (e.g. uterine sarcoma), transitional cell carcinoma, vaginal cancer, vulvar cancer, mesothelioma, squamous cell or epidermoid carcinoma, bronchial adenoma, choriocarinoma, head and neck cancers, teratocarcinoma, or Waldenstrom's macroglobulinemia.

[00183] Dosage and frequency may vary depending on the half-life of the agent in the patient. It will be understood by one of skill in the art that such guidelines will be adjusted for the molecular weight of the active agent, the clearance from the blood, the mode of administration, and other pharmacokinetic parameters. The dosage may also be varied for localized administration, e.g. intranasal, inhalation, etc., or for systemic administration, e.g. i.m., i.p., i.v., oral, and the like.

[00184] In some embodiments, the dosage may range from about 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the host body weight. For example dosages can be 1 mg/kg body weight or 10 mg/kg body weight or within the range of 1-10 mg/kg. An exemplary treatment regime entails administration once per every two weeks or once a month or once every 3 to 6 months. Therapeutic entities of the present invention are usually administered on multiple occasions. Intervals between single dosages can be weekly, monthly or yearly. Intervals can also be irregular as indicated by measuring blood levels of the therapeutic entity in the patient. Alternatively, therapeutic entities of the present invention can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the polypeptide in the patient. [00185] Methods of the present invention include treating, reducing or preventing tumor, growth, metastasis or tumor invasion of carcinomas, e.g. colorectal carcinoma.

[00186] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

Example 1 .

R-spondins (RSPOs) amplify WNT signaling during development and regenerative responses.

[00187] R-spondins (RSPOs) amplify WNT signaling during development and regenerative responses. We previously demonstrated that RSPOs 2 and 3 potentiate WNT^-catenin signaling in cells lacking leucine-rich repeat-containing G-protein coupled receptors (LGRs) 4, 5 and 6 (Lebensohn and Rohatgi, 2018). We now show that heparan sulfate proteoglycans (HSPGs) act as alternative co-receptors for RSP03 using a combination of ligand mutagenesis and ligand engineering. Mutations in RSP03 residues predicted to contact HSPGs impair it’s signaling capacity. Conversely, the HSPG-binding domains of RSP03 can be entirely replaced with a heterologous antibody that recognizes heparan sulfate (HS) chains attached to multiple HSPGs without diminishing WNT-potentiating activity in cultured cells and intestinal organoids. A genome wide screen for mediators of RSP03 signaling in cells lacking LGRs 4, 5 and 6 failed to reveal other receptors. We find that HSPGs are RSPO co-receptors that potentiate WNT signaling in the presence and absence of LGRs, implicating them in RSPO-regulated developmental and regenerative processes.

[00188] It was found that RSP02 and RSP03 can potentiate WNT signaling in the absence of LGRs 4, 5 and 6, previously considered obligate high-affinity receptors for RSPOs. RSP02 and RSP03 can still potentiate WNT signaling in LGR4/5/6^KO human haploid cells. Similarly, RSP02 can potentiate WNT signaling in 293T cells lacking LGR4. This “LGR-independent” mode of signaling is particularly relevant to RSPO-related birth defects because it appears to be the dominant mode of signaling during development of the limbs, lungs and cardiovascular system. Mice carrying loss-of-function mutations in LGRs 4, 5 and 6 ( Lgr4/5/& ^/_) do not show either the limb truncations, lung hypoplasia or craniofacial abnormalities observed in humans and mouse embryos lacking Rspo2, or the vascular and associated placental defects observed in mouse embryos lacking Rspo3.

[00189] These results raise a pressing question: are there alternative receptors that transduce RSPO signals in the absence of LGRs? RSPOs contain tandem furin-like repeats, FU1 and FU2, that interact with ZNRF3/RNF43 and LGRs, respectively. They also contain a thrombospondin type 1 (TSP) domain and a basic region (BR) (we refer to them together as the TSP/BR domain) that interacts with heparin and heparan sulfate proteoglycans (HSPGs). The TSP/BR domain has been considered dispensable to potentiate nnNT/b-catenin signaling, but we previously demonstrated that it is essential in the absence of LGRs (Lebensohn and Rohatgi, 2018). We also proposed that glypicans (GPCs), syndecans (SDCs) or other HSPGs may mediate LGR- independent signaling through interactions between their heparan sulfate (HS) chains and the TSP/BR domain of certain RSPOs.

[00190] In this study, we introduced structure-based mutations into the TSP/BR domain of RSP03, engineered RSP03 ligands fused to a non-native HSPG-binding single-chain variable fragment (scFv) and disrupted different classes of HSPGs to demonstrate that the interaction between RSP03 and the HS chains of HSPGs is necessary and sufficient to transduce signals in cells lacking LGRs. We find that the interaction with HSPGs also contributes substantially to LGR- dependent signaling in cultured cells and in small intestinal organoids, showing that HSPGs play an important role in promoting RSP03 signaling in most contexts.

Results

[00191] The interaction between the TSP/BR domain of RSP03 and HSPGs is necessary to potentiate WNT signaling. We previously reported that disruption of EXTL3, the gene encoding a glycosyltransferase required for the biosynthesis of all HS chains, reduces LGR-independent RSP03 signaling by -80%. However, depleting either all SDCs (1 -4) or PIGL, an enzyme required for the biogenesis of all GPCs, failed to reduce LGR-independent signaling by RSP03. These observations suggested functional redundancy between GPCs, SDCs and perhaps other HSPGs in mediating LGR-independent signaling, making loss-of-function genetic experiments challenging. Our previous experiments also left open the possibility that reducing the levels of all HSPGs by depleting EXTL3 may change the cell-surface abundance of a different co-receptor for RSPOs that mediates LGR-independent signaling. [00192] We previously showed that deletion of the complete TSP/BR domain of RSP03 eliminated its ability to potentiate WNT signaling in LGR4/5/6^KO cells. Deleting the entire TSP/BR domain is a drastic alteration that prevents binding to heparin, but could also disrupt interactions with other potential binding partners. To design more precise mutations that selectively disrupt the putative HS-binding surface of RSP03, we created a structural model of the RSP03 TSP/BR domain based on its homology to 26 of the most similar proteins in the Protein Data Bank (Figure 1A). Analogous to other TSP domains, the model shows an elongated three-stranded fold stabilized by disulfide bonds at both ends. Two positively charged grooves (Site-1 and Site-2 in Figure 1 A) on one face of the model suggested potential sites of interaction with negatively charged FIS polymers.

[00193] Based on this model, we mutated six (out of sixteen) lysine (K) and four (out of nine) arginine (R) residues in these grooves to charge-reversing glutamic acid (E) residues, thereby altering the electrostatic surface of the TSP/BR domain so that it should not be able to interact properly with FIS chains. We refer to this mutant protein as RSP03 TSP/BR (K/R E) (Figure 1 A and B). We produced a tagged version of RSP03 TSP/BR (K/R E) and purified it under physiological conditions using a one-step affinity purification strategy described in our previous Elite paper. We have demonstrated that tagged RSPO proteins produced and purified in this manner preserve the signaling properties of their untagged counterparts. A coomassie blue- stained gel of purified wild type (WT) RSP03, RSP03 TSP/BR (K/R E) and RSP03 ATSP/BR, a control construct lacking the TSP/BR domain entirely (Figure 1 B), is shown in Figure 1C.

