Modulation of WNT signaling in gastrointestinal disorders

Abstract

The present disclosure provides methods for treating gastrointestinal disorders with engineered WNT agonists and modulators of the WNT signaling pathway. In one embodiment, the present disclosure encompasses engineered WNT agonists comprising: (a) one or more binding domains that bind to one or more FZDs; and (b) one or more binding domains that bind to LRP5, LRP6, or both LRP5 and LRP6, comprising a polypeptide sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOs: 1-18, or a polypeptide sequence disclosed in any one of SEQ ID NOs: 1-25, FIG. 2, FIG. 6, Table 1, or Table 3, or a functional fragment or variant thereof, such as a binding fragment thereof, such as a VHH domain, a heavy chain variable domain, or a light chain variable domain.

Description

[Technical field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 159,010, filed March 10, 2021, U.S. Provisional Patent Application No. 63 / 190,535, filed May 19, 2021, and U.S. Provisional Patent Application No. 63 / 247,151, filed September 22, 2021, which are incorporated by reference in their entireties herein.

[0002] Sequence Listing This application has been filed electronically via EFS-Web and contains an electronically submitted Sequence Listing in .txt format. The .txt file contains a Sequence Listing entitled SRZN_020_03WO_ST25.txt, was created on Mar. 7, 2022, and is 80 kilobytes in size. The Sequence Listing contained in this .txt file is a part of the present specification and is incorporated herein by reference in its entirety.

[0003] FIELD OF THEINVENTION The present disclosure provides WNT signaling modulators as treatments for gastrointestinal disorders, particularly inflammatory bowel disease. [Background technology]

[0004] background WNT proteins form a family of highly conserved secreted signaling molecules that regulate cell-cell interactions during embryogenesis. WNT genes and WNT signaling are also implicated in cancer. Insights into the mechanism of WNT action have arisen from several systems: genetics in Drosophila and Caenorhabditis elegans; biochemistry in cell culture and ectopic gene expression in Xenopus embryos. Many WNT genes are mutated in mice, leading to highly specific developmental defects. As currently understood, WNT proteins bind to receptors of the Frizzled family on the cell surface. Through several cytoplasmic relay components, the signal is transmitted to beta-catenin, which then enters the nucleus and forms a complex with TCF to activate the transcription of WNT target genes. Expression of WNT proteins is variable but is often associated with developmental processes, for example in embryonic and fetal tissues.

[0005] The exploration of the physiological functions of WNT proteins in adult organisms is hindered by functional redundancy and the need for conditional inactivation strategies. Dickkopf-1 (Dkk1) was recently identified as a founding family member of secreted proteins that potently antagonize WNT signaling (see Glinka et al. (1998) Nature 391: 357-62; Fedi et al. (1999) J Biol Chem 274: 19465-72; and Bafico et al. (2001) Nat Cell Biol 3: 683-6). Dkk1 associates with both the WNT coreceptors LRP5 and LRP6 and the transmembrane protein Kremen, and the resulting ternary complex leads to rapid internalization of LRP6 and impaired WNT signaling in the absence of a functional Frizzled / LRP6 WNT receptor complex (see, e.g., Mao et al. (2001) Nature 411: 321-5; Semenov et al. (2001) Curr Biol 11: 951-61; and Mao et al. (2002) Nature 417: 664-7).

[0006] Transgenic mice with a knockout of the Tcf locus show a loss of the proliferative stem cell compartment in the small intestine during late embryogenesis. However, the knockout is lethal and has therefore not been tested in adults. In chimeric transgenic mice that allow adult analysis, expression of a constitutively active NH2-truncated β-catenin stimulated proliferation in small intestinal crypts, but also induced increased crypt apoptosis by either NH2-truncated β-catenin or a Lef-1 / β-catenin fusion. The cause of the intestinal stem cell defect is unclear, as β-catenin / Lef / Tcf-dependent transcription is regulated by diverse factors, including non-Frizzled GPCRs and PTEN / PI-3-kinase.

[0007] The adult intestinal epithelium is characterized by continuous replacement of epithelial cells through stereotyped cycles of cell division, differentiation, migration, and exfoliation that occur during the crypt-villus transition period of 5-7 days. Although putative growth factors regulating proliferation within the adult intestinal stem cell niche have not yet been fully identified, multiple studies suggest a cell-intrinsic action of β-catenin / Lef / Tcf signaling within the proliferative crypt compartment.

[0008] Many pathological conditions affect the cells of the intestine. Inflammatory bowel disease (IBD) involves either or both the small and large intestine. Crohn's disease and ulcerative colitis are the most well-known forms of IBD, and both fall into the category of "idiopathic" inflammatory bowel disease because their etiology is unknown. "Active" IBD is characterized by acute inflammation. "Chronic" IBD is characterized by architectural changes in the crypts, with distortion and scarring. Crypt abscesses can occur in many forms of IBD.

[0009] Crohn's disease may involve any part of the gastrointestinal tract, but most frequently involves the distal small bowel and colon. Inflammation is typically transmural and can result in anything from small ulcers spanning lymphoid follicles (aphthous ulcers) to deep fissuring ulcers leading to transmural scarring and chronic inflammation. One-third of cases have granulomas, and extracolonic sites such as lymph nodes, liver, and joints may also have granulomas. Transmural inflammation leads to the development of fistulas between bowel loops and other structures. Inflammation is typically segmental, with areas of involved bowel separated by uninvolved bowel. The etiology is unknown, but infectious and immunological mechanisms have been proposed. Ulcerative colitis (UC) involves the colon as a distally predominant diffuse mucosal disease. The rectum is virtually always involved, and additional portions of the colon may be involved, spreading proximally in a continuous pattern from the rectum. The etiology of UC is unknown. Patients with long-standing UC are at increased risk for developing colon cancer. Patients with UC are also at risk for developing liver disease, including sclerosing cholangitis and cholangiocarcinoma. Currently, all of the therapeutic agents used in the clinic for the treatment of UC and the majority of those under development are focused on reducing inflammation and do not directly induce epithelial healing, highlighting the unmet need for therapeutic agents that promote epithelial repair. [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] Mao et al., Nature (2001) 411:321-5 [Non-Patent Document 2] Semenov et al., Curr Biol (2001) 11:951–61 [Non-Patent Document 3] Mao et al., Nature (2002) 417:664-7 Summary of the Invention [Means for solving the problem]

[0011] It is interesting for clinical purposes to develop pharmacological agents for regulating intestinal epithelium growth.However, the search for WNT agonists as pharmacological agents is hindered in part by the fact that WNT agonists are not naturally soluble and are diffusible molecules.The present disclosure provides methods and compositions for specifically modulating WNT signaling through specific FZD receptors using engineered soluble WNT agonists.Such engineered WNT agonists can, for example, achieve epithelium-specific transient Wnt signaling activation, thereby driving robust epithelium regeneration and barrier restoration, ultimately leading to reduced inflammation and reversal of colitis.

[0012] Summary of the Invention In various aspects, the disclosure provides engineered WNT agonists and related pharmaceutical compositions and methods of use.

[0013] In one aspect, the disclosure encompasses engineered WNT agonists comprising: (a) one or more binding domains that bind one or more FZDs; and (b) one or more binding domains that bind LRP5, LRP6, or both LRP5 and LRP6, wherein the engineered WNT agonist comprises a polypeptide sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOs: 1-18, or a polypeptide sequence disclosed in any one of SEQ ID NOs: 1-25, FIG. 2, FIG. 6, Table 1, or Table 3, or a functional fragment or variant thereof, e.g., a binding fragment thereof, e.g., a VHH domain, a heavy chain variable domain, or a light chain variable domain. In certain embodiments, the one or more binding domains that bind one or more FZDs bind: i) FZD5; ii) FZD8; iii) FZD1; iv) FZD2; v) FZD7; vi) FZD5 and FZD8; vii) FZD1, FZD2, and FZD7; viii) FZD1, FZD2, FZD7, FZD5 and FZD8; ix) FZD4; x) FZD9; or xi) FZD10. In certain embodiments, the engineered WNT agonist comprises one or more (e.g., two) polypeptide sequences having at least 90%, at least 95%, %, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 1-18 or 19-25 or the sequences disclosed in Table 3. In certain embodiments, the engineered WNT agonist comprises: (a) one or more (e.g., two) polypeptide sequences having at least 90%, or at least 95%, homology to SEQ ID NO:1 and one or more (e.g., two) polypeptide sequences having at least 90%, or at least 95%, homology to SEQ ID NO:2; (b) one or more (e.g., two) polypeptide sequences having at least 90%, or at least 95%, homology to SEQ ID NO:3 and one or more (e.g., two) polypeptide sequences having at least 90%, or at least 95%, homology to SEQ ID NO:4;(c) one or more (e.g., two) polypeptide sequences having at least 80%, at least 90%, or at least 95% homology to SEQ ID NO:5 and one or more (e.g., two) polypeptide sequences having at least 80%, at least 90%, or at least 95% homology to SEQ ID NO:6; (d) one or more (e.g., two) polypeptide sequences having at least 90% or at least 95% homology to SEQ ID NO:7 and one or more (e.g., two) polypeptide sequences having at least 90% or at least 95% homology to SEQ ID NO:8; (e) one or more (e.g., two) polypeptide sequences having at least 90% or at least 95% homology to SEQ ID NO:9 and one or more (e.g., two) polypeptide sequences having at least 90% or at least 95% homology to SEQ ID NO:10; (f) one or more (e.g., two) polypeptide sequences having at least 90% or at least 95% homology to SEQ ID NO:7. (g) one or more (e.g., two) polypeptide sequences having at least 90% or at least 95% homology to SEQ ID NO:11 and one or more (e.g., two) polypeptide sequences having at least 90% or at least 95% homology to SEQ ID NO:12; (h) one or more (e.g., two) polypeptide sequences having at least 90% or at least 95% homology to SEQ ID NO:13 and one or more (e.g., two) polypeptide sequences having at least 90% or at least 95% homology to SEQ ID NO:14; (i) one or more (e.g., two) polypeptide sequences having at least 90% or at least 95% homology to SEQ ID NO:15 and one or more (e.g., two) polypeptide sequences having at least 90% or at least 95% homology to SEQ ID NO:16;or (j) one or more (e.g., two) polypeptide sequences having at least 90%, or at least 95%, homology to SEQ ID NO: 17 and one or more (e.g., two) polypeptide sequences having at least 90%, or at least 95%, homology to SEQ ID NO: 18. In certain embodiments, the polypeptide comprises CDRs present in any one of SEQ ID NOs: 1-18 or 19-25. In certain embodiments of the engineered WNT agonist, one or more binding domains that bind LRP5, LRP6, or both LRP5 and LRP6 are humanized. In certain embodiments, the engineered WNT agonist comprises a modified Fc domain, the modified Fc domain comprising a LALAPG or N297G modification. In certain embodiments, the WNT agonist has any of the structures or formats disclosed herein, including any of the various antibody-related structures or formats. Examples of suitable formats include, but are not limited to, monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, nanobodies, diabodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, including but not limited to scFv, Fab, and Fab2, so long as they exhibit the desired biological activity, e.g., WNT agonist activity. In a particular embodiment, the WNT agonist is R2M13-h26. R2M13 is a humanized form of the parent R2M13-26, which also contains a LALAPG substitution in the Fc domain. R2M13-h26 is sometimes referred to herein as R2M13-h26-LALAPG, R2M13-26 humanized LALAPG, or humanized LALPG.;

[0014] In a related aspect, the present disclosure provides a pharmaceutical composition comprising an engineered WNT agonist disclosed herein and a pharma- ceutically acceptable carrier, diluent, or excipient.

[0015] In a further related aspect, the present disclosure provides a method for treating a disease or disorder suitable for treatment by increased WNT pathway signaling in a subject, comprising administering to the subject an engineered WNT agonist or pharmaceutical composition disclosed herein. In certain embodiments, the disease or disorder is a gastrointestinal disorder, such as inflammatory bowel disease. In certain embodiments, the disease or disorder is selected from the group consisting of Crohn's disease (CD), CD with fistulization, and ulcerative colitis (UC). In certain embodiments, the engineered WNT agonist is administered orally, or parenterally, for example, intravenously, intraperitoneally, or subcutaneously. In certain embodiments, the WNT agonist is R2M13-h26. In certain embodiments, the WNT agonist is administered intravenously, for example, as a bolus injection. In certain embodiments, the WNT agonist is administered at least once per week. In certain embodiments, the subject is administered about 0.5 to about 100 mg of WNT agonist per kg of body weight, or about 2 to about 50 mg of WNT agonist per kg of body weight, e.g., about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, or about 50 mg / kg of WNT agonist. In certain embodiments, the subject is administered about 3 to about 30 mg of R2M13-h26 per kg of body weight intravenously at least once per week, wherein R2M13-h26 comprises two polypeptides of SEQ ID NO: 9 and two polypeptides of SEQ ID NO: 10 linked by disulfide bonds.

[0016] In another related aspect, the present disclosure provides a method for increasing WNT signaling in a cell, comprising contacting the cell with an engineered WNT agonist disclosed herein. In certain embodiments, the WNT agonist is R2M13-h26.

[0017] In another related aspect, the disclosure provides a method of modulating expression of a WNT pathway molecule in one or more tissues and / or cells of a subject having a gastrointestinal disorder, comprising administering to the subject an engineered WNT agonist or pharmaceutical composition disclosed herein. In certain embodiments, the WNT pathway molecule is a gene or protein listed in any one of Tables 4-7. In certain embodiments, the WNT pathway molecule is selected from the group consisting of RNAse4, angiogenin, Gsta3, Rnf43, Axin2, or any of the genes or proteins listed in Table 7. In certain embodiments, after administration of the engineered Wnt agonist, expression of WNT pathway molecules (genes or proteins) in one or more tissues and / or cells of the subject is increased by at least 20%, at least 50%, at least 80%, at least 1.1-fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, 2-fold, at least 5-fold, at least 10-fold, or at least 20-fold, or is decreased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%. In certain embodiments, the tissue is an epithelial tissue. In certain embodiments, the cells are gastrointestinal epithelial cells, optionally stem cells, TA1, TA2, basal goblet cells, injury-induced alternative progenitor cells (AltEnteroPC), injury-induced alternative enterocyte (AltEntero), enterocyte precursor (EnteroPrecur), goblet cell 1, goblet cell 2, or enteroendocrine cells or tuft cells. In certain embodiments, the WNT agonist is R2M13-h26. In certain embodiments, the WNT agonist is administered intravenously, for example, as a bolus injection. In certain embodiments, the WNT agonist is administered at least once per week.In certain embodiments, the subject is administered about 0.5 to about 100 mg of WNT agonist per kg of body weight, or about 2 to about 50 mg of WNT agonist per kg of body weight, e.g., about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, or about 50 mg / kg of WNT agonist. In certain embodiments, the subject is administered about 3 to about 30 mg of R2M13-h26 per kg of body weight intravenously at least once per week, wherein R2M13-h26 comprises two polypeptides of SEQ ID NO: 9 and two polypeptides of SEQ ID NO: 10 linked by disulfide bonds.

[0018] In another related aspect, the disclosure provides a method of stimulating tissue repair in a subject having a gastrointestinal disorder, comprising administering to the subject an engineered WNT agonist or pharmaceutical composition disclosed herein. In certain embodiments, tissue repair is stimulated by (or the method results in the modulation of) at least one WNT pathway molecule selected from the group consisting of genes associated with the cell cycle, genes associated with stem and progenitor cell renewal and differentiation, genes associated with epithelial cell repair and barrier restoration, and / or any of the genes listed in any of Tables 4-8. In certain embodiments, the cell cycle-related genes are selected from those provided in Table 4, or Aurka, Aurkb, Ccna2, Ccnb1, Ccnb2, Ccnd2, Ccne1, Cdc45, Cdk1, Cdkn3, Cenpm, Cenpp, Cenpq, Cenpu, Hells, Mcm4, Mcm5, Mcm6, Mcm7, Myc, Pbk, Plk1, Rrm1, and Rrm2. In certain embodiments, the stem cell and progenitor cell renewal and differentiation-related genes are selected from those provided in Table 8, and Axin2, Id1, Hmga2, Nhp2, Foxq1, and Adh1. In certain embodiments, the gene related to epithelial cell repair and barrier restoration is selected from those provided in Table 6, or Apex1, Agr2, B3gnt7, Fcgbp, Muc2, Muc3, Tff3, Zg16, and Sprr2a3. In certain embodiments, after administration of engineered Wnt agonist, the expression of gene in one or more tissues and / or cells of subject is increased by at least 20%, at least 50%, at least 80%, at least 2-fold, at least 5-fold, at least 10-fold, or at least 20-fold, or is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%. In certain embodiments, WNT agonist is administered intravenously, for example, as a bolus injection. In certain embodiments, WNT agonist is administered at least once per week.In certain embodiments, the subject is administered about 0.5 to about 100 mg of WNT agonist per kg of body weight, or about 2 to about 50 mg of WNT agonist per kg of body weight, e.g., about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, or about 50 mg / kg of WNT agonist. In certain embodiments, the subject is administered about 3 to about 30 mg of R2M13-h26 per kg of body weight intravenously at least once per week, wherein R2M13-h26 comprises two polypeptides of SEQ ID NO: 9 and two polypeptides of SEQ ID NO: 10 linked by disulfide bonds.

[0019] In another related aspect, the present disclosure provides a method for reducing inflammation in a subject (or tissue or cell of the subject) having a gastrointestinal disorder, comprising administering to the subject an engineered WNT agonist or pharmaceutical composition disclosed herein.In certain embodiments, inflammation is reduced (or the method results in the modulation of) at least one WNT pathway molecule selected from the group consisting of the genes provided in Table 5, or Adamdec1, Atf3, Gpx2, Gsta3, Gstm1, Gstm3, Gdf15, Ihh, Il18, Lyz2, Nox1, Reg4, Sycn, Selenbp1, Tgfbr2, and Timp3.In certain embodiments, inflammation is reduced in gastrointestinal tissue, optionally epithelial tissue. In certain embodiments, inflammation is reduced in gastrointestinal epithelial cells, epithelial stem cells, TA1, TA2, basal goblet cells, Alt progenitor, Alt Enterocyte, EnteroPrecur, goblet cell 1, goblet cell 2, or enteroendocrine or tuft cells. In certain embodiments, following administration of the engineered Wnt agonist, expression of WNT pathway molecules in one or more tissues and / or cells of the subject is increased by at least 20%, at least 50%, at least 80%, at least 1.1-fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, 2-fold, at least 5-fold, at least 10-fold, or at least 20-fold, or is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%. In certain embodiments, the WNT agonist is administered intravenously, for example, as a bolus injection. In certain embodiments, the WNT agonist is administered at least once per week.In certain embodiments, the subject is administered about 0.5 to about 100 mg of WNT agonist per kg of body weight, or about 2 to about 50 mg of WNT agonist per kg of body weight, e.g., about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, or about 50 mg / kg of WNT agonist. In certain embodiments, the subject is administered about 3 to about 30 mg of R2M13-h26 per kg of body weight intravenously at least once per week, wherein R2M13-h26 comprises two polypeptides of SEQ ID NO: 9 and two polypeptides of SEQ ID NO: 10 linked by disulfide bonds.