[00194] We tested the capacity of these recombinant RSP03 proteins to potentiate signaling induced by a low dose of WNT3A conditioned medium (CM) in WT HAP1 -7TGP or in LGR4/5/6^KO cells. HAP1-7TGP is a human haploid cell line harboring a fluorescent transcriptional reporter for WNT^-catenin signaling that we have previously established as a valid and genetically tractable system to study both WNT- and RSPO-dependent signaling. LGR4/5/6^KO cells are a clonal derivative of HAP1-7TGP in which LGRs 4, 5 and 6 were disrupted by CRISPR/Cas9-mediated genome editing. In LGR4/5/6^KO cells, the signaling response to low levels of WNT3A can be potentiated robustly by RSP02 and RSP03, but not by RSP01 or RSP04, making them a convenient system to study LGR-independent RSPO signaling.

[00195] Deleting the TSP/BR domain of RSP03 reduced its potency by ~80-fold in WT HAP1 -7TGP cells (Figure 1 D), and completely abolishes signaling in LGR4/5/6^KO cells (Figure 1 E). Similarly, the K/R E mutations we introduced into the TSP/BR domain of RSP03 reduced its potency by

~ 13-fold in WT HAP1-7TGP cells (Figure 1 D) and almost completely abolished its ability to potentiate WNT signaling in LGR4/5/6^KO cells (Figure 1 E). Our data supports the model that interactions between basic K and R residues in the TSP/BR domain of RSP03 and the HS chains of HSPGs contribute substantially to potentiating WNT signaling in the presence of LGRs. In the absence of LGRs, the RSP03-HSPG interaction becomes essential to potentiate WNT signaling.

[00196] The TSP/BR domain of RSP03 can be replaced by a non-native HS-binding domain without compromising activity. While the positively charged grooves we mutated in the TSP/BR domain (Figure 1A) are known to interact with HS chains in other TSP-containing proteins, it remained possible that the K/R E mutations disrupted the interaction of RSP03 with a different, unidentified receptor. To resolve this issue, we sought to provide the HSPG-binding functionality of the TSP/BR domain through an entirely different protein. We engineered a synthetic RSP03 protein in which we replaced the entire TSP/BR domain with HS20, an scFv antibody selected by phage display to bind to the HS chains of GPC3 (Figure 2A). We refer to this protein as RSP03 ATSP/BR HS20. Due to the presence of the HS20 scFv in the RSP03 ATSP/BR HS20 fusion protein, we excluded the Fc tag that we had used in other RSP03 constructs to promote their solubility. We retained an N-terminal HA tag and a small C-terminal 1 D4 tag for affinity purification of the fusion protein (Figure 2A and B).

[00197] Replacing the TSP/BR domain with HS20 in RSP03 ATSP/BR HS20 rescued the ability of RSP03 to signal both in the presence and absence of LGRs (Figure 2C and D). Importantly, mutations in the antigen-binding third complementarity-determining region (CDR3) of HS20 (RSP03 ATSP/BR HS20 (GS) and RSP03 ATSP/BR HS20 (A) in Figure 2A and B) entirely eliminated the WNT-potentiating activity of RSP03 ATSP/BR HS20, both in the presence and absence of LGRs (Figure 2E and F). The fact that HS20, a structurally unrelated protein specifically selected for HS-binding activity, can functionally substitute for the TSP/BR domain of RSP03 provides strong support for our model that interactions with HSPGs are required to potentiate WNT signaling in cells lacking LGRs, and also contribute significantly in cells containing LGRs.

[00198] What are the relative contributions of ZNRF3/RNF43, LGRs and HSPGs to signaling by RSP03 ATSP/BR HS20? In order to measure the contribution of FU1 domain interactions with ZNRF3/RNF43 and FU2 domain interactions with LGRs, we individually mutated the FU1 and FU2 domains of RSP03 ATSP/BR HS20. Point mutations (R67A/Q72A) in the FU1 domain of RSP03 ATSP/BR HS20 (RSP03 ATSP/BR HS20 (R67A/Q72A) in Figure 2A and B) known to weaken the interaction with ZNRF3/RNF43, reduced signaling potency by 10-fold in WT HAP1- 7TGP cells (Figure 2G) and abolished signaling in LGR4/5/6^KO cells (Figure 2H). Point mutations (F106E/F110E) in the FU2 domain of RSP03 ATSP/BR HS20 (RSP03 ATSP/BR HS20 (F106E/F110E) in Figure 2A and B) that weaken interactions with LGRs reduced signaling efficacy by 80% and potency by 3-fold in WT HAP1-7TGP cells (Figure 2G). As expected, disrupting the LGR-binding FU2 domain in RSP03 ATSP/BR HS20 (F106E/F110E) did not reduce signaling in LGR4/5/6^KO cells (Figure 2H).

[00199] We conclude that interactions with ZNRF3/RNF43, LGRs and HSPGs all contribute to the potentiation of WNT signaling by RSP03 ATSP/BR HS20 in the presence of LGRs. Interactions with ZNRF3/RNF43 and HSPGs are required for signaling in the absence of LGRs. The simplest model to explain these results is that RSP03 can bind simultaneously to LGR and HSPG co receptors, which together present the ligand to ZNRF3/RNF43, the key effectors that transmit the signal across the membrane. Without LGRs, RSP03 is presented by HSPGs alone, explaining the significantly lower potency of both WT RSP03 and RSP03 ATSP/BR HS20 in LGR4/5/6^KO compared to WT HAP1-7TGP cells (Figures 1 D, 1 E, 2C and 2D). Binding assays and structural studies with purified proteins will be required to test for the formation of a ternary RSP03-LGR- HSPG complex.

[00200] Signaling by RSP03 causes internalization and degradation of RNF43 in the presence or absence of LGRs. We tested whether the mechanism whereby RSP03 ATSP/BR HS20 potentiates WNT responses is equivalent to that of WT RSP03. LGR-dependent RSPO signaling triggers internalization and lysosomal degradation of the E3 ubiquitin ligases ZNRF3 and RNF43, promoting the stabilization of WNT receptors. Whether LGR-independent signaling by RSP03 proceeds through a similar mechanism is not known. We used a surface biotinylation assay to measure RSP03-mediated internalization and degradation of RNF43-Flag in WT HAP1-7TGP and in LGR4/5/6^KO cells. Treatment of either WT HAP1-7TGP or LGR4/5/6^KO cells with WT RSP03 led to the depletion of RNF43-Flag from the cell surface and a reduction in its total cellular level (Figure 3A and B), suggesting similar signaling mechanisms for LGR-dependent and LGR- independent potentiation of WNT signaling. These results are consistent with our previous mutational analysis demonstrating that the ZNRF3/RNF43-interacting FU1 domain is essential for RSP03 signaling in cells lacking LGRs.