[0020] In certain embodiments of any of the disclosed methods, the engineered Wnt agonist is R2M13-h26 or comprises a functional variant or fragment thereof. In certain embodiments of any of the disclosed methods, the subject is a mammal, optionally a human.

[0021] In another related aspect, the disclosure provides a method of restoring gastrointestinal epithelial barrier in a subject having damaged epithelium, comprising administering to the subject an engineered WNT agonist or pharmaceutical composition disclosed herein. In certain embodiments, the WNT agonist is administered intravenously, e.g., as a bolus injection. In certain embodiments, the WNT agonist is administered at least once per week. In certain embodiments, the subject is administered about 0.5 to about 100 mg of WNT agonist per kg of body weight, or about 2 to about 50 mg of WNT agonist per kg of body weight, e.g., about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, or about 50 mg / kg of WNT agonist. In certain embodiments, a subject is administered about 3 to about 30 mg / kg body weight of R2M13-h26 intravenously at least once per week, wherein R2M13-h26 comprises two polypeptides of SEQ ID NO: 9 and two polypeptides of SEQ ID NO: 10 linked by disulfide bonds. In some embodiments, the gastrointestinal epithelial barrier is restored by modulation of at least one WNT pathway molecule selected from the group consisting of genes associated with the cell cycle, genes associated with stem and progenitor cell renewal and differentiation, genes associated with epithelial cell repair and barrier restoration, and / or any of the genes listed in any of Tables 4, 5, 6, 7, 8, and 11. Genes associated with the cell cycle may be selected from those provided in Table 4, or Aurka, Aurkb, Ccna2, Ccnb1, Ccnb2, Ccnd2, Ccne1, Cdc45, Cdk1, Cdkn3, Cenpm, Cenpp, Cenpq, Cenpu, Hells, Mcm4, Mcm5, Mcm6, Mcm7, Myc, Pbk, Plk1, Rrm1, and Rrm2. Genes associated with the renewal and differentiation of stem and progenitor cells may be selected from those provided in Table 8, as well as Axin2, Id1, Hmga2, Nhp2, Foxq1, and Adh1.Genes associated with epithelial cell repair and barrier restoration may be selected from those provided in Table 6, or Apex1, Agr2, B3gnt7, Fcgbp, Muc2, Muc3, Tff3, Zg16, and Sprr2a3.

[0022] In some embodiments, the gastrointestinal epithelial barrier is restored by modulation of at least one WNT pathway molecule, where expression of the WNT pathway molecule in one or more tissues and / or cells of the subject is increased by at least 20%, at least 50%, at least 80%, at least 1.1-fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 2-fold, at least 5-fold, at least 10-fold, or at least 20-fold, or reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% after administration of the engineered Wnt agonist. In some embodiments, the gastrointestinal epithelial barrier is restored by modulation of at least one WNT pathway molecule, where expression of the WNT pathway molecule in one or more tissues and / or cells of the subject is increased within about 24 hours after administration of the engineered Wnt agonist. In some embodiments, the damaged epithelium of the subject is substantially restored within about 6 days after administration of the engineered Wnt agonist. In some embodiments, administration of the engineered Wnt agonist to a subject does not induce hyperproliferation of normal epithelium.

[0023] In another related aspect, the disclosure provides a method of inducing epithelial progenitor cell differentiation in a subject with a gastrointestinal disorder, comprising administering to the subject an engineered WNT agonist or pharmaceutical composition disclosed herein. In certain embodiments, the WNT agonist is administered intravenously, e.g., as a bolus injection. In certain embodiments, the WNT agonist is administered at least once per week. In certain embodiments, the subject is administered about 0.5 to about 100 mg of WNT agonist per kg of body weight, or about 2 to about 50 mg of WNT agonist per kg of body weight, e.g., about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, or about 50 mg / kg of WNT agonist. In certain embodiments, a subject is administered about 3 to about 30 mg / kg body weight of R2M13-h26 intravenously at least once per week, wherein R2M13-h26 comprises two polypeptides of SEQ ID NO: 9 and two polypeptides of SEQ ID NO: 10 linked by disulfide bonds. In some embodiments, epithelial cell differentiation is induced by modulation of at least one WNT pathway molecule selected from the group consisting of genes associated with the cell cycle, genes associated with stem and progenitor cell renewal and differentiation, genes associated with epithelial cell repair and barrier restoration, and / or any of the genes listed in any of Tables 4, 5, 6, 7, 8, and 11. Genes associated with the cell cycle may be selected from those provided in Table 4, or Aurka, Aurkb, Ccna2, Ccnb1, Ccnb2, Ccnd2, Ccne1, Cdc45, Cdk1, Cdkn3, Cenpm, Cenpp, Cenpq, Cenpu, Hells, Mcm4, Mcm5, Mcm6, Mcm7, Myc, Pbk, Plk1, Rrm1, and Rrm2. Genes associated with the renewal and differentiation of stem and progenitor cells may be selected from those provided in Table 8, as well as Axin2, Id1, Hmga2, Nhp2, Foxq1, and Adh1.Genes associated with epithelial cell repair and barrier restoration may be selected from those provided in Table 6, or Apex1, Agr2, B3gnt7, Fcgbp, Muc2, Muc3, Tff3, Zg16, and Sprr2a3.

[0024] In some embodiments, epithelial cell differentiation is induced by modulation of at least one WNT pathway molecule, where expression of the WNT pathway molecule in one or more tissues and / or cells of the subject is increased by at least 20%, at least 50%, at least 80%, at least 1.1-fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, 2-fold, at least 5-fold, at least 10-fold, or at least 20-fold, or is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, following administration of the engineered Wnt agonist. In some embodiments, epithelial cell differentiation is induced by modulation of at least one WNT pathway molecule, where expression of the WNT pathway molecule in one or more tissues and / or cells of the subject is increased within about 24 hours of administration of the engineered Wnt agonist.

[0025] In some embodiments, administering engineered Wnt agonist induces progenitor cell differentiation into enterocyte, goblet cell, enteroendocrine or tuft cell in subject.In some embodiments, administering engineered Wnt agonist induces substantial progenitor cell differentiation in subject within about 48 hours.In some embodiments, administering engineered Wnt agonist to subject does not induce hyperproliferation of normal epithelium. [Brief description of the drawings]

[0026] [Figure 1] Figure 1 provides an exemplary structure of one embodiment of an engineered WNT agonist. The R2M13 anti-Fzd5,8 antibody comprises two heavy chains and two light chains, with each light chain also comprising an anti-LRP6 VHH fused via a tag to its N-terminus.

[0027] [Diagram 2] Figure 2A provides an amino acid sequence alignment of the parental LRP6-binding VHH, VHH26, with the closest human germline gene. The CDR H1, H2, and H3 loop residues defined by the Kabat scheme are identified by thick lines at the top. Sequence alignment was performed using Clustal-Omega (https: / / www.ebi.ac.uk / Tools / msa / clustalo / ). Figure 2B provides an amino acid sequence alignment of the parental VHH26 with six different humanized variants thereof. The CDR H1, H2, and H3 loop residues defined by the Kabat scheme are identified by thick lines at the top. Sequence alignment was performed using Clustal-Omega (https: / / www.ebi.ac.uk / Tools / msa / clustalo / ).

[0028] [Diagram 3] Figures 3A-3B show the biophysical characterization of six humanized VHH26 variants (H1-H6). Figure 3A shows SDS-PAGE of Ni-pulldown elution fractions from metal-affinity chromatography. SEC and Octect-BLI profiles of VHH26-H1, VHH26-H2, VHH26-H3, VHH26-H4, VHH26-H5, and VHH26-H6 humanized variants are summarized in the table in Figure 3B. % monomer is based on the SEC profile of humanized VHH26 after ProA purification. ND = not determined.

[0029] [Figure 4] FIG. 4 shows the EC50 for binding to LRP5 or LRP6 of the indicated parent and variant VHH domains in the fully engineered Wnt agonist format.

[0030] [Diagram 5]5A-5D show the in vitro activity of the Fzd5,8 subfamily-specific Wnt mimetic R2M13-26. FIG. 5A is a graph showing the binding affinity of R2M13-26 Fzd5,8 binding factor IgG to its target Fzd5 CRD, as measured by Octet. FIG. 5B is a graph showing the binding affinity of R2M13-26 Fzd5,8 binding factor IgG to its target Fzd8 CRD, as measured by Octet. FIG. 5C is a graph showing the binding specificity of R2M13-26 Fzd5,8 binding factor IgG to each of the ten Fzd CRDs, as investigated by Octet. FIG. 5D is a graph showing dose-dependent STF activity of R2M13-26, the Fzd1,2,7 specific mimetic 1RC07-26, and the Fzd1,2,5,7,8 pan-specific mimetic R2M3-26 in the presence of 20 nM RSPO2 measured in Huh-7 cells.

[0031] [Figure 6] Figure 6 provides the heavy and light chain sequences present in the engineered WNT agonist, R2M13-h26. The heavy and light chain VH and VL domains are underlined, the VHH26 domain is italicized, and the CDR residues are bolded.

[0032] [Figure 7] FIG. 7 provides a schematic of the DSS model of acute colitis and serum antibody exposure resulting after treatment with various non-humanized and humanized versions, including R2M13-03-LALAPG (non-humanized), R2M13-26-LALAPG (non-humanized), R2M13-36-LALAPG (non-humanized), R2M13-humanized-03-LALAPG, R2M13-humanized-26-LALAPG, R2M13-humanized-36-LALAPG, R2M13-humanized-03-N297G, and R2M13-humanized-36-N297G.

[0033] [Figure 8]FIG. 8 provides a graph showing the Disease Activity Index for animals treated with various non-humanized and humanized versions, including R2M13-03-LALAPG (non-humanized), R2M13-26-LALAPG (non-humanized), R2M13-36-LALAPG (non-humanized), R2M13-humanized-03-LALAPG, R2M13-humanized-26-LALAPG, R2M13-humanized-36-LALAPG, R2M13-humanized-03-N297G, and R2M13-humanized-36-N297G. At 10 days, the lines on the graph correspond, from top to bottom, to: R2M13-h03-LALAPG, anti-GFP, R2M13-h03-N297G, R2M13-03-LALAPG, R2M13-36-LALAPG, R2M13-h36-N297G (after R2M13-h36-LALAPG), R2M13-h36-LALAPG, R2M13-h26-LALAPG, and no DSS, where "h" indicates humanization.

[0034] [Figure 9] FIG. 9 provides a graph showing cytokine levels in animals treated with various controls and non-humanized and humanized versions including, from left to right, no DSS, anti-GFP, parental R2M13-03-LALAPG (non-humanized), parental R2M13-26-LALAPG (non-humanized), parental R2M13-36-LALAPG (non-humanized), R2M13-humanized-03-LALAPG, R2M13-humanized-26-LALAPG, R2M13-humanized-36-LALAPG, R2M13-humanized-03-N297G, and R2M13-humanized-36-N297G.

[0035] [Figure 10]FIG. 10 provides a graph showing levels of lipocalin 2 in animals treated with various control and non-humanized and humanized versions including no DSS, anti-GFP, parental R2M13-03-LALAPG (non-humanized), parental R2M13-26-LALAPG (non-humanized), parental R2M13-36-LALAPG (non-humanized), R2M13-humanized-03-LALAPG, R2M13-humanized-26-LALAPG, R2M13-humanized-36-LALAPG, R2M13-humanized-03-N297G, and R2M13-humanized-36-N297G.

[0036] [Figure 11] Figure 11 provides photomicrographs showing restoration of epithelial tight junction marker ZO-1 following treatment with engineered WNT agonists in vivo in a DSS model of acute colitis. Brightly stained areas are ZO-1.

[0037] [Figure 12] FIG. 12 provides photomicrographs showing repair of damaged colonic epithelium following treatment with the engineered WNT agonist R2M13-h26-LALPG in vivo, compared to control anti-GFP, in a DSS model of acute colitis.

[0038] [Figure 13] FIG. 13 provides micrographs showing restoration of epithelial cell lineages including colonocytes, goblet cells, and tuft cells following treatment with the engineered WNT agonist R2M13-h26-LALPG in vivo, compared to control anti-GFP, in a DSS model of acute colitis.

[0039] [Figure 14] FIG. 14 provides graphs and tables showing the pharmacokinetics (PK) of parental R2M13-26-LALAPG and humanized R2M13-26-LALAPG after intravenous injection, as determined by measuring the amount of antibody in the serum at various time points after administration to rats and comparing with data obtained from mice.

[0040] [Figure 15] FIG. 15 provides a schematic diagram of the acute chronic colitis DSS animal model system.

[0041] [Figure 16] 16 provides a graph showing the Disease Activity Index (DAI) of animals treated with R2M13-h26-LALAPG (R2M13-h26) or R2M13-26-LALAPG (R2M13-26). At day 10, the lines on the graph correspond, from top to bottom, to the following: anti-GFP, cyclosporine A, R2M13-h26 (2mpk x 1), R2M13-h26 (20mpk x 1), R2M13-h26 (1mpk x 2), R2M13-h26 (6mpk x 1), R2M13-26 (3mpk x 2), R2M13-h26 (10mpk x 2), R2M13-26 (10mpk x 2), and no DSS.

[0042] [Figure 17] FIG. 17 shows cross sections of the transverse colon with H&E staining of animals treated with R2M13-h26 compared to anti-GFP or cyclosporine A.

[0043] [Figure 18] FIG. 18 provides a schematic of the chronic DSS colitis animal model.

[0044] [Figure 19] FIG. 19 shows photomicrographs of transverse colon sections after the indicated treatments.

[0045] [Figure 20] FIG. 20 provides graphs showing the histology scores and global disease activity index after the indicated treatments.

[0046] [Figure 21] FIG. 21 provides graphs showing lipocalin-2 and IL-6 expression after the indicated treatments.

[0047] [Figure 22] FIG. 22 is a diagram of the chronic DSS colitis animal model.

[0048] [Diagram 23] FIG. 23 provides a graph showing the disease activity index for animals treated with R2M13-h26 or IL12 / 23p40.

[0049] [Figure 24] FIG. 24 provides graphs showing expression of the indicated cytokines in animals treated with R2M13-h26 or IL12 / 23p40.

[0050] [Diagram 25] FIG. 25 provides graphs showing expression of Axin2 and Ki67 following the indicated treatments with R2M13-26-LALAPG (R2M13-26).

[0051] [Figure 26-1] Figures 26A-26C show the different cell types detected in the colon with scRNA-seq for non-injured and DSS-treated mice: Figure 26A is a schematic showing the experimental design of the scRNA-seq experiment. Figure 26B is a plot of the first two principal components: lineages / tissue layers are shown, with three groups radiating from the center. Figure 26C provides graphs showing the strong impact of DSS injury on a number of differential genes expressed in different tissue layers / lineages. The graph on the left shows the number of differentially expressed genes from each tissue layer of DSS mice compared to non-injured mice on days 5 and 6; the graph on the right shows the number of differentially expressed genes from each tissue layer of treatment with R2M13-26 compared to anti-GFP on days 5 and 6. Tissues / lines correspond from top to bottom of each bar to epithelium, immune, and stromal, and at day 5 are almost entirely epithelium following treatment with R2M13-26-LALAPG (R2M13-26). [Figure 26-2] Same as above.

[0052] [Figure 27-1] Figures 27A-27C show that DSS affects all tissue layers by day 5, while the main effect of R2M13-26-LALAPG (R2M13-26) at day 5, 24 hours post-treatment, is on the epithelium. Figures 27A-27C show that R2M13-26-LALAPG (R2M13-26) increased Wnt target and cell cycle gene expression and expanded progenitor cells in the injured epithelium. Figure 27A is a table listing the top selected gene sets (from GSEA) enriched in DSS-injured epithelium treated with R2M13-26 compared to DSS-injured epithelium treated with anti-GFP. Figures 27B and 27C show validation of scRNA-seq analysis in tissues. Figure 27B shows RNA in situ hybridization of two Wnt target genes, Axin2 and Cdkn3, in non-injured, DSS / anti-GFP and DSS / R2M13-26 treated groups (day 5); nuclei labeled with DAPI. Scale bar represents 100 microns. Figure 27C shows immunohistochemistry for MKI67, a proliferative cell marker, in non-injured, DSS / anti-GFP and DSS / R2M13-26 treated colon samples (day 6); nuclei labeled with DAPI. Scale bar represents 100 microns. [Figure 27-2] Same as above.

[0053] [Figure 28-1]Figures 28A-28E show that R2M13-26-LALAPG (R2M13-216) treatment caused accelerated proper differentiation in the DSS model: Figures 28A-D provide graphs showing uniform manifold approximate projection (UMAP) plots of epithelial cells. Figure 28A is a graph showing UMAP of epithelial cells colored by cluster / cell type. Figure 28B is a graph showing UMAP colored by experimental condition of cells. Figure 28C is a graph showing minimum spanning tree of cluster medoids, connecting clusters based on similarity. Only cell types in which injured cells were almost exclusively absent were included. Stem cells and TA2 cell types were merged and set as starting clusters. FIG. 28D is a graph showing completed slingshot predicted lineage trajectories showing the transition from stem cells / TA cells to EnteroPrecur cells (upwards) on the way to immature and mature enterocytes; and the branching from stem cells / TA cells down towards either brush cells or goblet cells and enteroendocrine cells with a second branching from goblet progenitor cell types in between. FIG. 28E provides a histogram of the number of cells from the indicated treatment groups at the indicated positions along the pseudo-time series or lineage trajectory axis derived from the enterocyte lineage shown in FIG. 28D at 48 hours / 6 days. The vertical dashed red lines indicate the same position along the axis in all three plots and the distribution shows how many cells are present at that position. The order of the pseudo-time series (x-axis) is the same in each plot and is ordered from left to right. FIG. 28E shows that progression towards the enterocyte lineage was increased with R2M13-26-LALPG (R2M13-26) treatment. [Figure 28-2] Same as above. [Figure 28-3] Same as above.

[0054] [Figure 29]Figures 29A-29L and 29A'-29L' show that Frizzled family receptors showed differential expression patterns in the small intestinal epithelium. Figures 29A-29L provide graphs showing the expression of each of the 10 Fzd receptors (Fzd1-10), Axin2, and Lgr5 in normal duodenum, respectively, as determined by RNAscope in situ hybridization. Figures 29A'-29L' provide graphs that are enlarged photographs showing Fzd expression in small intestinal crypts. The arrow in panel E' indicates an intestinal stem cell.

[0055] [Diagram 30] Figures 30A-30T show that Frizzled family receptors were expressed at different levels in the colon. Figures 30A-30J provide graphs showing colon expression of 10 Fzd receptors in naive mice, as examined by RNAscope in situ hybridization. Figures 30K-30T provide graphs showing colon expression of 10 Fzd receptors in mice treated with 4% DSS for 7 days.

[0056] [Diagram 31] Figure 31 shows reduced inflammation due to reduced neutrophil infiltration. S100A9 is a marker for neutrophil infiltration and CD45 is a marker for activated inflammatory cells.

[0057] [Diagram 32] FIG. 32 provides a graph showing the increase in serum ALP following administration of the indicated doses of R2M13-h26.

[0058] [Diagram 33] FIG. 33 is a schematic diagram showing the pharmacokinetic assay used to measure the mean serum concentrations of R2M13-h26.

[0059] [Diagram 34] FIG. 34 provides a graph showing the mean serum concentrations of R2M13-h26 in groups 2-4.