[00201] Having established the mechanism of LGR-independent signaling by RSP03, we tested whether RSP03 ATSP/BR HS20 potentiates WNT responses through an equivalent mechanism. Deletion of the TSP/BR domain of RSP03 (RSP03 ATSP/BR in Figure 3A and B) prevented the internalization and degradation of RNF43-Flag in both WT HAP1-7TGP and LGR4/5/6^KO cells, consistent with the reduced capacity of RSP03 ATSP/BR to promote LGR-dependent (Figure 1 D) and LGR-independent (Figure 1 E) signaling. In contrast, as with WT RSP03, addition of RSP03 ATSP/BR HS20 to either WT HAP1-7TGP (Figure 3A) or LGR4/5/6^KO (Figure 3B) cells resulted in the depletion of RNF43-Flag from the cell surface and a reduction in its total cellular level. The effects of RSP03 ATSP/BR HS20 were dependent on the interaction between HS20 and HS chains, since the RSP03 ATSP/BR HS20 (GS) mutant, which carries mutations in the HS-binding CDR3 loop of HS20, failed to promote internalization and degradation of RNF43-Flag (Figure 3A and B).

[00202] In summary, replacing the HS-binding TSP/BR domain of RSP03 with HS20, an unrelated protein that binds FIS chains, can rescue signaling in the absence of LGRs and substantially enhance signaling in their presence by promoting the internalization and degradation of RNF43. We propose that HSPGs represent the third major class of RSPO co-receptors, in addition to LGRs and ZNRF3/RNF43.

[00203] HSPG specificity of RSP03 signaling. Are specific HSPGs required for RSPO signaling? Cell-surface HSPGs comprise six glypicans (GPC1-6), which are anchored to the membrane by a glycophosphatidylinositol (GPI) linkage, and four syndecans (SDC1-4), which are single pass transmembrane proteins. Evidence has been presented that RSP03 can use the HS chains on multiple HSPGs to potentiate WNT signaling. However, HS20 is an scFv selected to bind to GPC3, raising the possibility that the synthetic RSP03 ATSP/BR HS20 ligand may have a narrower HSPG selectivity. In a purified binding assay, HS20 bound equally well to GPC3 and GPC4 (Figure 4A), the two GPCs expressed most highly in HAP1 cells: RPKM values for GPC3 and GPC4 were 157 and 220, respectively). HS20 failed to bind to mutant GPC3 and GPC4 proteins that cannot be modified by HS chains (GPC3AHS and GPC4AHS in Figure 4A). These results support a prior conclusion that HS20 recognizes the HS side-chain structure shared by multiple HSPGs, rather than binding to the protein core of a specific GPC.

[00204] To test the specificity of RSP03 ATSP/BR HS20 more thoroughly in cells, we used CRISPR/Cas9 methods to generate a series of clonal cell lines (see Materials and methods) with increasingly severe deficits in their HSPG composition: 1 . GPC3^K0, 2. GPC4^K0, 3. PIGL^K0 (lacking an enzyme required for GPI anchor biosynthesis and hence for the cell surface expression of all GPCs), 4. SDC1/2/3/4^KO (lacking all four SDCs) and 5. EXTL3^K0 (lacking a glycosyltransferase required for the synthesis of HS chains attached to all HSPGs, including all GPCs and SDCs, but not for the synthesis of other glycosaminoglycans or proteoglycans). Each of these mutations were introduced into LGR4/5/6^KO cells, in which the fusion of HS20 to RSP03 ATSP/BR is required for signaling (Figure 2D), and hence the effect of eliminating different HSPGs can be measured most accurately. We also tested the specificity of WT RSP03, containing the native HSPG-binding TSP/BR domain, in these cell lines.

[00205] Disrupting GPC3 in LGR4/5/6^KO cells had no significant effect on signaling induced by either WT RSP03 or RSP03 ATSP/BR HS20 (Figure 4B and C), consistent with the finding that HS20 can bind with equivalent affinity to the HS chains of other GPCs such as GPC4 (Figure 4A). Disrupting GPC4 or PIGL modestly reduced the potency (i.e. increased the EC₅o) of both WT RSP03 and RSP03 ATSP/BR HS20 (Figure 4B and C). Combined disruption of all SDCs - SDC1 , SDC2, SDC3 and SDC4 - surprisingly resulted in an increase in the potency (ie. lower ECso) of both WT RSP03 and RSP03 ATSP/BR HS20 (Figure 4B and C). This increase in potency may be related to the previous observation that some SDCs inhibit WNT^-catenin signaling through regulation of LRP6 and RSP03. Finally, disrupting EXTL3, predicted to globally block FIS chain biosynthesis, impaired signaling by both proteins to the greatest extent (Figure 4B and C).

[00206] Overall, the effects of disrupting different HSPGs led to a qualitatively similar effect on signaling by WT RSP03 and RSP03 ATSP/BR HS20, with GPC3 disruption having little effect, GPC4 or PIGL disruption having an intermediate effect and EXTL3 disruption having the greatest effect. These results are most consistent with the model that RSP03 ATSP/BR HS20, like WT RSP03, can signal in a redundant manner via either GPCs, SDCs or another HSPG by engaging their FIS chains rather than their protein cores.

[00207] HSPGs potentiate RSP03-supported small intestinal organoid growth. Given the substantial contribution made by the interaction between the TSP/BR domain (or HS20) and HSPGs to LGR-dependent signaling in haploid cells (Figures 1 D and 2C), it was important to test whether this interaction also mediates RSPO signaling in a more physiological context. The growth of mouse small intestinal organoids is strictly dependent on exogenously-supplied RSPOs (Sato et al., 2009) and on the expression of LGRs. In addition, studies in mice have shown that depletion of HS chains in the intestinal epithelium compromise WNT-dependent crypt renewal and regeneration following radiation-induced injury. We used this organoid culture system in combination with our mutant and engineered RSP03 ligands to test the importance of RSPO- HSPG interactions for supporting the crypt-villus architecture of the intestine. WT RSP03 promoted the growth of intestinal organoids with multiple budded structures at concentrations down to 0.5 nM (Figure 5A and B). The bud sites represent crypt domains, since they are enriched in markers of proliferating cells (Ki67) and of Paneth cells (lysozyme) (Figure 5C). RSP03 ATSP/BR was markedly less potent in this assay, enabling only a few organoids to grow at a concentration of 10 nM (Figure 5A and B). While the FU1 and FU2 domains present in RSP03 ATSP/BR were sufficient to promote organoid growth, our titration experiment revealed that abolishing HSPG interactions by deleting the TSP/BR domain sharply reduced ligand potency.

[00208] To determine if the positive effect of the TSP/BR domain in promoting organoid growth is due to its interactions with HSPGs, we tested the organoid-supporting properties of RSP03 ATSP/BR HS20. Not only was RSP03 ATSP/BR HS20 able to fully support organoid growth, with budding of normal crypt domains, but it was more potent than WT RSP03, supporting some organoid growth even at concentrations as low as 0.1 nM (Figure 5A and B). Organoids grown in the presence of RSP03 ATSP/BR HS20 were indistinguishable from those grown in the presence of WT RSP03, with crypt domains containing proliferating cells and Paneth cells (Figure 5C). The capacity of RSP03 ATSP/BR HS20 to promote organoid growth required the interaction between HS20 and FIS chains, since RSP03 ATSP/BR HS20 (GS), carrying mutations in the HS-binding CDR3 loop (Figure 2A) failed to promote organoid growth at concentrations below 10 nM, similar to RSP03 ATSP/BR (Figure 5A and B).