[0060] [Diagram 35] Figure 35 provides a graph showing individual serum R2M13-h26 concentrations measured after the first dose. The arrows indicate two animals in the 30 mg / kg dose group that began to experience accelerated clearance 3 days after dosing.

[0061] [Diagram 36] Figures 36A-36B provide a pair of graphs showing the increase in ALP on days 0-7 (Figure 36A) and days 28-42 (Figure 36B) for different dose groups of R2M13-h26.

[0062] [Figure 37] FIG. 37 provides a graph showing the mean serum R2M13-h26 concentrations following a single dose of R2M13-h26.

[0063] [Figure 38] FIG. 38 provides a table showing the PK parameters for R2M13-h26 following a single dose of R2M13-h26. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0064] Detailed Description As used in this specification, including the appended claims, singular words such as "a," "an," and "the" include their corresponding plural referents unless the context clearly dictates otherwise.

[0065] All references cited herein are incorporated by reference to the same extent as if each individual publication, patent application, or patent was specifically and individually indicated to be incorporated by reference.

[0066] I. Definition The "activity" of a molecule can describe or refer to the binding of the molecule to a ligand or receptor, for example, for catalytic activity, for the ability to stimulate gene expression, for antigenic activity, for modulating the activity of other molecules. The "activity" of a molecule can also refer to the activity in modulating or maintaining cell-cell interactions, for example, adhesion, or in maintaining the structure of a cell, for example, the cell membrane or cytoskeleton. "Activity" can also refer to specific activity, for example, [catalytic activity] / [mg protein], or [immunological activity] / [mg protein], etc.

[0067] The term "administering" or "introducing" or "providing" as used herein refers to delivering composition to a cell, a plurality of cells, tissue, tissue organoid, and / or organ of a subject, or to a subject.Such administering or introducing can be carried out in vivo, in vitro or ex vivo.

[0068] As used herein, the term "antibody" refers to an isolated or recombinant binding agent that contains the necessary variable region sequence to specifically bind to an antigen epitope. Thus, an antibody is any form of antibody or fragment thereof that exhibits the desired biological activity, such as binding to a specific target antigen. Thus, antibody is used in the broadest sense, specifically covering monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, VHH antibodies, camelid antibodies, nanobodies, diabodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, including but not limited to scFv, Fab and Fab2, provided that they exhibit the desired biological activity.

[0069] An "antibody fragment" includes a portion of an intact antibody, such as the antigen-binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (e.g., Zapata et al., Protein Eng. 8(10): 1057-1062 (1995)); single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments. Papain digestion of an antibody produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment (a designation reflecting the ability to crystallize readily). Pepsin treatment produces an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen.

[0070] The term "antigen" refers to a molecule or a portion of a molecule that can be bound by a selective binding agent such as an antibody and can be used in an animal to generate antibodies capable of binding to the epitope of the antigen. In certain embodiments, a binding agent (e.g., an engineered WNT agonist or binding region thereof, or a WNT antagonist) is said to specifically bind to an antigen if it preferentially recognizes its target antigen in a complex mixture of proteins and / or macromolecules.

[0071] The term "antigen-binding fragment" as used herein refers to a polypeptide fragment containing at least one CDR of an immunoglobulin heavy and / or light chain, or of a Nanobody® (Nab), that binds to an antigen of interest, particularly one or more FZD receptors, or LRP5 and / or LRP6. In this regard, the antigen-binding fragment of an antibody described herein can contain one, two, three, four, five, or all six CDRs of the VH and VL from an antibody that binds to one or more FZD receptors, or LRP5 and / or LRP6.

[0072] As used herein, the terms "biological activity" and "biologically active" refer to the activity ascribed to a particular biological element in a cell. For example, the "biological activity" of a WNT agonist, or a fragment or variant thereof, refers to the ability to mimic or enhance WNT signaling. As another example, the biological activity of a polypeptide or a functional fragment or variant thereof refers to the ability of the polypeptide or a functional fragment or variant thereof to perform its native function, such as binding, enzymatic activity, etc. In some embodiments, a functional fragment or variant retains at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% of the activity of the corresponding native protein or nucleic acid. As a third example, the biological activity of a gene regulatory element, such as a promoter, enhancer, Kozak sequence, etc., refers to the ability of the regulatory element or a functional fragment or variant thereof to regulate the expression of a gene to which it is operably linked, i.e., to promote, enhance, or activate the translation of the gene, respectively.

[0073] The term "bifunctional antibody" as used herein refers to an antibody that contains a first arm that has specificity for one antigenic site and a second arm that has specificity for a different antigenic site, i.e., a bifunctional antibody that has dual specificity.

[0074] "Bispecific antibodies" are used herein to refer to full-length antibodies generated by quadroma technology (see Milstein et al., Nature, 305 (5934): 537-540 (1983)), by chemical conjugation of two different monoclonal antibodies (see Staerz et al., Nature, 314 (6012): 628-631 (1985)), or by knobs-into-holes or similar techniques that introduce mutations into the Fc region, thereby resulting in multiple different immunoglobulin species, only one of which is a functional bispecific antibody (see Holliger et al., Proc. Natl. Acad. Sci. USA, 90 (14): 6444-6448 (1993)). A bispecific antibody binds one antigen (or epitope) with one of its two binding arms (one HC / LC pair) and a different antigen (or epitope) with a second arm (a different HC / LC pair). By this definition, a bispecific antibody has two separate antigen-binding arms (in terms of both specificity and CDR sequences) and is monovalent for each antigen to which it binds.

[0075] "Comprising" means that the recited elements are required, e.g., for a composition, method, kit, etc., but that other elements may be included, e.g., to form a composition, method, kit, etc. within the scope of the claims. For example, an expression cassette that "comprises" a gene encoding a therapeutic polypeptide operably linked to a promoter is an expression cassette that may include other elements in addition to the gene and promoter, e.g., polyadenylation sequences, enhancer elements, other genes, linker domains, etc.

[0076] "Consisting essentially of" means that the scope of a described, e.g., composition, method, kit, etc., is limited to the specified materials or steps that do not substantially affect the basic and novel feature(s) of the, e.g., composition, method, kit, etc. For example, an expression cassette "consisting essentially of" a gene encoding a therapeutic polypeptide operably linked to a promoter and polyadenylation sequence may contain additional sequences, e.g., linker sequences, so long as they do not substantially affect the transcription or translation of the gene. As another example, a variant, or mutant, polypeptide fragment "consisting essentially of" a recited sequence has the amino acid sequence of the recited sequence at the border of the sequence based on the full-length native polypeptide from which it is derived, plus or minus about 10 amino acid residues, e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 less residues than the recited binding amino acid residues, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 more residues than the recited binding amino acid residues.

[0077] "Consisting of" means that any element, step or ingredient not specified in the claim is excluded from the composition, method or kit. For example, a polypeptide or polypeptide domain "consisting of" a recited sequence contains only the recited sequence.

[0078] A "control element" or "control sequence" is a nucleotide sequence involved in molecular interactions that contribute to the functional regulation of a polynucleotide, including the replication, duplication, transcription, splicing, translation or degradation of the polynucleotide. This regulation can affect the frequency, speed or specificity of the process, and can be of an enhancing or inhibitory nature. Control elements known in the art include, for example, transcriptional regulatory sequences, such as promoters and enhancers. A promoter is a DNA region that, under certain conditions, can bind RNA polymerase and initiate transcription of the coding region that is usually located downstream (in the 3' direction) from the promoter.

[0079] An "epitope" is a specific region on an antigen that an antibody recognizes and binds to, also called an "antigenic determinant." Epitopes are on the surface of a protein and are usually 5-8 amino acids long. Proteins have a three-dimensional folded structure, and epitopes can only be recognized in the form they exist in solution, or in their native form. If an epitope is made up of amino acids held together by a three-dimensional structure, it is a conformational or discontinuous epitope. If an epitope exists on a single polypeptide chain, it is a continuous or linear epitope. Depending on the epitope it recognizes, an antibody may only bind to a fragment or denatured segment of a protein, or it may be able to bind to the native protein.

[0080] The part of an antibody or antibody fragment that recognizes an epitope is called the "epitope binding domain" or "antigen binding domain". The epitope or antigen binding domain of an antibody or antibody fragment resides in the Fab fragment, while the effector function resides in the Fc fragment. Six segments known as complementarity determining regions (CDRs) in the variable regions of the heavy and light chains (VH and VL) loop out from the remaining framework (FR) globular structure of the antibody and interact to form an exposed surface at one end of the molecule. This is the antigen binding domain. Generally, four to six of the CDRs are directly involved in binding to the antigen, although fewer CDRs may provide the primary binding motif.

[0081] "Expression vector" includes vectors, such as plasmids, minicircles, viral vectors, liposomes, etc., as discussed herein or known in the art, that contain a region encoding a gene product of interest and are used to cause expression of the gene product in an intended target cell. Expression vectors also include control elements, such as promoters, enhancers, UTRs, miRNA targeting sequences, etc., operably linked to the coding region to facilitate expression of the gene product in the target. The combination of control elements and operably linked gene(s) for expression is sometimes referred to as an "expression cassette," many of which are known and available in the art, or can be readily constructed from components available in the art.

[0082] As used herein, the term "FR set" refers to four adjacent amino acid sequences that frame the CDRs of a CDR set of a heavy or light chain V region. Some FR residues may contact bound antigen; however, FRs, especially those immediately adjacent to CDRs, are primarily responsible for folding the V region into the antigen-binding site. Within FRs, certain amino acid residues and certain structural features are highly conserved. In this regard, all V region sequences contain an internal disulfide loop of approximately 90 amino acid residues. When the V region folds into the binding site, the CDRs are displayed as protruding loop motifs that form an antigen-binding surface. It is generally understood that there are conserved structural regions of FRs that influence the folding shape of the CDR loops to be in a certain "canonical" structure regardless of the exact CDR amino acid sequence. In addition, certain FR residues have been found to participate in non-covalent interdomain contacts that stabilize the interaction of the heavy and light chains of an antibody.

[0083] A "humanized" antibody or fragment thereof refers to an antibody or fragment thereof from a non-human species in which the protein sequence has been modified to increase similarity to the antibody variants naturally produced in humans. The process of "humanization" is typically applied to monoclonal antibodies developed for administration to humans.

[0084] The terms "individual," "host," "subject," and "patient" are used interchangeably herein and refer to mammals, including, but not limited to, humans and non-human primates, including monkeys and humans, mammalian sport animals (e.g., horses), mammalian farm animals (e.g., sheep, goats, etc.), mammalian pets (e.g., dogs, cats, etc.); and rodents (e.g., mice, rats, etc.).

[0085] "Monoclonal antibody" refers to a homogeneous antibody population, which is composed of amino acids (naturally occurring and non-naturally occurring) involved in selective binding of an epitope. Monoclonal antibodies are highly specific and directed to a single epitope. The term "monoclonal antibody" encompasses intact and full-length monoclonal antibodies, as well as fragments thereof (e.g., Fab, Fab', F(ab')2, Fv), single chain (scFv), Nanobodies®, variants thereof, fusion proteins comprising antigen-binding fragments of monoclonal antibodies, humanized monoclonal antibodies, chimeric monoclonal antibodies, and any other modified configurations of immunoglobulin molecules that contain an antigen-binding fragment (epitope recognition site) with the required specificity and ability to bind to the epitope, including engineered WNT agonists disclosed herein. It is not intended to be limited as to the source of the antibody or the manner in which the antibody is made (e.g., by hybridoma, phage selection, recombinant expression, transgenic animals, etc.). The term encompasses whole immunoglobulins as well as fragments and the like described herein or falling under the definition of "antibody."

[0086] The term "native" or "wild type" as used herein refers to a nucleotide sequence, e.g., a gene or gene product, e.g., RNA or protein, present in a wild type cell, tissue, organ or organism. The term "variant" as used herein refers to a variant of a reference polynucleotide or polypeptide sequence, e.g., a native polynucleotide or polypeptide sequence, i.e., a variant having less than 100% sequence identity with a reference polynucleotide or polypeptide sequence. In other words, a variant comprises at least one amino acid difference (e.g., amino acid substitution, amino acid insertion, amino acid deletion) compared to a reference polynucleotide sequence, e.g., a native polynucleotide or polypeptide sequence. For example, a variant can be a polynucleotide having 50% or more, 60% or more, or 70% or more sequence identity with a full-length native polynucleotide sequence, e.g., 75% or 80% or more, e.g., 85%, 90% or 95% or more identity, e.g., 98% or 99% identity with a full-length native polynucleotide sequence. In another example, a variant can be a polypeptide that has 70% or more sequence identity with a full-length native polypeptide sequence, for example, 75% or 80% or more, for example, 85%, 90% or 95% or more, for example, 98% or 99% identity with a full-length native polypeptide sequence. A variant can also include a variant fragment of a reference, e.g., native, sequence that shares 70% or more sequence identity with a fragment of the reference, e.g., native, sequence, for example, 75% or 80% or more, for example, 85%, 90% or 95% or more, for example, 98% or 99% identity with the native sequence.

[0087] "Operably linked" or "operably linked" refers to the juxtaposition of genetic elements, where these elements are in a relationship that allows them to act in the expected manner. For example, a promoter is operably linked to a coding region if it helps initiate transcription of the coding sequence. There may be intervening residues between the promoter and the coding region, provided that this functional relationship is maintained.

[0088] As used herein, the terms "polypeptide," "peptide," and "protein" refer to polymers of amino acids of any length. These terms also encompass amino acid polymers that have been modified to include, for example, disulfide bond formation, glycosylation, lipidation, phosphorylation, or conjugation with a labeling component.

[0089] The term "polynucleotide" refers to a polymeric form of nucleotides of any length, including either deoxyribonucleotides or ribonucleotides, or analogs thereof. A polynucleotide may include modified nucleotides, such as methylated nucleotides and nucleotide analogs, and may be interrupted by non-nucleotide components. If present, modifications to the nucleotide structure may be added before or after assembly of the polymer. The term polynucleotide, as used herein, refers interchangeably to double-stranded and single-stranded molecules. Unless otherwise specified or required, any embodiment of the invention disclosed herein that is a polynucleotide encompasses both the double-stranded form and each of the two complementary single-stranded forms that are known or predicted to form a double-stranded form.

[0090] A polynucleotide or polypeptide has a certain percentage of "sequence identity" with another polynucleotide or polypeptide, where "sequence identity" means that the percentage of bases or amino acids are the same when the two sequences are aligned and then compared. As used herein, the terms "identity" and "identical" refer to the percentage of residues that perfectly match in the alignment of the target sequence with a reference sequence, such as an alignment generated by the BLAST algorithm, with respect to a polypeptide or polynucleotide sequence of interest. Identity is calculated over the entire length of the reference sequence, unless otherwise specified. Thus, a target sequence "shares at least x% identity" with a reference sequence if, when the reference sequence (as the query sequence) is aligned with the target sequence (as the subject sequence), at least x% (truncated) of the residues in the subject sequence are aligned as exact matches with the corresponding residues in the query sequence. Here, the denominator is the entire length of the reference sequence, plus the length of any gaps inserted in the reference sequence by the alignment of the reference sequence with the target sequence. When the subject sequence has a variable position (eg, a residue designated X), any alignment with that residue in the query sequence is counted as a match.

[0091] Sequence similarity can be determined in several different ways.To determine sequence identity, sequences can be aligned using methods and computer programs, including BLAST available on the World Wide Web at ncbi.nlm.nih.gov / BLAST / .Sequence alignment can be performed using the NCBI Blast service (BLAST+ version 2.12.0) or another program that produces the same results.Unless otherwise indicated, sequence identity is determined using the BLAST algorithm (e.g., bl2seq) with default parameters.

[0092] Another alignment algorithm is FASTA, available in the Genetics Computing Group (GCG) package, a wholly owned subsidiary of Oxford Molecular Group, Inc., Madison, Wis., USA. Other techniques for alignment are described in Methods in Enzymology, vol. 266: Computer Methods for Macromolecular Sequence Analysis (1996), ed. Doolittle, Academic Press, Inc., a division of Harcourt Brace & Co., San Diego, Calif., USA. Alignment programs that allow gaps in sequences are of particular interest. Smith-Waterman is one type of algorithm that allows gaps in sequence alignment. See Meth. Mol. Biol. 70: 173-187 (1997). Also, the GAP program, which uses the Needleman and Wunsch alignment method, can be utilized to align sequences. See J. Mol. Biol. 48: 443-453 (1970).

[0093] The BestFit program is of interest, which uses the local homology algorithm of Smith and Waterman (Advances in Applied Mathematics 2: 482-489 (1981)) to determine sequence identity. Gap creation penalties generally range from 1 to 5, usually from 2 to 4, but in many embodiments are 3. Gap extension penalties generally range from about 0.01 to 0.20, but in many instances are 0.10. The program has default parameters determined by the input sequences being compared. Preferably, sequence identity is determined using the default parameters determined by the program. This program is also available from the Genetics Computing Group (GCG) package, Madison, Wis., USA.

[0094] Another program of interest is the FastDB algorithm. FastDB is described in Current Methods in Sequence Comparison and Analysis, Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp. 127-149, 1988, Alan R. Liss, Inc. Percent sequence identity is calculated by FastDB based on the following parameters: mismatch penalty: 1.00; gap penalty: 1.00; gap size penalty: 0.33; and joining penalty: 30.0.

[0095] "Promoter" as used herein includes a DNA sequence that directs the binding of RNA polymerase and thereby promotes RNA synthesis, i.e., a minimal sequence sufficient to direct transcription. A promoter and corresponding protein or polypeptide expression can be universal, meaning that it is strongly active in a wide range of cells, tissues and species, or the promoter and expression can be cell type-specific, tissue-specific or species-specific. A promoter can be "constitutive", meaning that it is continuously active, or a promoter can be "inducible", meaning that it can be activated or inactivated by the presence or absence of a biotic or non-biotic factor. Enhancer sequences, which may or may not be contiguous with the promoter sequence, are also included in the nucleic acid construct or vector of the present invention. Enhancer sequences affect promoter-dependent gene expression and can be located in the 5' or 3' region of the native gene.

[0096] "Recombinant," when applied to a polynucleotide, means that the polynucleotide is the product of any combination of cloning, restriction, or ligation steps and other procedures that result in a construct that is distinct from polynucleotides found in nature.

[0097] The terms "treatment", "treating" and the like are used herein to generally mean obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, in terms of completely or partially preventing a disease or its symptoms, e.g., reducing the likelihood that a disease or its symptoms will occur in a subject, and / or may be therapeutic, in terms of partially or completely curing a disease and / or the deleterious effects caused by the disease. "Treatment", as used herein, covers any treatment of a disease in a mammal, including: (a) preventing the disease from occurring in a subject who is 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. Therapeutic agents may be administered before, during, or after the onset of the disease or injury. Treatment of ongoing disease, where the treatment stabilizes or reduces the undesirable clinical symptoms of the patient, is particularly targeted. Such treatment is desirably performed before complete loss of function in the affected tissue. The subject therapy is desirably administered during, and in some cases after, the symptomatic stage of the disease.