[00209] We conclude from these experiments that the interaction between the TSP/BR domain, or HS20, and HSPGs contributes substantially to the capacity of RSP03 to support the growth of mouse intestinal organoids. Multi-omics data shows that GPC4, SDC1 and SDC4 are expressed at detectable levels in mouse intestinal organoids, making them candidate receptors for RSP03 in this tissue.

[00210] A haploid genetic screen for RSP03 receptors in cells lacking LGRs. While our results demonstrate that the interaction between RSP03 and the FIS chains of HSPGs is necessary to promote LGR-independent signaling in cells, and can significantly enhance LGR-dependent signaling in cells and intestinal organoids, they do not preclude the existence of another co receptor capable of mediating these effects. We previously reported the results of a genome wide, haploid genetic screen for regulators of RSP01 -potentiated, low-level WNT signaling in WT HAP1-7TGP cells. We attempted to identify any additional co-receptors required for LGR- independent signaling through a similar haploid genetic screen for regulators of RSP03- potentiated, low-level WNT signaling in cells lacking LGRs 4, 5 and 6 (see Materials and methods). To distinguish genes required for LGR-independent signaling from those required for LGR-dependent signaling, we compared the results of this screen to our previous screen for RSP01 -driven signaling in WT HAP-7TGP cells. The prior screen was focused on LGR- dependent signaling, since RSP01 (unlike RSP03) requires LGR receptors to potentiate WNT signaling.

[00211] The RSP03 screen in LGR4/5/6^KO cells revealed a subset of the WNT signaling regulators identified in the RSP01 screen in WT HAP1-7TGP cells (Figure 6A-C), including the WNT co receptor LRP6, members of the TCF/LEF transcriptional complex such as CTNNB1 , CREBBP, BCL9, and DOT1 L, the ubiquitin ligase RNF146, which ubiquitinates poly-ADP-ribosylated AXIN for proteasomal degradation, and a truncated form of AXIN2 that represses WNT signaling through an unknown mechanism. LGR4, which was the top hit of the RSP01 screen in WT HAP1 - 7TGP cells, was not a significant hit in the RSP03 screen in LGR4/5/6^KO cells, in which LGR4 was disrupted by CRISPR/Cas9-mediated genome editing (Figure 6A-C).

[00212] The RSP03 screen in LGR4/5/6^KO cells failed to reveal any unique significant hits compared to the RSP01 screen in WT HAP1-7TGP cells (Figure 6A-C), suggesting there were no genes selectively required for LGR-independent potentiation of WNT signaling by RSP03. While several components of the GPI-anchor biosynthetic machinery, including PIGP, PIGS, PIGU, PIGL and DPM3, as well as GPC4 were hits in both screens, we have previously shown that their presence as significant hits in the RSP01 screen in WT HAP1-7TGP cells was due to the requirement of GPI-anchored GPCs for low-level WNT signaling, independently of RSPOs. In summary, haploid genetic screens did not reveal an additional co-receptor required for LGR- independent potentiation of WNT signaling by RSP03.

[00213] The TSP/BR domain of RSPOs is conserved across vertebrates, primitive chordates and hemichordates, suggesting a functional role in signaling by these ligands. The TSP/BR domain can bind to FIS chains, but its role in amplifying WNT^-catenin signaling has remained uncertain. Initial studies showed that this domain was dispensable, since a fragment of RSPOs that contains only the FU1 and FU2 domains can amplify WNT^-catenin signaling in vitro. The data presented herein shows a clear function for the TSP/BR domain of RSP03 in WNT^-catenin signaling in vitro and ex vivo : (1) it is essential for signaling in the absence of LGRs and (2) it enhances signaling potency in the presence of LGRs in both cultured cells and intestinal organoids. RSP02 (but not RSP01 or RSP04) can also amplify WNT^-catenin signaling in the absence of LGRs. The TSP/BR domain may be critical for certain RSPOs to potentiate WNT signaling in vivo, especially under regimes of limiting ligand concentration in tissues.

[00214] Until this study, it has been challenging to disentangle the role of HSPGs in mediating signaling through the core WNT^-catenin pathway from their contribution to the amplification of WNT signaling by RSPOs. HSPGs and HS chains were initially implicated in the distribution, stability and potency of WNT ligands in studies from Drosophila, which lacks an RSPO signaling module for WNT amplification. In those studies, HSPGs promoted WNT^-catenin signaling by directly binding to WNT ligands. However, depletion of HS chains by the genetic disruption of enzymes required for their synthesis has also been shown to impair WNT^-catenin signaling and associated responses in the mouse intestine, the mouse mammary gland and in multiple myeloma cells, all contexts in which RSPOs play prominent roles. Thus, a reduction in RSPO-potentiated WNT signaling caused by depletion of HSPGs can be due to a defect in WNT reception, RSPO reception or both. The present RSPO mutagenesis and engineering experiments, which leave the HSPG repertoire of cells intact and their role in WNT reception undisturbed, cleanly demonstrate that interactions with HSPGs are also required for RSPO reception.

[00215] How do HSPGs function during reception of RSPO ligands? HSPGs have been implicated in the function of many ligands, including chemokines and growth factors that activate receptor tyrosine kinases, such as fibroblast growth factor (FGF). In these cases, HSPGs can function as co-receptors by either templating a ligand-receptor interaction (such as the one between FGF and its receptor) or by inducing a conformational change in the ligand that increases its ability to interact with receptors (such as the one induced in anti-thrombin). In both of these cases, exogenously added heparin (untethered to the cell surface, in contrast to the HS chains of HSPGs) can promote receptor-ligand interactions. Alternatively, HSPGs can function as endocytic receptors to mediate ligand uptake and degradation in the lysosome. Our results are more consistent with HSPGs functioning as endocytic receptors in RSPO signaling. First, exogenously added heparin inhibits, rather than promotes, RSPO signaling. Second, the TSP/BR domain can be replaced by a synthetic HS-binding scFv, making steric effects or conformational changes unlikely. Instead, the mere interaction between RSPOs and HSPGs seems to be sufficient for signaling. Our results directly demonstrate that both WT RSP03 and RSP03 ATSP/BR HS20 can cause internalization and degradation of RNF43-Flag in cells lacking LGRs (Figure 3B). Since ZNRF3 and RNF43 are E3 ubiquitin ligases that negatively regulate WNT reception, their clearance from the cell surface would be sufficient to enhance WNT signaling.