[0098] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology techniques, microbiology, biochemistry and immunology, which are within the skill of the art. Such techniques are described in “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook et al., 1989); “Oligonucleotide Synthesis” (MJ Gait, ed., 1984); “Animal Cell Culture” (RI Freshney, ed., 1987); “Methods in Enzymology” (Academic Press, Inc.); “Handbook of Experimental Immunology” (DM Weir & CC Blackwell, eds.); “Gene Transfer Vectors for Mammalian Cells” (JM Miller & MP Calos, eds., 1987); “Current Protocols in Molecular Biology” (FM Ausubel et al., eds., 1987); “PCR: The Polymerase Chain Reaction”, (Mullis et al., eds., 1994); and “Current Protocols in Immunology“ (JE Coligan et al., eds., 1991), each of which is expressly incorporated herein by reference.

[0099] Some aspects of the present invention are described below in conjunction with application examples for illustration. It should be understood that numerous specific details, relationships and methods are set forth to provide a thorough understanding of the present invention. However, those skilled in the art of the relevant art will readily recognize that the present invention can be implemented without one or more of the specific details or with other methods. The present invention is not limited by the illustrated acts or sequence of events. Because some acts can be performed in different sequences and / or in conjunction with other acts or events. Moreover, not all illustrated acts or events are required to implement the methodology according to the present invention.

[0100] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present invention. As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms unless the context clearly dictates otherwise. Furthermore, when the terms "including," "includes," "having," "has," "including," or variations thereof are used in either the detailed description and / or the claims, such terms are intended to be inclusive in the same manner as the term "comprising."

[0101] The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which acceptable error range depends in part on the constraints of the method by which the value is measured or determined, i.e., the measurement system. For example, "about" may mean within a range of 1 or more than 1 standard deviation, according to the practice in the art. Alternatively, "about" may mean within a range of up to 20%, preferably up to 10%, more preferably up to 5%, and even more preferably up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term may mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold of a value. When a particular value is described in the present application and claims, unless otherwise stated, the term "about" should be assumed to mean within an acceptable error range for the particular value.

[0102] 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. In case of conflict, it is to be understood that the present disclosure supersedes the disclosure of any incorporated publications.

[0103] It is further noted that the claims may be construed to exclude any optional element, and thus, this statement is intended to serve as a predicate for use of exclusive terminology such as "solely," "solely," and the like, or use of a "negative" limitation in connection with the recitation of claim elements.

[0104] Unless otherwise indicated, all terms used herein have the same meaning as they would to one of ordinary skill in the art and the practice of the present invention employs conventional techniques of microbiology and recombinant DNA technology that are within the knowledge of those skilled in the art.

[0105] II.General The present invention provides compositions and methods for modulating WNT signaling to ameliorate a variety of diseases and disorders that may benefit from modulation of the WNT signaling pathway, such as gastrointestinal disorders, including but not limited to inflammatory bowel diseases, including but not limited to Crohn's disease, Crohn's disease with fistulization, and ulcerative colitis.

[0106] WNT ("Wingless-related integration site" or "Wingless and Int-1" or "Wingless-Int") ligands and their signals play important roles in controlling the development, homeostasis and regeneration of many important organs and tissues, including bone, liver, skin, stomach, intestine, kidney, central nervous system, mammary gland, taste buds, ovary, cochlea, lung and many other tissues (reviewed, for example, by Clevers, Loh, and Nusse, 2014; 346:1248012). Modulation of the Wnt signaling pathway has potential for the treatment of degenerative diseases and tissue injury.

[0107] One of the challenges for modulating WNT signaling as a therapeutic agent is the existence of multiple WNT ligands and WNT receptors, Frizzled 1-10 (FZD1-10), with many tissues expressing multiple overlapping FZDs. In addition to FZDs, canonical WNT signaling also involves low-density lipoprotein (LDL) receptor-related protein 5 (LRP5) and / or low-density lipoprotein (LDL) receptor-related protein 6 (LRP6) as co-receptors that are widely expressed in a variety of tissues. LRP5 and LRP6 are collectively referred to as LRP5 / 6, and references to "LRP5 / 6 binding" or the like refer to binding to LRP5 and / or LRP6.

[0108] R-spondins 1-4 (RSPO1-4) are a family of ligands that amplify WNT signals. Each of the R-spondins functions through a receptor complex that contains zinc and ring finger 3 (ZNRF3) or ring finger protein 43 (RNF43) at one end and leucine-rich repeat-containing G protein-coupled receptor 4-6 (LGR4-6) at the other (e.g., reviewed by Knight and Hankenson 2014, Matrix Biology; 37: 157-161). R-spondins may also function through additional mechanisms of action. ZNRF3 and RNF43 are two membrane-bound E3 ligases that specifically target WNT receptors (FZD1-10 and LRP5 or LRP6) for degradation. Binding of R-spondin to ZNRF3 / RNF43 and LGR4-6 causes clearance or sequestration of the ternary complex, thereby removing E3 ligase from the WNT receptor and stabilizing the WNT receptor, resulting in enhanced WNT signaling. Each R-spondin contains two furin domains (1 and 2), with furin domain 1 binding to ZNRF3 / RNF43 and furin domain 2 binding to LGR4-6. A fragment of R-spondin containing furin domains 1 and 2 is sufficient to amplify WNT signaling. Although R-spondin effects are dependent on WNT signaling, the effects of R-spondin are not tissue specific, since both LGR4-6 and ZNRF3 / RNF43 are widely expressed in various tissues.

[0109] Activation of WNT signaling by WNT agonists can be used to treat various diseases and disorders, including gastrointestinal disorders. Similarly, amplification of WNT signaling by RSPO or RSPO mimics can be used to treat various diseases and disorders, including gastrointestinal disorders. Previous studies in the literature suggest that RSPO can be used to treat experimental colonic colitis (J. Zhao et. al., 2007). WNT agonist molecules can also be used to treat gastrointestinal disorders. In particular, active WNT signaling can provide key stem cell maintenance signals and play an important role in regulating the regeneration of intestinal epithelium in homeostasis and injury.

[0110] The two intestinal epithelial lineages, absorptive and secretory, define the two main functions of the gastrointestinal organ. Secretory cells secrete hormones and also provide an important barrier against food-borne microbes, toxins, and antigens, mainly by secreting mucus and antimicrobial peptides. In contrast, absorptive cells are mainly located at the tips of the villi or at the top of the colonic crypts in the small intestine and therefore constitute the majority of luminal cells over the intestinal surface area, and therefore are responsible for the uptake of dietary nutrients (see, for example, Santos, et. al (2018) Trends in Cell Biol. In press, https: / / doi.org / 10.1016 / j.tcb.2018.08.001). Under homeostatic conditions, all cells in the intestinal epithelium are renewed in 3–10 days.

[0111] Different niche factors maintain intestinal stem cell (ISC) activity, and distinct non-epithelial and / or epithelial cells generate various signals that make up the cellular niche. Such niche factors include canonical signals such as WNT, R-spondin, Notch, and Bone Morpohogenetic Protein (BMP), but also inflammation and dietary influences. Upon injury, the ISC niche is adapted to transcend its homeostatic state and interpret pathogenic stimuli and convert them into epithelial regeneration. This regeneration is mediated by either surviving Lgr5+ISCs or other mature cell types such as enterocytes, enteroendocrine, or Paneth cells that can convert back to Lgr5+ISCs to support epithelial regeneration (Beumer and Clevers (2016), Development 143: 3639-3649).

[0112] ISCs, also known as columnar base cells (CBCs), reside at the base of intestinal crypts and are intercalated by WNT-secreting Paneth cells (Cheng and Leblond (1974) Am. J. Anat. 141: 537-561). Mesenchymal cells surrounding the intestinal epithelium also secrete some WNT proteins and perform redundant stem cell niche functions in vivo (Farin, el al. (2012) Gastroenterol. 143: 1518-1529). In the presence of WNT signaling, ISCs divide to give rise to self-renewing stem cells and differentiated daughter cells that first undergo several rapid transit amplifying (TA) divisions before differentiating into functional cell types. Intestinal crypts also contain a quiescent stem cell population, +4 cells, which can contribute to epithelial regeneration if the CBC is damaged (Tian, ​​el al. (2011) Nature 478: 255-259). Lineage commitment and terminal differentiation occur as TA cells migrate along the crypt-villus axis and away from WNT-producing cells.

[0113] III. Engineered WNT agonists The present disclosure provides engineered WNT agonists and contemplates the use of engineered WNT agonists to stimulate, agonize, or promote WNT signaling, for example, through the canonical WNT / β-catenin signaling pathway. Such engineered WNT agonists are sometimes referred to as WNT / β-catenin signaling agonists or Wnt mimetics.

[0114] There are several challenges in engineering Wnt proteins for clinical application. First, Wnt proteins are difficult to produce and do not have typical drug-like properties. Second, it has been reported that in vivo overexpression of RSPO or application of exogenous RSPO, which amplifies Wnt signaling, helps regenerate the intestinal epithelium in various injury models (Zhao et al., 2007), but also induces increased proliferation of normal intestinal epithelium (Yan Kelley S. et al., 2017).

[0115] The present disclosure addresses the first challenge by providing synthetic Wnt mimetics with drug-like properties, particularly in the form of recombinant bispecific antibodies that mimic endogenous Wnt ligands and bring together Fzd and Lrp to stimulate signaling. The Wnt mimetics of the present disclosure can diffuse freely and access damaged tissues, guiding tissue repair where Wnt signals are needed.

[0116] The present disclosure addresses the second challenge by providing Wnt mimetics that can repair damaged intestinal epithelium without the need for combination with RSPO. Unlike RSPO, the Wnt mimetics of the present disclosure do not induce hyperproliferation of normal intestinal epithelium.

[0117] The Wnt mimetics of the present disclosure have the desirable property of restoring diseased intestinal tissue to normal physiological function. In some embodiments, the Wnt mimetics of the present disclosure induce rapid restoration of damaged epithelial tissue. In some embodiments, the damaged epithelial barrier can be restored within about 10 days, within about 8 days, within about 7 days, within about 6 days, or within about 5 days after treatment with the Wnt mimetics of the present disclosure. In some embodiments, the damaged epithelial barrier can be restored within about 6 days after treatment with the Wnt mimetics of the present disclosure. In some embodiments, the Wnt mimetics of the present disclosure induce the expression of Wnt target genes in damaged epithelial cells within about 12 hours, within about 24 hours, within about 36 hours, or within about 48 hours. In some embodiments, the Wnt target genes induced by the Wnt mimetics of the present disclosure include Axin2, Rnf43, and Cdkn3. In some embodiments, expression of Axin2 in injured epithelial cells is induced by a Wnt mimetic of the present disclosure within about 24 hours.

[0118] In some embodiments, the Wnt agonist disclosed herein supports the proliferation and differentiation of stem cells in the damaged intestine or colon crypt of patients with moderate to severe IBD.In some embodiments, the Wnt agonist disclosed herein has the potential to accelerate the repair of intestinal barrier, thereby reducing bacterial invasion through intestinal epithelium and reducing immune cell activation and inflammation, and thus can treat inflammatory bowel disease.

[0119] In some embodiments, the Wnt agonists disclosed herein have several simultaneous beneficial effects: activating the Wnt signaling pathway in intestinal stem and progenitor cells, resulting in proliferation and differentiation; restoring intestinal barrier function and tissue architecture; reducing tissue inflammation; and reducing disease activity in moderate to severe IBD.

[0120] In some embodiments, the Wnt agonist disclosed herein is a bispecific antibody that targets Fzd5 / 8 and Lrp6. Fzd5 has previously been reported to be highly expressed in intestinal mucosal cells from IBD patients. Fzd5 is also highly expressed in mouse models of colitis induced by dextran sulfate sodium (DSS). In some embodiments, the Wnt agonist disclosed herein binds to DSS-injured intestinal cells and stimulates Wnt signaling as measured by the expression of Axin2, a downstream target gene in the Wnt pathway. In some embodiments, the Wnt agonist disclosed herein binds to Fzd5 / 8 and Lrp6 on intestinal stem cells and activates Wnt signaling.

[0121] In some embodiments, administration of the Wnt agonist disclosed herein improves disease activity index, or DAI, in DSS models. DAI is a composite score that is composed of weight change, diarrhea, and bloody stool, and is frequently used to quantify disease severity in preclinical rodent models and in clinics. In some embodiments, administration of the Wnt agonist disclosed herein leads to a dose-dependent decrease in DAI. In some embodiments, treatment with the Wnt agonist disclosed herein is superior to treatment with cyclosporine, anti-TNF antibody, or anti-IL12 / 23 antibody. In some embodiments, administration of the Wnt agonist disclosed herein improves DAI in both chronic and acute DSS models.

[0122] In some embodiments, the Wnt mimic of the present disclosure expands the progenitor cell population in epithelium.In some embodiments, the Wnt mimic of the present disclosure expands the progenitor cell population by increasing the expression of cell cycle genes in said cell population.Progenitor cell population can include, for example, normal progenitor cells that respond to injury, and progenitor cells that have altered cell state, for example, dedifferentiated.In some embodiments, the Wnt mimic of the present disclosure substantially expands the progenitor cell population in epithelium within about 24 hours.

[0123] In some embodiments, the Wnt mimetics disclosed herein accelerate the differentiation of progenitor cells into mature cell types. In some embodiments, the Wnt mimetics disclosed herein accelerate the differentiation of progenitor cells, such as gastrointestinal progenitor cells, into enterocytes, goblet cells, enteroendocrine, or tuft cells. In some embodiments, the Wnt mimetics disclosed herein accelerate the differentiation of progenitor cells into enterocytes. In some embodiments, the substantial differentiation of progenitor cells into mature cell types occurs within about 24 hours, about 36 hours, about 48 hours, or about 60 hours after treatment with the Wnt mimetics disclosed herein. In some embodiments, the substantial differentiation of progenitor cells into mature cell types occurs within about 48 hours after treatment with the Wnt mimetics disclosed herein. In some embodiments, the Wnt mimetics disclosed herein accelerate the differentiation of progenitor cells into mature cell types while simultaneously reducing high levels of inflammatory gene expression.

[0124] In some embodiments, the breakdown of the intestinal barrier induces the influx of luminal pathogens and inflammatory responses, which leads to further tissue damage.Disease modification in IBD can be measured by the level of inflammatory cytokines present in injured tissue and in serum.In some embodiments, treating epithelial tissue injury with the Wnt mimic of the present disclosure reduces the production of inflammatory cytokines.In some embodiments, treating injured epithelial tissue with the Wnt mimic of the present disclosure reduces inflammatory cytokine production by at least about 1%, at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% compared to treatment without the Wnt mimic of the present disclosure or compared to no treatment.

[0125] In some embodiments, the present disclosure provides a Wnt mimetic that can effectively repair damaged epithelium without inducing hyperproliferation of normal epithelium. In some embodiments, the Wnt mimetic of the present disclosure alone does not affect the proliferation of normal epithelium. In some embodiments, the epithelium is colon or small intestine epithelium. In some embodiments, the present disclosure provides a Wnt mimetic that can repair damaged epithelium with higher efficacy than treatments that include RSPO. In some embodiments, the present disclosure provides a Wnt mimetic that can repair damaged epithelium with higher efficacy than treatments that include RSPO and the Wnt mimetic of the present disclosure. In some embodiments, a Wnt mimetic of the disclosure improves damaged epithelium with at least about 1%, at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% better efficacy than a treatment comprising RSPO, or a treatment comprising RSPO and a Wnt mimetic of the disclosure. The efficacy of repair of damaged epithelium may be determined by a histological severity score, with higher scores indicating more severe damage, or by a Disease Activity Index (DIA), which may be calculated based on the average scores of weight loss, stool consistency, and the extent of intestinal bleeding.

[0126] In some embodiments, the present disclosure provides Fzd5, 8 and Lrp6 specific Wnt mimics (e.g., R2M13-26 or R2M13-h26). In some embodiments, the present disclosure's Fzd5, 8 and Lrp6 specific Wnt mimics can activate Wnt signaling in epithelial cells. Activation of Wnt signaling can be measured by gene expression using scRNA-seq (single cell RNA sequencing) methods known in the art and described in the present disclosure. In some embodiments, the epithelium cell is a colon or small intestine epithelium cell. In some embodiments, the epithelium cell comprises a plurality of stem cells or progenitor cells.

[0127] The engineered WNT agonist comprises one or more binding domains that bind one or more FZDs or epitopes thereof, and one or more binding domains that bind LRP5 and / or LRP6, or one or more epitopes within LRP5 and / or LRP6. In certain embodiments, the engineered WNT agonist specifically binds to a cysteine-rich domain (CRD) within the human frizzled receptor(s) to which it binds.

[0128] In certain embodiments, engineered WNT agonist may comprise one or more additional binding domains.For example, engineered WNT agonist may comprise one or more binding domains that bind to one or more specific epitopes of E3 ligase, ZNRF3 / RNF43, or E3 ligase.In certain embodiments, E3 ligase binding domain comprises R-SPO or a fragment thereof.

[0129] In certain embodiments, an engineered WNT agonist may contain one or more tissue-specific or cell type-specific binding domains that specifically bind to a target tissue or cell type.

[0130] In one aspect, the disclosure provides a VHH domain that binds to LRP5 and / or LRP6. Exemplary sequences of these VHH domains are provided in Table 1. The VHH binding domain may be derived from any of the disclosed sequences. In certain embodiments, the VHH binding domain is humanized. The disclosure contemplates engineered WNT agonists that include any of the humanized VHH domains disclosed herein, as well as functional fragments and variants of such VHH domains that have at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to any of the VHH sequences disclosed herein. In certain embodiments, the VHH domain comprises three CDR sequences: GRIFAIYDIA, IRPVVTEIDYADSVKG, and RPWGSRDEY. In certain embodiments, the engineered Wnt agonist comprises a VHH domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 19-25. In certain embodiments, the engineered Wnt agonist is a bispecific antibody-like molecule comprising an IgG structure comprising two heavy chains and two light chains, with a VHH domain fused to the N-terminus of each light chain present in the antibody-like molecule. In certain embodiments, the heavy chains are effector-less, e.g., contain a LALAPG mutation.

[0131] In another aspect, the present disclosure provides FZD binding domains that bind to one or more FZDs. Exemplary sequences of these FZD binding domains are provided in Table 3 for the VH and VL domains derived from anti-FZD antibody R2M13. The present disclosure contemplates engineered WNT agonists that include one or more of the VH or VL domains disclosed herein, as well as functional fragments and variants of such VH or VL domains that have at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to any of the VH or VL sequences disclosed herein. Additionally, the present disclosure contemplates engineered WNT agonists that include one or more of the heavy or light chain sequences provided in Table 3, as well as functional fragments and variants of such heavy or light chains that have at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to any of the heavy or light chain sequences disclosed herein. In certain embodiments, the FZD-binding domain comprises three light chain CDR sequences: RASQSISSYLN (CDRL1), AASSLQS (CDRL2), and QQSYSTPLT (CDRL3), and / or three heavy chain and light chain CDR sequences: GGTFTYRYLH (CDRH1), GIIPIFGTGNYAQKFQG (CDRH2), and SMVRVPYYYGMDV (CDRH3), any of the CDRs provided herein.

[0132] In related embodiments, the present disclosure contemplates engineered WNT agonists that include one or more CDRs present in the FZD-binding domain or LRP5 / 6-binding domain disclosed herein: for example, one or more (e.g., two or three) of the VHH CDRs shown in Figure 6 or Table 1; one or more (e.g., two or three) of the CDRs present in the heavy or light chains disclosed herein. In certain embodiments, the engineered WNT agonist includes four, five, or all six of the CDRs shown for the FZD-binding domain disclosed herein, for example, in Figure 6 or Table 3. In certain embodiments, the engineered WNT agonist includes six, seven, eight, or all nine of the CDRs shown for the engineered WNT agonist disclosed herein, for example, in Figure 6 or Table 3.

[0133] The disclosure also provides polypeptides comprising or consisting of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the binding domains provided herein, as well as polypeptides comprising two or more, such as three, of the CDR sequences disclosed herein, such as the following CDRs: GRIFAIYDIA, IRPVVTEIDYADSVKG, and RPWGSRDEY (VHHs, respectively). and CDRs 1-3) and bind to LRP5 or LRP6; or a polypeptide comprising the following CDRs: RASQSISSYLN (CDRL1), AASSLQS (CDRL2), and QQSYSTPLT (CDRL3), and in combination with a heavy chain, binds to one or more FZDs; or a polypeptide comprising the following CDRs: GGTFTYRYLH (CDRH1), GIIPIFGTGNYAQKFQG (CDRH2), and SMVRVPYYYGMDV (CDRH3), and in combination with a light chain, binds to one or more FZDs. The disclosure also encompasses an FZD-binding domain comprising two heavy chains and two light chains, each heavy chain comprising two or more of the following CDRs: GGTFTYRYLH (CDRH1), GIIPIFGTGNYAQKFQG (CDRH2), and SMVRVPYYYGMDV (CDRH3), and each light chain comprising two or more of the following CDRs: RASQSISSYLN (CDRL1), AASSLQS (CDRL2), and QQSYSTPLT (CDRL3), which bind to one or more FZDs. In certain embodiments, the FZD-binding domain is an antibody, and the heavy chain further comprises an Fc domain, e.g., an IgG1 Fc domain, which may be modified. The disclosure further provides polypeptides comprising or consisting of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to a variable heavy or variable light domain disclosed herein, e.g., in SEQ ID NOs: 1-25, FIG. 6, or Table 3.The disclosure further provides polypeptides comprising or consisting of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to a VHH domain disclosed herein, e.g., in SEQ ID NOs: 1-25, FIG. 6, or Table 3. The disclosure further provides polypeptides comprising or consisting of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to a heavy chain or light chain or fusion polypeptide disclosed herein, e.g., in SEQ ID NOs: 1-25, FIG. 6, or Table 3. In any embodiment of the polypeptide variants disclosed herein, the CDRs are unaltered compared to the original or parent sequence.

[0134] Additionally, the disclosure provides polynucleotide sequences encoding any of the polypeptides described herein, as well as functional fragments and variants thereof, such as functional fragments and variants that bind to one or more FZDs or LRP5 / 6, VH domains, and VL domains.

[0135] In certain embodiments, the engineered WNT agonists disclosed herein comprise an Fc domain (e.g., as part of a heavy chain). In certain embodiments, the Fc domain is engineered to contain specific amino acid substitutions, including those corresponding to LALAPG or N297G.

[0136] In certain embodiments of engineered WNT agonists, one or more LRP5 / 6 binding domains disclosed herein (e.g., any of VHH26-H1-H6) are fused to one or more of the light or heavy chains of a FZD binding domain disclosed herein (e.g., a FZD binding domain from R2M13), e.g., directly or via a linker, e.g., a peptide linker. However, in other embodiments, any LRP5 / 6 binding domain disclosed herein can be fused or complexed with a different FZD binding domain to achieve an engineered WNT agonist, and any FZD binding domain disclosed herein can be fused or complexed with a different LRP5 / 6 binding domain to achieve an engineered WNT agonist. Various anti-FZD or anti-LRP antibodies that may be present in whole or in part in the engineered WNT agonists disclosed herein include those described in U.S. Patent No. 7,462,697, PCT Publication No. WO2019 / 126399, and PCT Publication No. WO2019 / 126401. Exemplary formats and sequences are also provided in PCT Publication No. WO2019 / 126398. Each of these patent documents is incorporated herein in its entirety.

[0137] The engineered WNT agonist may adopt a variety of different structural conformations, each of which comprises one or more, for example, two FZD binding domains and one or more, for example, two LRP5 / 6 binding domains.The FZD binding domain(s) and the LRP5 / 6 binding domain(s) may be directly fused to each other, or may be fused via a linker, for example, a peptide linker.Alternatively, the FZD binding domain(s) and the LRP5 / 6 binding domain(s) may form a complex with each other.

[0138] In certain embodiments, the engineered WNT agonist comprises two heavy chains and two light chains, where the light chains comprise a fused VHH and adopt an antibody-like conformation, the two heavy chains are linked to each other by disulfide bonds, and the two light chains are linked to the heavy chains by disulfide bonds.

[0139] Engineered WNT agonists may adopt other antibody-like structures or conformations, including those found in various functional fragments, including but not limited to any of those disclosed herein.

[0140] As is well known in the art, an antibody is an immunoglobulin molecule capable of specifically binding to a target, e.g., carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least an epitope binding domain located in the variable region of the immunoglobulin molecule. As used herein, the term encompasses not only intact polyclonal or monoclonal antibodies, but also fragments thereof containing the epitope-binding domain (e.g., dAbs, Fab, Fab', (F(ab')2, Fv, single chain (scFv), camelid antibodies, Nanobodies® (Nab; also known as sdAbs or VHH domains), DVD-Ig, synthetic variants thereof, naturally occurring variants, fusion proteins containing the epitope-binding domain, humanized antibodies, chimeric antibodies, and any other modified configurations of immunoglobulin molecules that contain an antigen-binding site or fragment (epitope recognition site) of the required specificity. "Diabodies", multivalent or multispecific fragments constructed by gene fusion (WO 94 / 13804; P. Holliger et al., Proc. Natl. Acad. Sci. USA 90 6444-6448, 1993) are also specific forms of antibodies contemplated herein. Minibodies comprising scFvs joined to CH3 domains are also included herein (S. Hu et al., Cancer Res., 56, 3055-3061, 1996). See, for example, Ward, ES et al., Nature 341, 544-546 (1989);Bird et al., Science, 242, 423-426, 1988;Huston et al., PNAS USA, 85, 5879-5883, 1988);PCT / US92 / 09965;WO94 / 13804;P. Holliger et al., Proc. Natl. Acad. Sci. USA 90 6444-6448, 1993;Y. Reiter et al., Nature Biotech, 14, 1239-1245, 1996; S. Hu et al., Cancer Res., 56, 3055-3061, 1996.

[0141] The proteolytic enzyme papain preferentially cleaves IgG molecules to produce several fragments, two of which (F(ab) fragments) each contain a covalently linked heterodimer with an intact antigen binding site. The enzyme pepsin can cleave IgG molecules to produce several fragments, including the F(ab')2 fragment, which contains both antigen binding sites. Fv fragments for use according to certain embodiments of the present disclosure can be produced by preferential proteolytic cleavage of IgM, and more rarely, by proteolytic cleavage of IgG or IgA immunoglobulin molecules. However, Fv fragments are more commonly derived by recombinant techniques known in the art. Fv fragments encompass non-covalent VH::VL heterodimers containing antigen binding sites that retain much of the antigen recognition and binding capacity of the native antibody molecule. Inbar et al. (1972) Proc. Nat. Acad. Sci. USA 69: 2659-2662; Hochman et al. (1976) Biochem 15: 2706-2710; and Ehrlich et al. (1980) Biochem 19: 4091-4096.

[0142] In certain embodiments, the antibodies and antigen-binding fragments thereof described herein comprise a set of heavy and light chain CDRs, each of which is located between a set of heavy and light chain framework regions (FRs) that provide support for the CDRs and define the spatial relationship of the CDRs to each other. As used herein, the term "CDR set" refers to the three hypervariable regions of the heavy or light chain V region. Proceeding from the N-terminus of the heavy or light chain, these regions are referred to as "CDR1", "CDR2", and "CDR3", respectively. Thus, an antigen-binding site comprises six CDRs, including a set of CDRs from each of the heavy and light chain V regions. A polypeptide comprising a single CDR (e.g., CDR1, CDR2 or CDR3) is referred to herein as a "molecular recognition unit". Crystallographic analysis of numerous antigen-antibody complexes has demonstrated that the amino acid residues of the CDRs form extensive contacts with the bound antigen, where the majority of extensive antigen contacts are with the heavy chain CDR3. Thus, the molecular recognition unit is primarily responsible for the specificity of the antigen-binding site.

[0143] As used herein, the term "FR set" refers to four adjacent amino acid sequences that frame the CDRs of a CDR set of a heavy or light chain V region. Some FR residues may contact bound antigen; however, FRs, especially those immediately adjacent to CDRs, are primarily responsible for folding the V region into the antigen-binding site. Within FRs, certain amino acid residues and certain structural features are highly conserved. In this regard, all V region sequences contain an internal disulfide loop of approximately 90 amino acid residues. When the V region folds into the binding site, the CDRs are displayed as protruding loop motifs that form an antigen-binding surface. It is generally understood that there are conserved structural regions of FRs that influence the folding shape of the CDR loops to be in a certain "canonical" structure regardless of the exact CDR amino acid sequence. In addition, certain FR residues have been found to participate in non-covalent interdomain contacts that stabilize the interaction of the heavy and light chains of an antibody.

[0144] "Monoclonal antibody" refers to a homogeneous antibody population, which is composed of amino acids (naturally occurring and non-naturally occurring) involved in selective binding of an epitope. Monoclonal antibodies are highly specific and directed to a single epitope. The term "monoclonal antibody" encompasses intact and full-length monoclonal antibodies, as well as fragments thereof (e.g., Fab, Fab', F(ab')2, Fv, etc.), single chain (scFv), Nanobodies®, variants thereof, fusion proteins comprising antigen-binding fragments of monoclonal antibodies, humanized monoclonal antibodies, chimeric monoclonal antibodies, and any other modified configurations of immunoglobulin molecules that contain an antigen-binding fragment (epitope recognition site) of the required specificity and binding ability to an epitope, including the engineered WNT agonists disclosed herein. It is not intended to be limited as regards to the source of the antibody or the manner in which it is made (e.g., by hybridoma, phage selection, recombinant expression, transgenic animals, etc.) The term includes whole immunoglobulins as well as fragments, etc., listed above under the definition of "antibody."

[0145] In certain embodiments, single chain Fv or scFV antibodies are intended for use in engineered Wnt agonists. For example, kappabodies (Ill et al., Prot. Eng. 10: 949-57 (1997)); minibodies (Martin et al., EMBO J 13: 5305-9 (1994)); diabodies (Holliger et al., PNAS 90: 6444-8 (1993)); or Janusins ​​(Traunecker et al., EMBO J 10: 3655-59 (1991) and Traunecker et al., Int. J. Cancer Suppl. 7: 51-52 (1992)) can be prepared using standard molecular biology techniques following the teachings of the present application for selecting antibodies with desired specificity. In yet other embodiments, bispecific or chimeric antibodies can be made that include the ligands of the present disclosure. For example, chimeric antibodies can be produced that contain CDRs and framework regions from different antibodies, but that bind to one or more FZD receptors through one binding domain and specifically bind to a second molecule through a second binding domain. These antibodies can be produced by recombinant molecular biology techniques or can be physically conjugated together.

[0146] Single-chain Fv (scFv) polypeptides are covalently linked VH::VL heterodimers and are expressed from gene fusions that include a VH-encoding gene and a VL-encoding gene linked by a peptide-encoding linker. Huston et al. (1988) Proc. Nat. Acad. Sci. USA 85 (16): 5879-5883. Several methods have been described for determining the chemical structure for converting the naturally assembled but chemically separate light and heavy polypeptide chains from an antibody V region into scFv molecules that fold into a three-dimensional structure substantially similar to that of an antigen-binding site. See, for example, U.S. Patent Nos. 5,091,513 and 5,132,405 to Huston et al.; and U.S. Patent No. 4,946,778 to Ladner et al.

[0147] In certain embodiments, the antibody described herein is in the form of a diabody.A diabody is a multimer of polypeptides, each polypeptide comprising a first domain comprising a binding region of an immunoglobulin light chain and a second domain comprising a binding region of an immunoglobulin heavy chain, the two domains being linked (e.g., by a peptide linker) but unable to associate with each other to form an antigen-binding site: the antigen-binding site is formed by the association of the first domain of one polypeptide in the multimer with the second domain of another polypeptide in the multimer (WO94 / 13804).

[0148] A dAb fragment of an antibody consists of one VH domain (Ward, ES et al., Nature 341, 544-546 (1989)).

[0149] When bispecific antibodies are used, they can be produced in a variety of ways (Holliger, P. and Winter G., Current Opinion Biotechnol. 4, 446-449 (1993)), for example, they can be traditional bispecific antibodies that can be prepared chemically or from hybrid hybridomas, or they can be any of the bispecific antibody fragments described above. Diabodies and scFvs can be constructed without an Fc region, using only variable domains, thereby potentially reducing the effects of anti-idiotypic reaction.

[0150] Bispecific diabodies can be particularly useful because, in contrast to whole bispecific antibodies, they can be easily constructed and expressed in E. coli. Diabodies (and many other polypeptides, such as antibody fragments) of appropriate binding specificity can be easily selected from libraries using phage display (WO94 / 13804). If one arm of the diabody is held constant, for example with specificity for antigen X, the other arm can be varied to create a library from which an antibody of appropriate specificity can be selected. Whole bispecific antibodies can be made by knob-into-hole engineering (JBB Ridgeway et al., Protein Eng., 9, 616-621 (1996)).

[0151] In certain embodiments, the antibodies described herein may be provided in the form of a UniBody®. A UniBody® is an IgG4 antibody with the hinge region removed (see GenMab Utrecht, The Netherlands; see also, e.g., US20090226421). This proprietary antibody technology creates a stable smaller antibody format with a larger predicted therapeutic window than current small antibody formats. IgG4 antibodies are inactive and therefore are not thought to interact with the immune system. Fully human IgG4 antibodies can be engineered by eliminating the hinge region of the antibody to obtain half-molecule fragments with distinct stability characteristics from the corresponding intact IgG4 (GenMab, Utrecht). By halving the IgG4 molecule, only one region remains on the UniBody® that can bind to the cognate antigen (e.g., disease target), and thus the UniBody® binds monovalently to only one site on the target cell.

[0152] In certain embodiments, the antibodies of the present disclosure may take the form of single domain (sdAb) or VHH antibody fragments (also known as Nanobodies®). sdAb or VHH technology was originally developed following the discovery and identification that camelids (e.g., camels and llamas) have fully functional antibodies that consist only of heavy chains and thus lack light chains. These heavy chain-only antibodies contain a single variable domain (VHH) and two constant domains (CH2, CH3). The cloned and isolated single variable domains have full antigen binding capacity and are highly stable. These single variable domains, together with their unique structural and functional properties, form the basis of "Nanobodies®". sdAbs or VHHs are encoded by a single gene and are efficiently produced in almost all prokaryotic and eukaryotic hosts, such as E. coli (see, e.g., U.S. Pat. No. 6,765,087), molds (e.g., Aspergillus or Trichoderma) and yeasts (e.g., Saccharomyces, Kluyvermyces, Hansenula or Pichia (see, e.g., U.S. Pat. No. 6,838,254). The production process is scalable and multi-kilogram quantities of Nanobodies® have been produced. sdAbs or VHHs can be formulated as ready-to-use solutions with a long shelf life. The Nanoclone® method (see, e.g., WO06 / 079372) is a proprietary method for generating Nanobodies® against desired targets based on automated high-throughput selection of B cells. sdAbs or VHHs are single-domain antigen-binding fragments of heavy-chain-only antibodies specific to camelids.

[0153] Another contemplated antibody fragment is the dual variable domain-immunoglobulin (DVD-Ig), an engineered protein that combines the functions and specificities of two monoclonal antibodies into one molecular entity. DVD-Ig is designed as an IgG-like molecule, except that instead of one variable domain in IgG, each light and heavy chain contains two variable domains in tandem with a short peptide linkage. The orientation of the fusion of the two variable domains and the selection of the linker sequence are crucial for the functional activity and efficient expression of the molecule. DVD-Ig can be produced by conventional mammalian expression systems as a single species for manufacturing and purification. DVD-Ig has the specificity of the parent antibody, is stable in vivo, and exhibits IgG-like physicochemical and pharmacokinetic properties. DVD-Ig and methods for making DVD-Ig are described in Wu, C., et al., Nature Biotechnology, 25: 1290-1297 (2007)).

[0154] In certain embodiments, the antibody or antigen-binding fragment thereof disclosed herein is humanized. This refers to a chimeric molecule, generally prepared using recombinant techniques, having an antigen-binding site derived from an immunoglobulin from a non-human species and the remaining immunoglobulin structure of the molecule based on the structure and / or sequence of a human immunoglobulin. The antigen-binding site may comprise either a complete variable domain fused to a constant domain, or only the CDRs grafted onto the appropriate framework regions in the variable domain. The epitope-binding site may be wild-type or modified by one or more amino acid substitutions. This eliminates the constant regions as immunogens in human individuals, but leaves open the possibility of an immune response to the foreign variable regions (LoBuglio, AF et al., (1989) Proc Natl Acad Sci USA 86: 4220-4224; Queen et al., PNAS (1988) 86: 10029-10033; Riechmann et al., Nature (1988) 332: 323-327).

[0155] Another approach focuses on not only providing constant regions of human origin, but also modifying variable regions and reshaping them as close as possible to human form.It is known that the variable regions of both heavy and light chains contain three complementarity determining regions (CDRs) that are relatively conserved in a given species and flanked by four framework regions (FRs) that are predicted to provide scaffolds for CDRs, and that vary depending on the epitope in question and determine binding ability.When a non-human antibody is prepared for a specific epitope, the variable region can be "reshaped" or "humanized" by grafting the CDRs from a non-human antibody into the FRs present in the human antibody to be modified. The application of this technique to various antibodies has been reported in Sato, K., et al., (1993) Cancer Res 53: 851-856;Riechmann, L., et al., (1988) Nature 332: 323-327;Verhoeyen, M., et al., (1988) Science 239: 1534-1536;Kettleborough, CA, et al., (1991) Protein Engineering 4: 773-3783;Maeda, H., et al., (1991) Human Antibodies Hybridoma 2: 124-134;Gorman, SD, et al., (1991) Proc Natl Acad Sci USA 88: 4181-4185;Tempest, PR, et al., (1991) Bio / Technology 9: 266-271; Co, MS, et al., (1991) Proc Natl Acad Sci USA 88: 2869-2873; Carter, P., et al., (1992) Proc Natl Acad Sci USA 89: 4285-4289; and Co, MS et al., (1992) J Immunol 148: 1149-1154. In some embodiments, the humanized antibody preserves all CDR sequences (e.g., a humanized mouse antibody that contains all six CDRs from the mouse antibodies).In other embodiments, a humanized antibody has one or more CDRs (one, two, three, four, five, six) that are altered with respect to the original antibody, also referred to as one or more CDRs "derived from" one or more CDRs from the original antibody.