[00216] Our work also sheds light on the relative roles of HSPGs and LGRs in mediating signaling by different RSPO family members. Previous studies have proposed that the simultaneous binding of RSPOs to LGRs through the FU2 domain and to ZNRF3/RNF43 through the FU1 domain triggers the ubiquitination, endocytosis and degradation of ZNRF3/RNF43. The affinity of the FU2 domain for LGRs is high (in the nanomolar range) for all RSPOs, while the affinity of the FU1 domain for ZNRF3/RNF43 is much higher for RSP02 and RSP03 compared to RSP01 and RSP04. At least for RSP03, our experiments suggest that the interaction with LGRs is not sufficient to drive the efficient endocytosis of RNF43. HSPG binding, provided by the native TSP/BR domain or by the heterologous HS20 scFv, is also required even in the presence of LGRs (Figure 3A). In the case of RSP03 and also RSP02, not only are LGRs insufficient to mediate RSPO signaling, but they are in fact dispensable. This is most likely due to the higher affinity of these RSPO ligands for ZNRF3/RNF43, since replacing the ZNRF3/RNF43-bindnig FU1 domain of RSP01 with that of RSP03 is sufficient to render RSP01 capable of signaling without LGRs. However, even when LGRs are present, HSPGs can markedly enhance the potency of RSP03 (Figure 1 D and Figures 5A and B) and potentially other RSPOs. One possibility is that HSPGs concentrate RSPOs near the cell surface, increasing their local concentration and the extent of their binding to LGRs. This model is supported by the observation that either the genetic or enzymatic depletion of HS chains, or the removal of the TSP/BR domain, reduces binding of RSPOs to the surface of multiple myeloma cells, while the genetic depletion of LGR4 has no effect. Alternatively, the simultaneous binding of RSP03 to LGRs and HSPGs may be required to trigger efficient endocytosis.

[00217] Disruption of GPC3, GPC4, all GPI-anchored GPCs or all SDCs either had no effect or only partially reduced the signaling potency of WT RSP03 and synthetic RSP03 ATSP/BR HS20 ligands (Figure 4B and C). Only the disruption of all HS-containing HSPGs reduced signaling by these RSPOs substantially, confirming the functional redundancy of HSPGs. However, specific HSPGs may be important in different tissues, stages of development, or pathological conditions. For instance, the interaction of SDC4 and RSP03 was shown to regulate WNT/planar cell polarity (POP) signaling during Xenopus gastrulation by a process that requires clathrin-dependent endocytosis, and SDC1 was shown to promote signaling in multiple myeloma by presenting WNTs and RSPOs. Our results strongly suggest that HSPGs such as GPCs or SDCs are the main co receptors, in addition to ZNRF3 or RNF43, that transduce RSPO signals in the presence and absence of LGRs.

Materials and methods

[00218] The following Materials and methods relevant to this manuscript have been described previously (Lebensohn et al., 2016; Lebensohn and Rohatgi, 2018): cell lines and growth conditions, preparation of WNT3A conditioned medium, construction of the HAP1-7TGP WNT reporter haploid cell line, construction of mutant HAP1-7TGP cell lines by CRISPR/Cas9- mediated genome editing, production of tagged RSPO proteins by transient transfection of 293T cells and immuno-affinity purification from conditioned media, quantification of tagged RSPO proteins, reporter-based forward genetic screens, and comparative analysis of significant hits across screens. Where applicable, modifications to these methods are described below.

[00219] Plasmids. pHLsec-HA-hRSP03-Tev-Fc-Avi-1 D4 and pHLsec-HA-hRSP03ATSP/BR-Tev- Fc-Avi-1 D4 (encoding proteins containing an N-terminal HA tag and C-terminal Fc and 1 D4 tags) were constructed as described previously (Lebensohn and Rohatgi, 2018). pHLsec-HA- hRSP03TSP/BR(K/R E)-Tev-Fc-Avi-1 D4 (Figure 1 B) was synthesized as a gBIock Gene

Fragment (Integrated DNA Technologies (IDT)) flanked at the 5’ and 3’ ends, respectively, by 24 base pair (bp) overhangs overlapping the sequence upstream of the unique Agel site and downstream of the unique Kpnl site in the pHLsec-HA-Tev-Fc-Avi-1 D4 vector. The gBIock was subcloned into pHLsec-HA-Tev-Fc-Avi-1 D4, previously digested with Agel and Kpnl, using the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs (NEB) Cat. # E2621 L).

[00220] pHLsec-HA-hRSPO3ATSP/BRHS20-Avi-1 D4, pHLsec-HA-hRSPO3ATSP/BRHS20(GS)-

Avi-1 D4, and pHLsec-HA-hRSPO3ATSP/BRHS20(A)-Avi-1 D4, all of which encode proteins containing an N-terminal HA tag and a C-terminal 1 D4 tag but lacking the dimerizing Fc tag (Figure 2A), were synthesized as gBIock Gene Fragments (IDT) flanked at the 5’ and 3’ ends, respectively, by 24 bp overhangs overlapping the sequence upstream of the unique Agel site and downstream of the unique Kpnl site in the pHLsec-HA-Avi-1 D4 vector described previously. The gBIock was subcloned into pHLsec-HA-Avi-1 D4, previously digested with Agel and Kpnl, using the NEBuilder HiFi DNA Assembly Master Mix (NEB Cat. # E2621 L).

[00221] pHLsec-HA-hRSPO3ATSP/BRHS20(R67A/Q72A)-Avi-1 D4 and pHLsec-HA- hRSPO3ATSP/BRHS20(F106E/F110E)-Avi-1 D4 were made by replacing the FU1 and FU2 domains in pHLsec-HA-hRSPO3ATSP/BRHS20-Avi-1 D4 with those from pHLsec-HA- hRSP03(R67A/Q72A)-Tev-Fc-Avi-1 D4 and pHLsec-HA-hRSPO3(F106E/F110E)-Tev-Fc-Avi- 1 D4, respectively. A fragment containing the FU1 and FU2 domains was subcloned by double digestion with Agel and Mfel followed by ligation.

[00222] RNF43-2xFlag-2xHA and N-terminal SNAP-tagged mouse FZD5 (Ala27-Val559) were described previously (Koo et al., 2012).

[00223] pVRC8400 was a gift from Dr. Gary J. Nabel, National Institute of Allergy and Infectious Diseases.

[00224] pSpCas9(BB)-2A-GFP (pX458) was a gift from Feng Zhang (Addgene plasmid # 48138; RRID:Addgene_48138).

[00225] pX458-mCherry was made by replacing the coding sequence of GFP in pX458 with that of a modified mCherry sequence. The WT mCherry sequence has a Bbsl cleavage site that makes it incompatible with the Bbsl restriction digestion required for cloning the single guide RNA (sgRNA) oligos into pX458. Therefore, a silent mutation was introduced into the mCherry sequence to eliminate the Bbsl cleavage site. Terminal EcoRI sites and a T2A sequence were appended to the modified mCherry sequence to obtain an EcoRI-T2A-mCherry-EcoRI fragment. The EcoRI-T2A-EGFP-EcoRI fragment in px458 was replaced with the EcoRI-T2A-mCherry- EcoRI fragment by EcoRI double digestion and ligation to obtain pX458-mCherry.

[00226] Antibodies. Primary antibodies: Rho 1 D4 purified monoclonal antibody (University of British Columbia, https://uilo.ubc.ca/rho-1d4-antibody), mouse anti-Flag M2 (MilliporeSigma Cat. # F3165), mouse anti-actin monoclonal (Clone C4, MP Biomedicals Cat. # 08691002), polyclonal rabbit anti-human lysozyme (Agilent Dako Cat. # A0099), rabbit anti-Ki67 (Abeam Cat. # ab15580). Secondary antibodies: chicken anti-rabbit Alexa Fluor 488 (Thermo Fisher Scientific Cat. # A21441), goat anti-mouse Alexa Fluor 680 (Thermo Fisher Scientific Cat. # A21057), goat anti-mouse IRDye800 (Rockland Immunochemicals Cat. # 610-132-121), goat anti-human Ig kappa chain FIRP conjugate (MilliporeSigma Cat. # AP502P). Staining reagents: phalloidin-TRITC (MilliporeSigma Cat. # P1951 ), DAPI (MilliporeSigma Cat. # D9542).

[00227] RSP03 modeling. A structural model of human RSP03 (residues valine 146 - isoleucine 232; UniProtKB Q9BXY4) was built using the HHpred web server (Max Planck Institute Bioinformatics Toolkit) based on homology to the 26 most similar protein structures in the Protein Data Bank. Polymers consisting of four disaccharide units of heparin (PDB ID 1 HPN) were docked to RSP03 using AutoDock Vina. Heparin was kept rigid during docking. Positively charged side chains of residues 161 , 162, 164, 169, 170, 198, 203-205, 208, 211 (Site-1) or 213, 214, 216, 218-222, 225, 229 (Site-2) were kept flexible during docking. The models depicted in Figure 1 A and the animation shown as Supplementary file 1 were rendered using PyMOL 2.3.2.

[00228] Production of tagged RSPO proteins. Tagged RSPO proteins were produced by transient transfection of 293T cells and immuno-affinity purification as described previously, with some modifications. The WT, mutant and truncated RSP03 proteins have an N-terminal HA tag and C-terminal Fc and 1 D4 tags; the engineered RSP03 ATSP/BR HS20 fusion proteins, and mutant derivatives thereof, have an N-terminal HA tag and a C-terminal 1 D4 tag. ~24 hr before transfection, 20x10⁶293T cells were seeded in a T-225 flask containing 55 ml of growth medium (DM EM, 10% FBS, 1 mM Sodium Pyruvate, 1.x MEM-NEAA and 2 mM L-G!utamine). Once they had reached -80% confluency, the cells in each flask were transfected with 1 ml of a transfection mix prepared as follows. 30 pg of DNA constructs encoding tagged WT, mutant or synthetic RSPO proteins were diluted in 910 mI of serum-free DMEM and 90 mI of polyethylenimine (PEI, linear, MW -25,000, Polysciences, Inc. Cat. # 23966) was added from a 1 mg/ml stock (prepared in sterile water, stored frozen and equilibrated to 37°C before use). The transfection mix was vortexed briefly, incubated for 15-20 min at room temperature (RT) and added to the cells without replacing the growth medium. -24 hr post-transfection, the cells were washed with 55 ml PBS and the medium was replaced with 55 ml of CD 293 medium (Thermo Fisher Scientific Cat. # 11913019) supplemented with 1x L-glutamine solution (stabilized, Gemini Bio Products Cat. # 400-106) and 2 mM valproic acid (MilliporeSigma Cat. # P4543, added from a 0.5 M stock prepared in water and sterilized by filtration through a 0.22 pm filter) to promote protein expression. ~96 hr post-transfection, the CM from each flask, containing secreted RSPO protein, was filtered through 0.45 pm filters (Nalgene aPES membrane, Thermo Fisher Scientific Cat. # 166-0045) to remove particulates and was reserved on ice. 300 mI of a -50% slurry of Rho 1 D4 resin was added to a 50 ml conical tube containing the RSPO CM and the suspension was incubated overnight, rocking at 4°C. Following binding and during all subsequent washes, the resin was collected by centrifugation for 5 min at 400 x g in a swinging bucket rotor. The beads were washed three times at RT with 25 ml PBS by resuspending them in buffer and mixing by inversion for -1 min. The resin was transferred to a 1.5 ml Eppendorf tube and washed three more times with 1 ml of PBS, 10% glycerol. Following the last wash, the buffer was aspirated and the resin was resuspended in 150 mI of 500 mM 1 D4 peptide ((NH3)-T-E-T-S-Q-V-A-P-A-(COOH)) prepared in PBS, 10% glycerol, to obtain a -50% slurry. Elution was carried out by rotating the tube horizontally overnight at 4°C. Following centrifugation of the resin, the eluate was recovered and reserved on ice. The resin was resuspended in another 150 mI of 500 mM 1 D4 peptide in PBS, 10% glycerol, and a second round of elution was carried out for 2 hr at RT. Following centrifugation of the resin, the second eluate was recovered and pooled with the first. The final eluate was centrifuged again to remove residual resin and the supernatant containing tagged RSPO proteins was aliquoted, frozen in liquid nitrogen and stored at -80°C. Tagged RSPO proteins were quantified as described previously.

[00229] Dose-response analysis of RSP03 effects on WNT reporter fluorescence. 20,000 cells were plated per well of a 96-well plate and grown in complete IMDM growth medium (Iscove's Modified Dulbecco's Medium (IMDM) with L-glutamine, with HEPES, without A!pha-Thiog!ycerol (GE Healthcare Life Sciences Cat. # SH30228.01); 1x GlutaMAX; 40 Units/ml Penicillin, 40 pg/ml Streptomycin; 10% FBS). -24 hr after plating, the cells were treated with complete IMDM growth medium containing 1.43% WNT3A CM and the indicated concentrations of RSPO proteins. After 24 hr of treatment, cells were trypsinized in 40 mI of Tryspin (0.05% in EDTA) and harvested in 160 mI of IMDM. WNT reporter (EGFP) fluorescence data for 2,500 singlet-gated cells was measured by flow cytometry on a BD Accuri RUO Special Order System (BD Biosciences). The median EGFP fluorescence from each well was depicted as circles in Figure 1 D and E and Figure 2C-H. Dose-response curves were fitted using the nonlinear regression (curve fit) analysis tool in GraphPad Prism 8 using the [agonist] vs. response - variable slope (four parameters) equation. Results presented are representative of experiments repeated at least twice. [00230] Cell surface RNF43 biotinylation assay. WT HAP1-7TGP or LGR4/5/6^KO cells were transfected with human RNF43-2xFlag-2xHA and SNAP-tagged mouse FZD5 using PEI. 24 hr after transfection, cells were washed with ice-cold PBS complete (containing Ca²⁺ and Mg²⁺) and kept on ice. 800 mM of EZ-Link Sulfo-NHS-SS-Biotin (Thermo Fisher Scientific Cat. # 21331) in PBS complete was added to each well and biotinylation was allowed to proceed for 30 min on ice. The medium was aspirated, cells were washed once with ice cold PBS complete and unreacted biotin was quenched with complete IMDM growth medium containing 50 mM glycine for 5 min on ice. Cells were washed with ice-cold PBS complete and lysed for 30 min on ice in lysis buffer (20 mM Tris pH 7.5, 150 mM NaCI, 1% TX-100, 1 mM EDTA, 1 mM PMSF, 10 pg/ml Leupeptin, 10 pg/ml Aprotinin). Cell lysates were centrifuged for 5 min at 14,000 x g at 4°C. An aliquot of the supernatant (“Total” in Figure 3A and B) was mixed with SDS sample buffer and the remaining supernatant was incubated with 25 pi of a 50% slurry of Pierce Streptavidin Agarose beads (Thermo Fisher Scientific Cat. # 20349) for 60 min at 4°C. The beads were washed with 0.1x PBS complete, and biotinylated proteins (“Surface” in Figure 3A and B) were eluted in 20 pi 2x SDS sample buffer, 40 mM DTT for 30 min at 37°C and analyzed by SDS-PAGE and Western blot. Western blotting was performed using standard procedures. Samples were resolved by SDS- PAGE, transferred to Immobilon-FL PVDF membranes (MiliporeSigma Cat. # IPFL00005), blocked with Odyssey blocking buffer (Li-Cor Cat. # 927-40000), incubated with the indicated primary and secondary antibodies, and imaged using the Amersham Typhoon NIR laser scanner (GE Healthcare).