[0156] In certain embodiments, the antibody of the present disclosure may be a chimeric antibody. In this regard, a chimeric antibody is composed of an antigen-binding fragment of an antibody operably linked or otherwise fused to a heterologous Fc portion of a different antibody. In certain embodiments, the heterologous Fc domain is of human origin. In other embodiments, the heterologous Fc domain may be from an Ig class different from the parent antibody, including IgA (including subclasses IgA1 and IgA2), IgD, IgE, IgG (including subclasses IgG1, IgG2, IgG3, and IgG4), and IgM. In further embodiments, the heterologous Fc domain may be composed of a CH2 domain and a CH3 domain from one or more of the different Ig classes. As described above with respect to humanized antibodies, the antigen-binding fragment of a chimeric antibody may include only one or more of the CDRs of the antibodies described herein (e.g., one, two, three, four, five, or six CDRs of the antibodies described herein), or may include the entire variable domain (VL, VH, or both).

[0157] The structures and locations of immunoglobulin CDRs and variable domains can be determined by reference to Kabat, EA et al., Sequences of Proteins of Immunological Interest. 4th Edition. US Department of Health and Human Services. 1987, and revised versions thereof, now available on the internet (immuno.bme.nwu.edu).

[0158] In some embodiments, the engineered WNT agonist comprises one or more Fabs or antigen-binding fragments thereof and one or more VHHs or sdAbs or antigen-binding fragments thereof (or one or more scFvs or antigen-binding fragments thereof). In certain embodiments, the Fabs specifically bind to one or more Fzd receptors and the VHHs or sdAbs (or scFvs) specifically bind to LRP5 and / or LRP6. In certain embodiments, the Fabs specifically bind to LRP5 and / or LRP6 and the VHHs or sdAbs (or scFvs) specifically bind to one or more Fzd receptors. In certain embodiments, the VHHs or sdAbs (or scFvs) are fused to the N-terminus of the Fab, while in some embodiments, the VHHs or sdAbs (or scFvs) are fused to the C-terminus of the Fab. In certain embodiments, the Fabs are present in a full IgG format and the VHHs or sdAbs (or scFvs) are fused to the N-terminus and / or C-terminus of the IgG light chain. In certain embodiments, the Fab is present in a full IgG format, with the VHH or sdAb (or scFv) fused to the N-terminus and / or C-terminus of the IgG heavy chain, in certain embodiments, two or more VHH or sdAb (or scFv) are fused to the IgG at any combination of these positions.

[0159] Fab can be converted to a complete IgG format containing both Fab and Fc fragments, for example, by using genetic engineering to generate a fusion polypeptide containing a Fab fused to an Fc region, i.e., the Fab is present in a complete IgG format. The Fc region of the complete IgG format can be derived from any of a variety of different Fcs, including, but not limited to, wild-type or modified IgG1, IgG2, IgG3, IgG4 or other isotypes, such as wild-type or modified human IgG1, human IgG2, human IgG3, human IgG4, human IgG4Pro (containing a mutation in the core hinge region that prevents the formation of half IgG4 molecules), human IgA, human IgE, human IgM, or a modified IgG1 called IgG1 LALAPG. The L235A, P329G (LALA-PG) variant has been shown to eliminate complement binding and fixation and Fc-gamma-dependent antibody-dependent cell-mediated cytotoxicity (ADCC) for both mouse IgG2a and human IgG1. These LALA-PG substitutions allow for more accurate translation of results achieved with the "effector-less" antibody framework scaffold between mice and primates. In certain embodiments of any of the IgGs disclosed herein, the IgG comprises one or more of the following amino acid substitutions: N297G, N297A, N297E, L234A, L235A, or P236G.

[0160] Non-limiting examples of bivalent and bispecific engineered WNT agonists that are bivalent for both one or more Fzd receptors and LRP5 and / or LRP6 are provided, including but not limited to those provided in Table 3. VHH or sdAb (or scFv) can be fused to the N-terminus of both light chains, the N-terminus of both heavy chains, the C-terminus of both light chains, or the C-terminus of both heavy chains. For example, it is further contemplated that VHH or sdAb (or scFv) can be fused to both the N-terminus and C-terminus of the heavy and / or light chains, the N-terminus of the light chain and the C-terminus of the heavy chain, or the C-terminus of the heavy chain and the N-terminus of the light chain. In other related embodiments, two or more VHH or sdAb (or scFv) can be fused together, optionally via a linker moiety, and fused to Fab or IgG at one or more of these positions. In related embodiments, the engineered WNT agonist has a hetero-IgG format, while the Fab is present as a half antibody and one or more VHH or sdAb (or scFv) are fused to one or more of the N-terminus of the Fc, the N-terminus of the Fab, the C-terminus of the Fc, or the C-terminus of the Fab. In certain embodiments, the Fab or antigen-binding fragment thereof (or IgG) is fused directly to the VHH or sdAb (or scFv) or antigen-binding fragment thereof, while in other embodiments, the binding regions are fused via a linker moiety.

[0161] In various embodiments, the engineered WNT agonist comprises one or more Fabs or antigen-binding fragments thereof that bind one or more FZD receptors, and one or more Fabs or antigen-binding fragments thereof that bind LRP5 and / or LRP6. In certain embodiments, the engineered WNT agonist comprises two Fabs or antigen-binding fragments thereof that bind one or more FZD receptors, and / or two Fabs or antigen-binding fragments thereof that bind LRP5 and / or LRP6. In certain embodiments, one or more of the Fabs are present in a full IgG format, and in certain embodiments, both Fabs are present in a full IgG format. In certain embodiments, a Fab in a full IgG format specifically binds one or more FZD receptors, and the other Fab specifically binds LRP5 and / or LRP6. In certain embodiments, a Fab specifically binds one or more FZD receptors, and a Fab in a full IgG format specifically binds LRP5 and / or LRP6. In certain embodiments, a Fab specifically binds LRP5 and / or LRP6, and a Fab in a full IgG format specifically binds one or more FZD receptors. In certain embodiments, the Fab is fused to the N-terminus of the IgG, for example, the heavy or light chain N-terminus, optionally via a linker. In certain embodiments, the Fab is fused to the N-terminus of the heavy chain of the IgG, and not to the light chain. In certain embodiments, the two heavy chains can be fused together directly or via a linker. In other related embodiments, two or more VHHs or sdAbs can be fused together, optionally via a linker moiety, and can be fused to the Fab or IgG at one or more of these positions. In related embodiments, the engineered WNT agonist has a hetero-IgG format, while one of the Fabs is present as a half antibody, and the other Fab is fused to one or more of the N-terminus of the Fc, the N-terminus of the Fab, or the C-terminus of the Fc.In certain embodiments, a Fab or antigen-binding fragment thereof is fused directly to another Fab or IgG or antigen-binding fragment thereof, while in other embodiments, the binding region is fused via a linker moiety.

[0162] In certain embodiments, the WNT agonists of the present invention may have, comprise, or consist of any of the sequences provided in any of the tables, figures, or examples herein, or a functional fragment or variant thereof.

[0163] In certain embodiments, the FZD binding domain, LRP5 / 6 binding domain, and / or engineered WNT agonist binds with a dissociation constant (KD) of about 1 μM or less, about 100 nM or less, about 40 nM or less, about 20 nM or less, or about 10 nM or less.For example, in certain embodiments, the FZD binding domain or antibody described herein that binds to more than one FZD binds to those FZDs with a KD of about 100 nM or less, about 20 nM or less, or about 10 nM or less.In certain embodiments, the binding domain binds to one or more of its target antigens with an EC50 of about 1 μM or less, about 100 nM or less, about 40 nM or less, about 20 nM or less, about 10 nM or less, or about 1 nM or less.

[0164] The engineered WNT agonist of the present invention, its binding domain, antibody or other agent can be assayed for specific binding by any method known in the art.The immunoassays that can be used include, but are not limited to, competitive and non-competitive assay systems using techniques such as BIAcore analysis, FACS analysis, immunofluorescence, immunocytochemistry, Western blot, radioimmunoassay, ELISA, "sandwich" immunoassay, immunoprecipitation assay, precipitation reaction, gel diffusion precipitin reaction, immunodiffusion assay, agglutination assay, complement fixation assay, immunoradiometric measurement, fluorescent immunoassay and protein A immunoassay.Such assays are conventional and well known in the art (see, for example, Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, which is incorporated herein by reference in its entirety).

[0165] For example, the specific binding of an antibody to a target antigen can be determined using ELISA. ELISA assays include preparing an antigen, coating the wells of a 96-well microtiter plate with the antigen, adding an antibody or other binding agent conjugated with a detectable compound, such as an enzyme substrate (e.g., horseradish peroxidase or alkaline phosphatase) to the wells, incubating for a period of time, and detecting the presence of the antigen. In some embodiments, the antibody or agent is not conjugated with a detectable compound, and instead, a second conjugated antibody that recognizes the first antibody or agent is added to the well. In some embodiments, instead of coating the well with an antigen, the well can be coated with an antibody or agent, and the antigen can be added to the coated well, followed by the addition of a second antibody conjugated with a detectable compound. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected as well as other art-known variations of ELISA (see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 11.2.1).

[0166] The binding affinity of an antibody or other agent to a target antigen and the off-rate of antibody-antigen interaction can be determined by competitive binding assay. One example of competitive binding assay is a radioimmunoassay, which includes incubating a labeled antigen (e.g., FZD, LRP) or a fragment or variant thereof with an antibody of interest in the presence of increasing amounts of unlabeled antigen, and then detecting the antibody bound to the labeled antigen. The affinity and binding off-rate of an antibody can be determined from the data by Scatchard plot analysis. In some embodiments, BIAcore kinetic analysis is used to determine the binding on-rate and off-rate of an antibody or agent. BIAcore kinetic analysis involves analyzing the binding and dissociation of an antibody to a chip with an antigen immobilized on its surface.

[0167] The engineered WNT agonist of the present invention is biologically active in terms of binding to one or more FZD receptors, and one or more of LRP5 and LRP6, and in terms of activating WNT signaling. The term "WNT agonist activity" refers to the ability of an agonist to mimic the effect or activity of WNT protein binding to frizzled protein and / or LRP5 or LRP6. The ability of the engineered WNT agonist disclosed herein to mimic the activity of WNT can be confirmed by several assays. WNT agonists typically initiate similar or identical reactions or activities to those initiated by the natural ligand of the receptor. In particular, the WNT agonists disclosed herein activate, enhance or increase the standard WNT / β-catenin signaling pathway. As used herein, the term "enhance" refers to a measurable increase in the level of WNT / β-catenin signaling compared to the level in the absence of the WNT agonist, for example, the engineered WNT agonist disclosed herein. In certain embodiments, the increase in the level of WNT / β-catenin signaling is, for example, at least 10%, at least 20%, at least 50%, at least 1.1-fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, or at least 100-fold, compared to the level of WNT / β-catenin signaling in the absence of the engineered WNT agonist in the same cell type. Methods for measuring WNT / β-catenin signaling are known in the art and include those described herein.

[0168] In certain embodiments, the engineered WNT agonists disclosed herein are bispecific, i.e., specifically bind to two or more different epitopes, e.g., one or more FZD receptors, and LRP5 and / or LRP6. In certain embodiments, the engineered WNT agonists bind to FZD5 and / or FZD8, and LRP5 and / or LRP6.

[0169] In certain embodiments, the engineered WNT agonists disclosed herein are multivalent, e.g., comprise two or more regions that each specifically bind to the same epitope, e.g., two or more regions that bind to an epitope in one or more FZD receptors, and / or two or more regions that bind to an epitope in LRP5 and / or LRP6. In certain embodiments, the engineered WNT agonists disclosed herein comprise two or more regions that bind to an epitope in one or more FZD receptors, and two or more regions that bind to an epitope in LRP5 and / or LRP6. In certain embodiments, the engineered WNT agonist comprises a ratio of the number of regions that bind one or more FZD receptors to the number of regions that bind LRP5 and / or LRP6 of about: 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 2:3, 2:5, 2:7, 7:2, 5:2, 3:2, 3:4, 3:5, 3:7, 3:8, 8:3, 7:3, 5:3, 4:3, 4:5, 4:7, 4:9, 9:4, 7:4, 5:4, 6:7, 7:6, 1:2, 1:3, 1:4, 1:5, or 1:6. In certain embodiments, the engineered WNT agonist is bispecific and multivalent.

[0170] In certain aspects, the present disclosure provides novel tissue-specific WNT signal enhancing molecules that can enhance WNT activity in a tissue- or cell-specific manner. They can be used alone or in combination with one or more engineered WNT agonists disclosed herein. In certain embodiments, the tissue-specific WNT signal enhancing molecules are bifunctional molecules that include a first domain that binds to one or more ZNRF3 and / or RNF43 ligases and a second domain that binds to one or more targeted tissues or cell types in a tissue- or cell-specific manner. Each of the first domain and the second domain can be any moiety that can bind to the ligase complex or the targeted tissue or cell, respectively. For example, each of the first domain and the second domain can be a moiety selected from, but not limited to, a polypeptide (e.g., an antibody or its antigen-binding fragment, or a peptide or polypeptide different from an antibody), a small molecule, and a natural ligand or its variant, fragment, or derivative. In certain embodiments, the natural ligand is a polypeptide, a small molecule, an ion, an amino acid, a lipid, or a sugar molecule. The first domain and the second domain can be the same type of moiety, or they can be different types of moieties.In certain embodiments, tissue-specific WNT signal enhancer molecule binds to tissue- or cell-specific cell surface receptor.In certain embodiments, tissue-specific WNT signal enhancer molecule increases or enhances WNT signaling, for example, by at least 50%, at least 2-fold, at least 3-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, or at least 50-fold, compared with negative control.

[0171] Tissue-specific WNT signal enhancing molecule can have different formats. In certain embodiments, tissue-specific WNT signal enhancing molecule is a fusion protein comprising a first polypeptide sequence that binds to ZNRF3 / RNF43 and a second polypeptide sequence that binds to one or more targeted tissues or cell types in a tissue- or cell-specific manner. In certain embodiments, the two polypeptide sequences can be directly fused or fused via a linker. In certain embodiments, tissue-specific WNT signal enhancing molecule comprises two or more polypeptides, for example, a dimer or multimer comprising two or more fusion proteins, each comprising a first domain and a second domain, and the two or more polypeptides are linked, for example, via a linker moiety or by a bond between amino acid residues in each of the two or more polypeptides, for example, an intermolecular disulfide bond between cysteine ​​residues.

[0172] In certain embodiments, the tissue-specific WNT signal enhancing molecule is an antibody (or an antigen-binding fragment thereof) that comprises an antibody heavy chain and a light chain, which constitute either a first domain or a second domain, and the other domain (i.e., the second domain or the first domain) is linked to the antibody heavy chain or light chain, either as a fusion protein or via a linker moiety. In certain embodiments, the other domain is linked to the N-terminus of the heavy chain, the C-terminus of the heavy chain, the N-terminus of the light chain, or the C-terminus of the light chain. Such structures may be referred to herein as additional IgG scaffolds or formats. For example, the tissue-specific WNT signal enhancing molecule may be an antibody that binds to ZNRF3 / RNF43, where a binding domain that binds to a tissue- or cell-specific receptor is fused or added to either the heavy chain or the light chain of the antibody that binds to ZNRF3 / RNF43. In another example, the tissue-specific WNT signal enhancing molecule can be an antibody that binds to a tissue- or cell-specific receptor, in which a binding domain that binds to ZNRF3 / RNF43 is fused or attached to either the heavy or light chain of the antibody that binds to the tissue- or cell-specific receptor.

[0173] In certain embodiments, the intestine-specific WNT signal enhancing molecule is an antibody or antigen-binding fragment thereof that binds to GPA33, CDH17, MUC-13, and the binding domain that binds to ZNRF3 / RNF43 is fused or appended to either the heavy or light chain of the antibody or antigen-binding fragment thereof. In certain embodiments, the binding domain that binds to ZNRF3 / RNF43 comprises Fu1 and Fu2 domains, and the Fu1 and Fu2 domains optionally comprise one or more amino acid modifications, including any of those disclosed herein, e.g., F105R and / or F109A.

[0174] In certain embodiments, tissue-specific WNT signal enhancing molecules include a first domain ("action module") that binds to ZNRF3 / RNF43, and a second domain ("targeting module") that binds to tissue- or cell-specific receptors, for example with high affinity. In certain embodiments, each of these two domains has substantially reduced activity or is inactive in enhancing WNT signaling by itself. However, when tissue-specific WNT signal enhancing molecules engage with target tissues that express tissue-specific receptors, E3 ligase ZNRF3 / RNF43 is recruited to a ternary complex with the tissue-specific receptor, thereby being sequestered and / or cleared from the cell surface by receptor-mediated endocytosis. The end result is the enhancement of WNT signaling in a tissue-specific manner.

[0175] In certain embodiments, the action module is a binding factor to ZNRF3 / RNF43 E3 ligase, and the action module can be designed based on R-spondin, for example, R-spondin-1-4, including but not limited to human R-spondin-1-4. In certain embodiments, the action module is an R-spondin, for example, wild-type R-spondin-1-4, optionally human R-spondin-1-4, or a variant or fragment thereof. In certain embodiments, it is a variant of any of R-spondin-1-4 that has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the corresponding wild-type R-spondin-1-4 sequence. In certain embodiments, the action module comprises or consists of the furin domain 1 of an R-spondin, for example, any of R-spondin-1-4, that binds to ZNRF3 / RNF43. Extended versions of furin domain 1 (including but not limited to those with mutant furin domain 2 that no longer binds to LGR4-6 or has reduced binding to LGR4-6), or engineered antibodies, or any other derivatives, or any engineered polypeptides different from antibodies that can specifically bind to ZNRF3 / RNF43, can also be used. In certain embodiments, the action module comprises one or more furin domains 1 of R-spondin.

[0176] In certain embodiments, the action module does not include the furin domain 2 of R-spondin, or it includes a modified or variant furin domain 2 of R-spondin, for example a furin domain 2 with reduced activity compared to wild-type furin domain 2. In certain embodiments, the action module includes the furin domain 1 of R-spondin, but does not include the furin domain 2. In certain embodiments, the action module includes two or more furin domains 1, or a multimer of furin domain 1. The action domain may include one or more wild-type furin domains 1 of R-spondin. In certain embodiments, the action module includes a modified or variant furin domain 1 of R-spondin with increased activity, for example binding to ZNRF3 / RNF43, compared to wild-type furin domain 1. Variants with increased binding to ZNRF3 / RNF43 can be identified, for example, by screening a phage or yeast display library that includes variants of R-spondin furin domain 1. Peptides or polypeptides that have increased binding to ZNRF3 / RNF43 but are unrelated to R-spondinfurin domain 1 can also be identified by screening. The action module can further comprise additional moieties or polypeptide sequences, for example, additional amino acid residues, to stabilize the structure of the action module or tissue-specific WNT signal enhancing molecule in which it is present.