[00231] Production of recombinant GPC mutant proteins without HS chains. The GPC3 and GPC4 mutant proteins without HS chains (GPC3AHS and GPC4AHS in Figure 4A) were constructed by replacing serine with alanine residues at the HS attachment site. The mutant constructs were PCR-amplified and inserted into a pVRC8400 expression vector as described previously. The IL-2 signal peptide sequence was added to the N-terminus and the human Fc sequence was added to the C-terminus of the mutant GPCs. The plasmids were transiently transfected into HEK-293T cells. The cell supernatant was harvested and the Fc-tagged GPC proteins were purified on a Protein A Hi-Trap column (GE Healthcare Cat. # 29048576) according to the manufacturer’s instructions. The quality and quantity of purified proteins were determined by SDS-PAGE and by measuring absorbance at 280 nm on a NanoDrop instrument (Thermo Fisher Scientific), respectively. [00232] Measurement of HS20 binding to GPCs by enzyme-linked immunosorbent assay (ELISA). Increasing concentrations of HS20 (serial 2-fold dilutions starting from 1 pg/ml) were added into a 96-well ELISA plate coated with 5 pg/ml of glycosylated GPC3, GPC4 (R&D Systems Cat. # 2119-GP and 9195-GP, respectively), GPC3AHS, GPC4AHS, or an irrelevant human Fc- fusion protein (CD276-hFc, labeled “Control” in Figure 4A) and incubated for 1 hr at RT. The plate was washed three times with PBST (PBS, 0.1% Tween-20) and incubated with 50 pi of goat anti human kappa chain HRP conjugate (1 :5000 dilution) for 1 hr at RT. After washing six times with PBST, 50 mI/well of 3,3',5,5'-tetramethylbenzidine detection reagent (Kirkegaard & Perry Laboratories Cat. # 95059-156) was added and incubated for 10 min at RT. Absorbance was read at 450 nm.

[00233] Construction of mutant HAP1-7TGP cell lines by CRISPR/Cas9-mediated genome editing. The LGR4/5/6^KO, LGR4/5/6^KO; PIGL^K0, LGR4/5/6^KO; SDC1/2/3/4^KO and LGR4/5/6^KO; EXTL3^K0 cell lines used in this study have been described previously. The LGR4/5/6^KO; GPC3^K0 and LGR4/5/6^KO; GPC4^K0 cell lines were constructed as described previously. Briefly, oligonucleotides encoding sgRNAs were selected from a published library and cloned into pX458- mCherry according to a published protocol. Clonal cell lines were established as described previously by transient transfection of LGR4/5/6^KO cells with pX458-mCherry containing the desired sgRNAs (since we used pX458-mCherry, there was no need to co-transfect with pmCherry), followed by single-cell sorting of mCherry-positive cells. Genotyping was done as described previously.

[00234] Growth of mouse small intestinal organoids. Small intestinal organoids were established and maintained as described, starting from isolated crypts collected from the entire length of the small intestine of B6 mice. The basic culture medium (advanced DMEM/F12, penicillin/streptomycin, 10 mM HEPES, 1x GlutaMAX, 1x N-2 supplement, 1x B-27 supplement (all from Thermo Fisher Scientific), 1 mM N-acetylcysteine (MilliporeSigma Cat. # 106425)), was supplemented with 50 ng/ml recombinant murine EGF (Peprotech Cat. # 315-09), and 1% v/v Noggin CM to obtain EN medium. Purified RSPO recombinant proteins were added to the basic culture medium as indicated. Organoids were cultured in matrigel droplets (Corning). The medium was refreshed 3 and 6 days after splitting. Images were captured 8 days after splitting using an EVOS M5000 imaging system (Thermo Fisher Scientific). [00235] Immunofluorescence microscopy of organoids. Organoids were carefully harvested from matrigel, collected by centrifugation for 5 min at 100 x g at 4°C and washed with ice-cold medium. Organoids were transferred to a m-slide 8 well (Ibidi Cat. # 80826) and fixed in paraformaldehyde (4%, diluted in 0.1 M sodium phosphate buffer pH 7.4) for 1 hr at RT. Paraformaldehyde was removed and 20 mM ammonium chloride (in PBS) was added for 10 min. Organoids were permeabilized in PBD 0.2 T buffer (1x PBS, 1% BSA, 1% DMSO, 0.2% TX-100) for 30 min at RT. Organoids were incubated with anti-human lysozyme or anti-Ki67 primary antibodies in PBD 0.2 T for 3 hr at RT, followed by anti-rabbit Alexa Fluor 488 secondary antibody containing 0.2 pg/ml DAPI and phalloidin-TRITC in PBD 0.2 T for 3 hr at RT. Organoids were mounted in Ibidi mounting medium (Ibidi Cat. # 50001) and images were acquired with a Zeiss LSM700 confocal microscope. Images were processed in ImageJ (National Institutes of Health).

[00236] Reporter-based forward genetic screens for regulators of RSP01 signaling in HAP1- 7TGP cells and for regulators of RSP03 signaling in LGR4/5/6KO cells. HAP1-7TGP or LGR4/5/6^KO cells were mutagenized with a GT-containing retrovirus as described previously. Six days following mutagenesis, the HAP1-7TGP or LGR4/5/6^KO cell populations were treated with a low dose (1 .04%) of WNT3A CM in complete IMDM growth medium, combined with a saturating dose (10 ng/ml) of recombinant human RSP01 (R&D Systems Cat. # 4645-RS) or RSP03 (R&D Systems Cat. # 3500-RS), respectively. A total of 1.25 x 10⁸ singlet-gated cells were sorted by fluorescence-activated cell sorting (FACS), gating for the lowest 10% WNT reporter (EGFP) fluorescence. A population of singlet-gated HAP1-7TGP or LGR4/5/6^KO cells (not gated based on EGFP fluorescence) was also sorted and carried along as a control to set FACS gates during the subsequent sort. Cells were expanded for 6 days to allow recovery and resetting of the WNT reporter, and a portion of the cells was used in a subsequent round of sorting, following the same treatment and FACS gating criteria. For both screens described, cells underwent two consecutive rounds of FACS sorting. We identified genes selectively enriched for GT integrations in the sorted cell populations as follows. 3 x 10⁷ cells expanded following the final sort for each screen were used to generate the dataset of GT insertions in the sorted cell populations, and an equivalent number of mutagenized but unsorted HAP1 -7TGP or LGR4/5/6^KO cells were used to generate the dataset of GT insertions in the respective control cell populations. Preparation of genomic GT insertion libraries for deep sequencing, mapping of sequencing reads and statistical analysis of GT insertion enrichment was conducted as described previously. We generated a ranked list of hits from each screen and used it to create circle plots depicting the data (Figure 6A and B). Note that the data presented here for the RSP01 screen in HAP1-7TGP cells is different from that presented in our previous report because in order to maintain a comparable degree of phenotypic enrichment between this screen and the RSP03 screen in LGR4/5/6^KO cells, we analyzed a cell population from our previous RSP01 screen in HAP1 -7TGP cells that had been sorted only twice (as opposed to four times) for the lowest 10% WNT reporter activity.

[00237] Comparative analysis of significant hits between screens. We used two metrics to compare the results of the RSP01 screen in HAP1-7TGP cells and the RSP03 screen in LGR4/5/6^KO cells. The FDR-corrected p-value (Figure 6A and B) tests for the enrichment of inactivating GT insertions in the sorted cell population over total GT insertions in the control cell population. While this is a good metric by which to compare the significance of hits in a given screen, it depends on many experimental variables affecting both the sorted and control cell datasets, making it an equivocal metric to directly compare hits between screens. We therefore used the Intronic GT Insertion Orientation Bias (IGTIOB) score we developed previously, which is based on the frequency of intronic sense vs. antisense GT insertions only in the sorted cell population from each screen, to directly compare significant hits between the RSP01 screen in HAP1-7TGP cells and the RSP03 screen in LGR4/5/6^KO cells. In order to avoid missing any potential hits unique to only one screen, we relaxed the GT enrichment FDR-corrected p-value threshold for inclusion in the comparative analysis from <10⁴ to <0.01. We generated a list of all genes with an FDR-corrected p-value<0.01 in at least one of the two screens and used the IGTIOB scores of those genes in both screens to build a heat map (Figure 6C) as described previously.

[00238] Statistical analysis. Data analysis and visualization for the dose-response analysis of RSP03 effects on WNT reporter fluorescence (Figure 1 D and E, Figure 2C-H) were performed in Graph Pad Prism 8. Dose-response curves were fitted with the nonlinear regression curve fit using the [agonist] vs. response - variable slope (four parameters) equation. All reported IC₅o values were obtained from these curve fits. The statistical significance of the differences in EC50 values for various RSPO variants and cell lines (Figure 4B and C) was determined by a two-tailed, unpaired t-test in GraphPad Prism 8. Statistical analysis of screen data was performed as described previously.

Example 2

[00239] Fusion of the anti-HSPG ABR improves the potency of a soluble WNT inhibitor. Shown in Figure 7(A) Diagrams of fusion proteins tested in the WNT signaling assay shown in (B). The anti HSPG ABR (HS20) is fused to the DKK1 c (Dickkopf WNT Signaling Pathway Inhibitor 1 ) protein, a known soluble inhibitor of WNT signaling. The inhibitory activity of the DKK1c-HS20 fusion is compared to two controls, HS20 alone and Dkklc alone. WNT signaling assay shows that fusion of the anti-HSPG ABR increases the inhibitory potency of DKK1c by 20-fold.

[00240] The sequences are SEQ ID NO:24, residues 1-29 signal peptide, residues 30-38 HA tag, residues 39-278 HS20, residues 279-286 linker, residues 287-302 Avi tag, residues 303-311 ID4 tag.

[00241] SEQ ID NO:25, residues 1-29 signal peptide, residues 30-38 HA tag, residues 39-130 Dkk1C, residues 131-138 linker, residues 139-160 Avi and ID4 tags.

[00242] SEQ ID NO:26 residues 1-29 signal peptide, residues 30-38 HA tag, residues 39-130 Dkk1C, residues 131-138 linker, residues 143-381 HS20, residues 382-389 linker, residues 390- 414 Avi and ID4 tags. The mature protein comprises residues 39-381 , Dkk1C - linker - HS20.

[00243] The preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present invention is embodied by the appended claims.

Claims

What is Claimed is:

1. An engineered WNT signaling agonist or antagonist protein, comprising: a WNT agonist or antagonist linked to an antigen binding region (ABR) that specifically binds to heparan sulfate chains.

2. The engineered protein of claim 1 , wherein the WNT agonist comprises an Rspondin protein sequence comprising at least FU1 and FU2 domains of RSPO.

3. The engineered RSPO protein of claim 2, wherein the RSPO protein is human RSP03.

4. The engineered RSPO protein of claim 2, wherein the RSPO protein is human RSP02.

5. The engineered RSPO protein of any of claims 2-4, wherein the RSPO protein sequence is truncated to delete the native TSP/BR domain.

6. The engineered RSPO protein of any of claims 2-4, wherein the RSPO protein comprises targeted mutations to reduce binding of the native TSP/BR domain to HSPG.

7. The engineered protein of any of claims 1 -6, wherein the ABR comprises variable heavy and light chain domains from an antibody.

8. The engineered protein of claim 7, wherein the ABR is a single chain variable region.

9. The engineered protein of claim 8, wherein the ABR specifically binds to heparan sulfate chains present on heparan sulfate proteoglycan (HSPG).

10. The engineered protein of claim 9, wherein the ABR specifically binds to the heparan sulfate chains present on human glypican.

11. The engineered protein of claim 10, wherein the ABR comprises the set of CDR sequences comprising amino acid residues 26-33, 51-58 and 97-105 of SEQ ID NO: 3 and residues 27-32, 50-52 and 89-97 of SEQ ID NO: 4.

12. The engineered protein of any of claims 1 -1 1 , wherein the WNT agonist or antagonist sequence is contiguously fused to the ABR.

13. The engineered protein of any of claims 1 -11 , wherein the WNT agonist or antagonist sequence is separated from the ABR by a linker.

14. The engineered protein of any of claims 1-11 , wherein an engineered RSPO protein enhances signaling potency of WNT proteins, relative to the corresponding native RSPO protein, on cells that have low or no expression of one or more of leucine-rich repeat-containing G-protein coupled receptors (LGRs) 4, 5 and 6.

15. The engineered protein of any of claims 1-1 , wherein an engineered RSPO protein enhances signaling potency of WNT proteins, relative to the corresponding native RSPO protein, on cells that have low or no expression of each of leucine-rich repeat-containing G-protein coupled receptors (LGRs) 4, 5 and 6.

16. The engineered protein of any of claims 1 -11 , wherein an engineered RSPO protein enhances signaling potency of WNT proteins, relative to the corresponding native RSPO protein, on cells that express leucine-rich repeat-containing G-protein coupled receptors (LGRs) 4, 5 and 6.

17. A pharmaceutical composition comprising an engineered WNT signaling agonist or antagonist protein according to any of claims 1-16.

18. A method of for activating, increasing or enhancing Wnt signaling, the method comprising: contacting a cell expressing a receptor for Wnt with an effective amount of an engineered RSPO protein of any of claims 1-16 at a concentration that is effective to increase signaling, relative to the signaling in the absence of the engineered RSPO protein.

19. The method of claim 18, wherein the engineered RSPO protein is provided in combination with provision of a wnt signaling agonist.

20. The method of claim 18 or 19, wherein the contacting is performed in vivo.

21 . The method of claim 18 or 19, wherein the contacting is performed in vitro.

22. The engineered protein of claim 1 , wherein the WNT antagonist comprises a human Dickopf (Dkk) protein.

23. The engineered protein of claim 22, wherein the WNT antagonist comprises DKK1 or DKK1c protein.