[0177] In further embodiments, the action module comprises another inhibitory moiety, such as a nucleic acid molecule that reduces or prevents ZNRF3 / RNF43 activity or expression, for example, an antisense oligonucleotide; a small interfering RNA (siRNA); a small hairpin RNA (shRNA); a microRNA (miRNA); or a ribozyme. As used herein, "antisense" refers to a nucleic acid sequence, regardless of length, that is complementary to a nucleic acid sequence. In certain embodiments, antisense RNA refers to a single-stranded RNA molecule that can be introduced into an individual cell, tissue, or subject and results in a reduction in the expression of a target gene by a mechanism that does not necessarily rely on an endogenous gene silencing pathway. Antisense nucleic acids may contain modified backbones, such as phosphorothioates, phosphorodithioates, or others known in the art, or may contain non-natural internucleoside linkages. Antisense nucleic acids may include, for example, locked nucleic acids (LNAs). In certain embodiments, the other inhibitory moiety is one that inhibits the activity of one or both of ZNRF3 / RNF43 or one that inhibits the gene, mRNA or protein expression of one or both of ZNRF3 / RNF43. In certain embodiments, the inhibitory moiety is a nucleic acid molecule that binds to the ZNRF3 / RNF43 gene or mRNA or its complement.

[0178] In certain embodiments, the targeting module specifically binds to a cell-specific surface molecule, e.g., a cell-specific surface receptor, and can be, e.g., a natural ligand, an antibody, or a synthetic chemical. In certain embodiments, the cell-specific surface molecule is preferentially expressed in a target organ, tissue, or cell type, e.g., an organ, tissue, or cell type, for which it is desired to enhance WNT signaling to treat or prevent a disease or disorder. In certain embodiments, the cell-specific surface molecule is increased or enhanced in expression in a target organ, tissue, or cell type, e.g., an organ, tissue, or cell type, for which it is desired to enhance WNT signaling to treat or prevent a disease or disorder, for example, compared to one or more other non-targeted organs, tissues, or cell types. In certain embodiments, the cell-specific surface molecule is preferentially expressed on the surface of a target organ, tissue, or cell type, respectively, compared to one or more other organs, tissues, or cell types. For example, in certain embodiments, a cell surface receptor is considered to be a tissue- or cell-specific cell surface molecule if it is expressed in a target organ, tissue or cell at a level at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 100-fold, at least 500-fold, or at least 1000-fold higher than its expression in one or more, five or more, all other organs, tissues or cells, or the average of all other organs, tissues or cells, respectively. In certain embodiments, a tissue- or cell-specific cell surface molecule is a cell surface receptor, e.g., a polypeptide receptor, that includes a region located within the cell surface membrane and an extracellular region to which a targeting module can bind.In various embodiments, the methods described herein may be carried out by specifically targeting a cell surface molecule that is expressed only in the target tissue or a subset of tissues that includes the target tissue, or by specifically targeting a cell surface molecule that has a high level of expression in the target tissue compared to all, a majority, or a substantial number of other tissues, e.g., higher expression in the target tissue than at least 2, at least 5, at least 10, or at least 20 other tissues.

[0179] Tissue-specific and cell-specific cell surface receptors are known in the art.Examples of tissue-specific and cell-specific cell surface receptors include, but are not limited to, GPA33, CDH17, and MUC-13.In certain embodiments, the targeting module comprises an antibody or its antigen-binding fragment that specifically binds to these intestinal specific receptors.

[0180] In certain embodiments, components of an engineered WNT agonist can be combined with a WNT signal enhancing molecule to confer greater tissue specificity.

[0181] The present invention is based in part on the use of engineered WNT agonists to regulate gastrointestinal epithelial proliferation, particularly in inflammatory bowel disease.

[0182] In one embodiment, the present invention provides a method of treating a subject suffering from a gastrointestinal disorder, comprising administering to the subject an engineered WNT signalling modulator.

[0183] In certain embodiments, the engineered WNT agonist comprises one or more binding domains that bind one or more FZD receptors (FZD1-10) and one or more binding domains that bind one or more LRP (LRP5-6) receptors. In yet further embodiments, the binding domains of the engineered WNT agonist comprise one or more binding domains that bind FZD5, FZD8, FZD1, FZD2, FZD7, FZD5,8, FZD1,2,7 or FZD1,2,7,5,8; FZD4; FZD9; or FZD10, and one or more binding domains that bind LRP5, LRP6, or LRP5 and 6. In further embodiments, the engineered WNT agonist comprises one or more binding domains that bind FZD5 and / or FZD8, and one or more binding domains that bind LRP5 and / or LRP6. In yet further embodiments, the engineered WNT agonist comprises binding domains that bind FZD5 and FZD8, and a binding domain that binds LRP6. In further embodiments, the WNT agonist comprises a heavy chain sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, or 17, or a variable heavy chain region derived therefrom; and a light chain sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, or 18, or a variable light chain region derived therefrom.

[0184] In some embodiments, the engineered WNT agonist comprises a tissue targeting molecule, hi further embodiments, the tissue targeting molecule is an antibody or fragment thereof that binds to a tissue-specific cell surface antigen. In some embodiments, the tissue targeting molecule is selected from the group consisting of cell surface A33 antigen (GPA33; a representative sequence is NCBI polypeptide reference sequence NP_005805.1), cadherin-17 (CDH17; a representative sequence is NCBI polypeptide reference sequence NP_004054.3), and mucin 13 (cell surface associated (Muc-13; a representative sequence is NCBI polypeptide reference sequence NP_149038.3), or a functional fragment or variant thereof. In certain embodiments, the WNT agonist is administered with a binding domain that specifically binds to an inflammatory molecule. In further embodiments, the binding domain that specifically binds to an inflammatory molecule is an antagonist of the inflammatory molecule. In further embodiments, the antagonist of the inflammatory molecule is an antagonist of TNFα, an antagonist of IL-12, an antagonist of IL-12 and IL-23, or an antagonist of IL-23.

[0185] In another related embodiment, the present disclosure provides a combination molecule comprising: a) an engineered WNT agonist disclosed herein; and b) an engineered WNT signal enhancing molecule comprising a first domain that binds to one or more E3 ubiquitin ligases and a second domain that binds to a tissue-specific receptor.

[0186] In related embodiments, the disclosure provides a polypeptide that specifically binds to Frizzled 5 (FZD5) and Frizzled 8 (FZD8), the polypeptide comprising one or more sequences having at least 80%, at least 90%, at least 95%, or at least 98% homology to any of the sequences set forth in SEQ ID NOs: 1-18. In some embodiments, the polypeptide comprises an antibody or antibody binding fragment, e.g., one or more variable heavy or variable light chains. In some embodiments, the antibody or antibody binding fragment comprises at least five or all six of the CDRs present in any of the following sequence combinations: SEQ ID NOs: 1 and 2; SEQ ID NOs: 3 and 4; SEQ ID NOs: 5 and 6; or SEQ ID NOs: 7 and 8, SEQ ID NOs: 9 and 10, SEQ ID NOs: 11 and 12, SEQ ID NOs: 13 and 14, SEQ ID NOs: 15 and 16, or SEQ ID NOs: 17 and 18. In some embodiments, the polypeptide comprises six of the CDRs present in any of these sequence combinations, where one or more of the CDRs comprises one, two, or three amino acid modifications, optionally a point mutation, an amino acid deletion, or an amino acid insertion.

[0187] The present disclosure also provides combination molecules comprising an engineered WNT agonist as disclosed herein; and an engineered WNT signal enhancing molecule comprising a first domain that binds to one or more E3 ubiquitin ligases and a second domain that binds to a tissue-specific receptor.

[0188] In some embodiments, the engineered WNT agonists of the present disclosure promote cell differentiation toward enterocytes (e.g., gastrointestinal cells, stem cells, and / or epithelial cells). In some embodiments, cell differentiation is determined based on the percentage of intestinal progenitor cells. In some embodiments, a timestamp-based method is used to determine cell differentiation toward enterocytes. In some embodiments, a lineage trajectory inference tool, slingshot, is used to complement the timestamp-based observations of cell differentiation. In some embodiments, slingshot predicts the direction of cell differentiation from an initial starting population. As an example, slingshot predicted cells progress to TA1, goblet, brush, and enteroendocrine in one direction and enterocyte in the other direction. In some embodiments, the predicted lineage trajectory pseudo time series values ​​show a higher percentage of engineered WNT agonist-treated samples that progress further along the enterocyte lineage trajectory compared to control-treated cells; FIG. 28E provides an example of predicted pseudo time series values. In some embodiments, this prediction for the intestinal cell lineage is consistent with the actual timestamp data.

[0189] In some embodiments, the reliable standard for verifying improved differentiation is that at a given number of days after inducing injury, the expression of mature, differentiated cell type markers in the engineered WNT agonist-treated group is more similar to that of naive, non-injured colon compared to the control group.In some embodiments, improved differentiation is observed after 6 days of treatment with engineered WNT agonist.In some embodiments, the engineered WNT agonist-treated sample comprises enterocytes, goblet cells, enteroendocrine, tuft cells, or combinations thereof.

[0190] The evidence disclosed herein shows that the Wnt mimetic molecules of the present disclosure have desirable properties, including the ability to restore diseased intestinal tissue to normal physiological function. In some embodiments, short-term treatment with the Wnt mimetics of the present disclosure (e.g., R2M13-26 or R2M13-h26) induces rapid restoration of epithelial tissue. As an illustration, in a severe DSS model, a single injection of R2M13-26 at various doses restores normal histology of damaged colonic tissue. Within 6 days of treatment, R2M13-h26 completely restores the severely damaged epithelial barrier in an acute DSS model.

[0191] In addition to barrier and colonic tissue restoration, in some embodiments, treatment with the Wnt mimics of the present disclosure shows a reduction in inflammatory cytokines and disease activity index, thereby eliminating the vicious circle of barrier damage, microbial pathogen invasion, tissue inflammation and damage.In some embodiments, the Wnt mimics of the present disclosure directly affect epithelial cells, expanding progenitor cell pools and accelerating differentiation into all mature differentiated cell types.In some embodiments, the Wnt mimics of the present disclosure restore Wnt signaling and stem cell niche in damaged colonic tissue, without further affecting crypts after repair.

[0192] In some embodiments, treatment with the Wnt mimic of the present disclosure alone does not affect normal intestinal epithelium.In such embodiments, RSPO can induce hyperplasia.Together, the present disclosure provides a Wnt activator with optimal tissue repair and bioactivity.

[0193] As an illustration, treatment with R2M13-26, an Fzd5,8-specific Wnt mimetic, resulted in rapid mucosal healing within days, with improved tissue histology and disease activity, and a concomitant reduction in inflammation and colitis symptoms. In this injury model, R2M13-26 primarily affected the epithelium immediately after administration. Wnt target genes, such as Axin2, were increased in the epithelium 24 hours after treatment, suggesting that exploiting FZD receptor specificity is a viable option for inducing tissue layer-specific pathway activation. Concomitant with induction of Wnt target genes, R2M13-26 also caused a robust increase in cell cycle gene expression in a wide range of progenitor cells, whether they were normal stem / progenitor cells in response to injury or in an altered cellular state consistent with dedifferentiation. These transcriptomic changes manifested as a transient expansion of the progenitor pool and accelerated differentiation of the appropriate secretory and absorptive lineages into colonic epithelium and reconstitution of the epithelial barrier. The direct effect on epithelial regeneration and barrier restoration secondarily led to a reduction in inflammatory signals and infiltrating immune cells.

[0194] The injury / damage situation may set the stage for epithelial progenitor cell expansion. As disclosed in the accompanying examples, injury triggers an inflammatory response in all tissue layers in addition to affecting developmental signaling pathways such as EGF and Notch. In epithelium, interferon gamma and NF-κB pathways become active after injury, and recent studies on other stem cell niches have shown that inflammatory signaling can promote the initial proliferative response to injury (M. Chen, Reed, & Lane, 2017; Kyritsis et al., 2012). Activation of NF-kB and Wnt pathways together may further promote the process of dedifferentiation towards progenitor cells in the intestine (Schwitalla et al., 2013). Illustratively, in the DSS model disclosed herein, Wnt signaling was dramatically reduced in colonic epithelium, likely due to reduced expression of certain Wnts and increased expression of some Wnt antagonists. In this instance, R2M13-h26 was able to overcome this defect in Wnt signaling. Thus, R2M13-h26-induced Wnt pathway activation may cooperate with these inflammatory signals to enhance progenitor cell proliferation, even if only transiently.

[0195] Unlike the effect of RSPO, which affects both non-injured and injured epithelium (Kim et al., 2005; Yan Kelley S. et al., 2017; Zhao et al., 2007), targeted receptor-level Wnt signaling agonism using the disclosed Wnt mimetics can promote specific crypt proliferation in the context of injured tissue. As an illustration, extensive proliferation in the small intestine and colon was observed when the Wnt mimetic R2M13-26 was introduced together with RSPO2 in a DSS model. Although reversal of DSS-induced colitis in mice by RSPO was observed, hyperproliferation also occurred with RSPO treatment. We surprisingly found that the Wnt mimetic itself can specifically induce the expression of β-catenin target genes and proliferation of epithelial cells in the injured colon.

[0196] As an illustration, Wnt pathway activation by R2M13-26 did not lead to crypt hyperproliferation or enlargement. This is in sharp contrast to the effects of inherited genetic variants as well as RSPO treatment. As previously reported, genetically ablation of the negative regulator Apc or expression of a constitutively active variant of beta-catenin resulted in crypts proliferating in an uncontrolled manner and failing to differentiate (Barker Nick et al., 2009; Krausova & Korinek, 2014; Mah, Yan, & Kuo, 2016). However, the disclosed Wnt mimics avoided these outcomes by mimicking endogenous Wnt signaling and initiating pathway activation at the receptor level, as opposed to permanent genetic modifications in which negative feedback is avoided. R2M13-26 allows for the negative feedback mechanism to be enabled by affecting the pathway at the receptor level. In this illustration, Axin2 was induced, which contributes to the destruction complex. Expression of the E3 ubiquitin ligase Rnf43, another Wnt target gene, was also increased, promoting the removal of FZD receptors from the cell surface, and in this instance, R2M13-26 increased the expression of several inhibitors of cyclin-dependent kinases, potentially limiting proliferation.

[0197] IV. Pharmaceutical Compositions Also disclosed is a pharmaceutical composition comprising the engineered WNT agonist molecule described herein and one or more pharma- ceutically acceptable diluents, carriers, or excipients. In another embodiment, the present disclosure provides a pharmaceutical composition comprising the polypeptide, engineered WNT agonist, or combination molecule disclosed herein and one or more pharma- ceutically acceptable diluents, carriers, or excipients.

[0198] In further embodiments, pharmaceutical compositions are also disclosed that include a polynucleotide comprising a nucleic acid sequence encoding a WNT agonist molecule (or a polypeptide chain thereof) described herein, and one or more pharma- ceutically acceptable diluents, carriers, or excipients. In certain embodiments, the polynucleotide is a DNA or mRNA, e.g., a modified mRNA. In certain embodiments, the polynucleotide is a modified mRNA that further comprises a 5' cap sequence and / or a 3' tailing sequence, e.g., a polyA tail. In other embodiments, the polynucleotide is an expression cassette that comprises a promoter operably linked to the coding sequence.

[0199] In some embodiments, the WNT agonist is an engineered recombinant polypeptide that incorporates different epitope-binding fragments that bind to different molecules in the WNT signaling pathway. For example, FZD and LRP antibody fragments (e.g., Fab, scFv, sdAb, VHH, etc.) can be joined together on one molecule directly or using linkers of different sizes.

[0200] Similarly, polypeptides such as RSPO can be engineered to contain an antibody or fragment thereof against a tissue-specific cell surface antigen, e.g., MUC-13. RSPO can also be administered simultaneously or sequentially with an enhancer of the E3 ligase ZNRF3 / RNF43. The E3 ligase enhancer can be an agonistic antibody or fragment that binds to ZNRF3 / RNF43 and enhances E3 ligase activity.

[0201] Conversely, WNT agonist can also be a recombinant polypeptide that incorporates epitope-binding fragments that bind to various molecules in the WNT signaling pathway and enhance WNT signaling.For example, WNT agonist can be an antibody or fragment thereof that binds to FZD receptor and / or LRP receptor and enhances WNT signaling.FZD and LRP antibody fragments (such as Fab, scFv, sdAb or VHH) can be joined together directly or by using linkers of various sizes on one molecule.

[0202] In further embodiments, pharmaceutical compositions are also disclosed that include an expression vector, e.g., a viral vector, comprising a polynucleotide that includes a nucleic acid sequence encoding a WNT agonist molecule described herein, and one or more pharma- ceutically acceptable diluents, carriers, or excipients.

[0203] The present disclosure also contemplates a pharmaceutical composition comprising a cell comprising an expression vector comprising a polynucleotide comprising a promoter operably linked to the nucleic acid encoding a WNT agonist molecule, and one or more pharma- ceutically acceptable diluents, carriers or excipients.In certain embodiments, the pharmaceutical composition further comprises a cell comprising an expression vector comprising a polynucleotide comprising a promoter operably linked to the nucleic acid sequence encoding a WNT agonist.In certain embodiments, the cell is a heterologous cell or an autologous cell obtained from the subject to be treated.

[0204] The molecules of interest, alone or in combination, can be combined with pharma- ceutically acceptable carriers, diluents, excipients and reagents useful in preparing formulations that generally contain safe, non-toxic, and desirable excipients that are acceptable for use in mammals, such as humans or primates. Such excipients can be solid, liquid, semi-solid, or, in the case of aerosol compositions, gaseous. Examples of such carriers, diluents and excipients include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Supplementary active compounds can also be incorporated into the formulation. The solution or suspension used in the formulation may include a sterile diluent, such as water for injection, saline solution, non-volatile oil, polyethylene glycol, glycerin, propylene glycol or other synthetic solvent; an antibacterial compound, such as benzyl alcohol or methylparaben; an antioxidant, such as ascorbic acid or sodium bisulfite; a chelating compound, such as ethylenediaminetetraacetic acid (EDTA); a buffer, such as acetic acid, citric acid or phosphoric acid; a detergent, such as Tween® 20 to prevent aggregation; and a compound for adjusting osmotic pressure, such as sodium chloride or dextrose. pH may be adjusted with an acid or base, such as hydrochloric acid or sodium hydroxide. In certain embodiments, the pharmaceutical composition is sterile.

[0205] The pharmaceutical compositions may further include sterile aqueous solutions or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, or phosphate buffered saline (PBS). In some cases, the composition may be fluid so that it can be drawn into a syringe or delivered from a syringe to a subject. In certain embodiments, it is stable under the conditions of manufacture and storage and is protected against the contaminating action of microorganisms such as bacteria and fungi. The carrier may be, for example, a solvent or dispersion medium containing water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and suitable mixtures thereof. The proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. Prevention of the action of microorganisms may be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it is preferable to include isotonic agents in the composition, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride, etc. Prolonged absorption of the internal composition can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

[0206] Sterile solution can be prepared by incorporating the engineered WNT agonist, for example, an antibody or its antigen-binding fragment (or the coding polynucleotide or the cell containing it) in the required amount in a suitable solvent with one or a combination of the above-listed components, and then optionally filter sterilization.Generally, dispersion is prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the other required components listed above.In the case of sterile powder for preparing sterile injectable solution, the method of preparation is vacuum drying and freeze-drying, which produces a powder of the active ingredient plus any additional desired ingredients from its previously sterile-filtered solution.

[0207] In one embodiment, pharmaceutical compositions such as controlled release formulations, including implants and microencapsulated delivery systems, are prepared using carriers that protect the antibody or its antigen-binding fragment against rapid elimination from the body.Biodegradable biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid, can be used.Methods for the preparation of such formulations are clear to those skilled in the art.These materials can also be obtained commercially.Liposomal suspensions can also be used as pharmaceutically acceptable carriers.These can be prepared according to methods known to those skilled in the art.

[0208] For ease of administration and uniformity of dosage, it may be advantageous to formulate pharmaceutical compositions in dosage unit form.Dosage unit form, as used herein, refers to a physically separate unit suitable as a unit dosage for the subject to be treated; each unit contains a predetermined amount of active antibody or its antigen-binding fragment calculated to produce a desired therapeutic effect in association with the required pharmaceutical carrier.The specification for dosage unit form is determined and directly depends on the unique characteristics of antibody or its antigen-binding fragment, and the specific therapeutic effect to be achieved, as well as the inherent limitations in the field of making such active antibody or its antigen-binding fragment for the treatment of individuals.

[0209] The pharmaceutical compositions may be included in a container, pack, or dispenser, such as a syringe, including a pre-filled syringe, together with instructions for administration.

[0210] The pharmaceutical compositions of the present disclosure include any pharma- ceutically acceptable salts, esters, or salts of such esters, or any other compounds capable of providing (directly or indirectly) a biologically active antibody or antigen-binding fragment thereof, upon administration to an animal, including a human.

[0211] The present disclosure includes pharmaceutically acceptable salts of the WNT agonist molecules described herein. The term "pharmaceutically acceptable salts" refers to physiologically and pharmaceutically acceptable salts of the compounds of the present disclosure: that is, salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects to it.Various pharmaceutically acceptable salts are known in the art and are described, for example, in "Remington's Pharmaceutical Sciences", 17th edition, Alfonso R. Gennaro (Ed.), Mark Publishing Company, Easton, PA, USA, 1985 (and its more recent editions), "Encyclopaedia of Pharmaceutical Technology", 3rd edition, James Swarbrick (Ed.), Informa Healthcare USA (Inc.), NY, USA, 2007 and J. Pharm. Sci. 66: 2 (1977). Also, for a review of suitable salts see "Handbook of Pharmaceutical Salts: Properties, Selection, and Use", Stahl and Wermuth (Wiley-VCH, 2002). Pharmaceutically acceptable base addition salts are formed with metals or amines, such as alkali metals and alkaline earth metals or organic amines.

[0212] Metals used as cations include sodium, potassium, magnesium, calcium, and the like. Amines include N-N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine (see, for example, Berge et al., "Pharmaceutical Salts," J. Pharma Sci., 1977, 66, 119). The base-addition salts of the above acidic compounds are prepared by contacting the free acid form with a sufficient amount of the desired base to produce the salt in a conventional manner. The free acid form may be regenerated by contacting the salt form with an acid and isolating the free acid in a conventional manner. The free acid forms differ somewhat from their respective salt forms in certain physical properties, such as solubility in polar solvents, but otherwise the salts are equivalent to their respective free acids for purposes of this disclosure.

[0213] In some embodiments, the pharmaceutical compositions provided herein comprise a therapeutically effective amount of a WNT agonist molecule described herein or a pharma- ceutically acceptable salt thereof in admixture with a pharma- ceutically acceptable carrier, diluent and / or excipient, such as saline, phosphate buffered saline, phosphate, and amino acids, polymers, polyols, sugars, buffers, preservatives, and other proteins. Exemplary amino acids, polymers, sugars, and the like are octylphenoxypolyethoxyethanol compounds, monostearate polyethylene glycol compounds, polyoxyethylene sorbitan fatty acid esters, sucrose, fructose, dextrose, maltose, glucose, mannitol, dextran, sorbitol, inositol, galactitol, xylitol, lactose, trehalose, bovine or human serum albumin, citrate, acetate, Ringer's and Hanks' solution, cysteine, arginine, carnitine, alanine, glycine, lysine, valine, leucine, polyvinylpyrrolidone, polyethylene, and glycol. Preferably, the formulation is stable at 4° C. for at least 6 months.

[0214] In some embodiments, the pharmaceutical compositions provided herein include a buffer, such as phosphate buffered saline (PBS) or sodium phosphate / sodium sulfate, Tris buffer, glycine buffer, sterile water and other buffers known to those of skill in the art, such as those described by Good et al. (1966) Biochemistry 5:467. The pH of the buffer can be in the range of 6.5 to 7.75, preferably in the range of 7 to 7.5, and most preferably in the range of 7.2 to 7.4.

[0215] V. How to use The present disclosure also provides methods for using engineered WNT agonists and / or tissue-specific WNT signal enhancing molecules, e.g., to modulate the WNT signaling pathway, e.g., to increase WNT signaling, as well as administration of engineered WNT agonists and / or tissue-specific WNT signal enhancing molecules in various therapeutic contexts.

[0216] Methods of treatment using engineered WNT agonist molecules and / or tissue-specific WNT signal enhancing molecules are also provided herein.

[0217] In certain embodiments, engineered WNT agonist can be used to increase Wnt signaling in tissue or cell.Thus, in some aspects, the present invention provides a method for increasing or enhancing Wnt signaling in tissue or cell, comprising contacting tissue or cell with an effective amount of engineered WNT agonist disclosed herein or its pharma-ceutically acceptable salt, wherein engineered WNT agonist is a Wnt signaling pathway agonist.In some embodiments, the contacting step is carried out in vitro, ex vivo, or in vivo.In certain embodiments, the cell is a cultured cell, and the contacting step is carried out in vitro.In certain embodiments, the method further comprises contacting tissue or cell with one or more Wnt polypeptides or Norrin polypeptides.

[0218] The engineered WNT agonist disclosed herein can be used to treat disease, disorder or condition, for example, by increasing Wnt signaling in targeted cells, tissues or organs.Thus, in some aspects, the present invention provides a method for treating a disease or condition, for example, a disease or disorder associated with reduced Wnt signaling or a disease or disorder in which increased Wnt signaling provides therapeutic benefit, in a subject in need thereof, comprising contacting the subject with an effective amount of the composition of the present disclosure.In certain embodiments, the composition is a pharmaceutical composition comprising any of the following: engineered WNT agonist; one or more polynucleotides, for example, DNA or mRNA, comprising a nucleic acid sequence encoding a liver engineered WNT agonist, optionally modified mRNA; a vector, for example, an expression vector or viral vector, comprising a nucleic acid sequence encoding an engineered WNT agonist; or a cell comprising a nucleic acid sequence encoding an engineered WNT agonist.In certain embodiments, the disease or condition is a pathological disease or disorder, or injury, for example, injury resulting from a wound.In certain embodiments, the wound can be the result of another therapeutic treatment. In certain embodiments, the disease or condition involves impaired tissue repair, healing or regeneration, or would benefit from increased tissue repair, healing or regeneration, hi some embodiments, the contacting step is performed in vivo, i.e., the subject composition is administered to the subject.

[0219] Wnt signaling plays an important role in developmental processes and the maintenance of stem cells. Reactivation of Wnt signaling is associated with the regeneration and repair of most tissues after injury and disease. Engineered WNT agonist molecules are expected to provide healing and tissue repair benefits in response to injury and disease. Causes of tissue damage and loss include, but are not limited to, aging, degeneration, genetic conditions, infection and inflammation, traumatic injury, toxin / metabolism-induced toxicity, or other pathological conditions. Wnt signals and enhancers of Wnt signals have been shown to activate adult tissue-resident stem cells. In some embodiments, the compounds of the present invention are administered for use in treating diseased or damaged tissues, for use in tissue regeneration and for use in cell growth and proliferation, and / or for use in tissue engineering.

[0220] Human diseases related to mutations in Wnt pathway provide strong evidence of the enhancement of Wnt signal in disease treatment and prevention.Preclinical in vivo and in vitro studies provide further evidence of the involvement of Wnt signal in many disease states, further supporting the use of engineered WNT agonists in various human diseases.For example, the composition of the present invention can be used to promote or increase bone growth or regeneration, bone grafting, fracture healing, osteoporosis and osteoporotic fractures, spinal fusion, spinal cord injury including vertebral compression fracture, preoperative spinal surgery optimization, orthopedic device osseointegration, tendon-osseointegration, tooth growth and regeneration, dental implantation, periodontal disease, maxillofacial reconstruction, and the treatment of osteonecrosis of the jaw. The compositions of the present invention may also be used to treat alopecia; enhance the regeneration of sensory organs, e.g., the treatment of hearing loss, including the regeneration of inner and outer hair cells, the treatment of reduced vestibular function, the treatment of macular degeneration, the treatment of retinopathies, including vitreous retinopathy, diabetic retinopathy, other diseases of retinal degeneration, Fuchs' dystrophy, other corneal diseases, and the like; the treatment of stroke, traumatic brain injury, Alzheimer's disease, multiple sclerosis, multiple dystrophies, muscle atrophy as a result of sarcopenia or cachexia, and other conditions which affect the degeneration or integrity of the blood-brain barrier.

[0221] In certain embodiments, the present invention provides a method for treating a subject with a disease or disorder that is related to the reduction of WNT signaling or that may benefit from the increase of Wnt signaling, comprising administering to the subject an effective amount of an engineered WNT agonist or a pharmaceutical composition that comprises an engineered WNT agonist.In certain embodiments, the disease or disorder is oral mucositis, short bowel syndrome, inflammatory bowel disease (IBD), graft-versus-host disease (GVHD), alcoholic hepatitis, short bowel syndrome, celiac disease, other gastrointestinal disorders, including but not limited to radiation-induced gastrointestinal mucositis and chemotherapy-induced gastrointestinal mucositis; metabolic syndrome, treatment of dyslipidemia, treatment of diabetes, pancreatitis, treatment of conditions in which pancreatic exocrine or endocrine tissue is damaged; conditions in which enhanced epidermal regeneration is desired, such as epidermal wound healing, diabetic foot ulcers, syndromes involving teeth, nails, or skin hypoplasia, and conditions in which angiogenesis is beneficial; myocardial infarction, coronary artery disease, heart failure; immune deficiency, graft-versus-host disease, acute kidney injury, The therapeutic agent is selected from the group consisting of chronic kidney disease, chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), cirrhosis, acute liver failure, chronic liver disease due to hepatitis C or B virus infection or following antiviral drug therapy, alcoholic liver disease, alcoholic hepatitis, non-alcoholic liver disease with steatosis or steatohepatitis, treatment of hearing loss including loss of inner and outer hair cells, vestibular hypofunction, macular degeneration, vitreous retinopathy, diabetic retinopathy, other diseases of retinal degeneration, Fuchs' dystrophy, treatment of other corneal diseases, stroke, traumatic brain injury, Alzheimer's disease, multiple sclerosis and other conditions affecting the blood-brain barrier; spinal cord injury, bone-related diseases, other spinal cord diseases, and alopecia.

[0222] The engineered WNT agonists and compositions of the invention may also be used to treat oral mucositis, inflammatory bowel disease (IBD), including short bowel syndrome, Crohn's disease (CD) and ulcerative colitis (UC), particularly CD associated with fistulization, other gastrointestinal disorders; metabolic syndrome, dyslipidemia, diabetes, pancreatitis, conditions in which exocrine or endocrine pancreatic tissue is damaged; conditions in which enhanced epidermal regeneration is desired, e.g., epidermal wound healing, diabetic foot ulcers, syndromes involving dental, nail, or skin hypoplasia, and other conditions in which angiogenesis is beneficial; myocardial infarction, coronary artery disease, heart failure; enhancing hematopoietic cell growth, e.g., enhancing hematopoietic stem cell transplantation from bone marrow, peripheral blood mobilization, immunodeficiency, graft versus host disease, and the like; treating acute kidney injury, chronic kidney disease; treating pulmonary disease, chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, including idiopathic pulmonary fibrosis, enhancing regeneration of lung tissue. The compositions of the present invention can also be used to regenerate hepatocytes, for example, to enhance liver regeneration, treat cirrhosis, enhance liver transplantation, treat acute liver failure, treat chronic liver disease due to hepatitis C or B virus infection or after antiviral drug therapy, alcoholic liver disease including alcoholic hepatitis, non-alcoholic liver disease with steatosis or steatohepatitis, etc. The compositions of the present invention can treat diseases and disorders including, but not limited to, conditions in which regenerative cell growth is desired.

[0223] Human genetics involving loss-of-function or gain-of-function mutations in Wnt signaling components provide strong evidence to support enhancing Wnt signaling for bone growth.Conditions in which enhanced bone growth is desired may include, without limitation, fractures, grafts, ingrowth around prosthetic devices, osteoporosis, osteoporotic fractures, spinal fusion, spinal compression fractures, preoperative optimization for spinal surgery, osteonecrosis of the jaw, dental implants, periodontal disease, maxillofacial reconstruction, etc.Engineered WNT agonists enhance and promote Wnt signaling, which is crucial in promoting bone regeneration.Methods for regenerating bone tissue benefit from the administration of the compounds of the present invention, which may be systemic or localized.In some embodiments, bone marrow cells are exposed to the molecules of the present invention, which results in activation of stem cells in bone marrow.

[0224] In some embodiments, bone regeneration is enhanced by contacting responsive cell populations, such as bone marrow, osteoprogenitor cells, osteoprogenitor cells, etc., with an effective dose of an engineered WNT agonist disclosed herein. Methods for regenerating bone tissue benefit from the administration of engineered WNT agonists, which may be systemic or localized. In some such embodiments, the contacting is performed in vivo. In other such embodiments, the contacting is performed ex vivo. Molecules can be localized to the site of action, for example, by loading onto a matrix that is optionally biodegradable and optionally provides sustained release of the active agent. Matrix carriers include, without limitation, absorbable collagen sponges, ceramics, hydrogels, polymeric microspheres, nanoparticles, bone cements, and the like.

[0225] In certain embodiments, compositions comprising one or more engineered WNT agonists disclosed herein (or polynucleotides encoding engineered WNT agonists, or vectors or cells comprising polynucleotides encoding engineered WNT agonists) are used to treat or prevent bone diseases or disorders, including, but not limited to, any of the following, or injuries associated with, including, but not limited to, any of the following: osteoporosis, osteoporotic fractures, bone fractures including vertebral compression fractures, nonunion fractures, delayed union fractures, spinal fusion, osteonecrosis, osteonecrosis of the jaw, hip joint, femoral head, etc., osseointegration of implants (e.g., accelerating recovery after partial or total knee or hip replacement), osteogenesis imperfecta, bone grafts, tendon repair, maxillofacial surgery, dental implants, any other bone disorder or defect due to genetic disease, degeneration, aging, drugs, or injury. In one embodiment, engineered WNT agonists that bind to Fzd1, Fzd2, and Fzd7 and also bind to LRP5 and / or LRP6 are used to treat or prevent any bone disease or disorder. In one embodiment, engineered WNT agonists that bind to Fzd1, Fzd2, Fzd5, Fzd7, and Fzd8 and also bind to LRP5 and / or LRP6 are used to treat or prevent any bone disease or disorder. Other Fzd molecules that bind to additional Fzd receptors can be used together with LRP5 and / or LRP6 binding agents.

[0226] In certain embodiments, the compositions and methods disclosed herein are used to increase bone mineral density, increase bone volume (e.g., tibia and / or femur volume), increase cortical thickness (e.g., trabecular area or femoral midshaft), increase bone mineralization rate, increase osteoblast number and / or decrease osteoclast number (e.g., in bone), increase bone stiffness, increase ultimate load for fracture, improve bone resistance to fracture, decrease bone resorption, reduce bone loss associated with osteoporosis, or increase bone biochemical strength in a subject. In one embodiment, engineered WNT agonists that bind Fzd1, Fzd2, and Fzd7 are used for any of these indicated uses. In one embodiment, engineered WNT agonists that bind Fzd1, Fzd2, Fzd5, Fzd7, and Fzd8 are used for any of these indicated uses.

[0227] The methods disclosed herein, including methods for treating or preventing bone diseases or disorders, include methods that include providing to a subject in need thereof both an engineered WNT agonist and a bone resorption inhibitor. In certain embodiments, the methods are used to treat osteoporosis, optionally postmenopausal osteoporosis.

[0228] The present disclosure also provides a method for inhibiting or reducing bone resorption in a subject in need thereof, comprising providing an effective amount of engineered WNT agonist to the subject, wherein the engineered WNT agonist is an agonist of Wnt signaling pathway.In certain embodiments, the method further comprises providing a bone resorption inhibitor to the subject.In certain embodiments, the subject is diagnosed with or at risk of osteoporosis, optionally postmenopausal osteoporosis.Various bone resorption inhibitors are known in the art, including but not limited to those disclosed herein.

[0229] When engineered WNT agonist is provided to subject in combination with another therapeutic agent, such as bone resorption inhibitor, the two agents can be provided in the same pharmaceutical composition or different pharmaceutical compositions.The two agents can be provided to subject at the same time, at different times, for example, simultaneously, continuously, or during overlapping or non-overlapping periods.In certain embodiments, the two agents are therapeutically active in subject during overlapping periods.

[0230] The composition comprising one or more engineered WNT agonists disclosed herein (or a polynucleotide encoding an engineered WNT agonist, or a vector or cell comprising a polynucleotide encoding an engineered WNT agonist) can be used for in vivo treatment of skeletal tissue defects. "Skeletal tissue defects" refers to defects in bone or other skeletal connective tissue at any site where it is desired to restore bone or connective tissue, regardless of the origin of the defect, for example, as a result of surgical intervention, tumor removal, ulceration, implant, fracture, or as a result of other traumatic or degenerative conditions. The composition of the present invention can be used to repair defects or lesions of cartilage tissue, such as degenerative wear and arthritis, trauma to tissue, replacement of torn meniscus, meniscectomy, joint dislocation due to torn ligament, joint malalignment, fracture, or genetic disease, as part of a regimen for restoring the cartilage function of connective tissue.

[0231] Engineered WNT agonists may also be used to treat periodontal disease. Periodontal disease is the leading cause of tooth loss and is associated with multiple systemic conditions. In some embodiments, tooth or underlying bone regeneration is enhanced by contacting with a responsive cell population. In some such embodiments, the contacting is performed in vivo. In other such embodiments, the contacting is performed ex vivo, followed by implantation of activated stem or progenitor cells. Molecules may be localized to the site of action, for example, by loading onto a matrix that is optionally biodegradable and optionally provides sustained release of the active agent. Matrix carriers include, without limitation, absorbable collagen sponges, ceramics, hydrogels, bone cements, polymeric microspheres, nanoparticles, and the like.

[0232] Several studies have shown that Wnt signaling and R-spondin biology can promote sensory hair cell regeneration in the inner ear after injury, aging, or degeneration.The composition of the present invention can also be beneficial for sensory hair cell loss in the inner ear that is involved in hearing loss or vestibular dysfunction.In the inner ear, the auditory organ contains mechanosensitive hair cells that are required to convert sound vibrations into electrical impulses.The vestibular organ, which is composed of the semicircular canal (SSC), utricle, and saccule, also contains sensory hair cells to detect head position and movement.The composition of the present invention can be used, for example, in injection; in matrix or other depot system; or in other external application to the ear, to enhance hearing regeneration.

[0233] The engineered WNT agonists can also be used to regenerate retinal tissue. In the adult mammalian retina, Müller glia cells can regenerate retinal cells, including photoreceptor cells, for example, after neurotoxic injury in vivo. Wnt signaling and enhancers of Wnt signals can promote the proliferation of Müller glia-derived retinal progenitor cells after injury or during degeneration. The compositions of the present invention can also be used to regenerate tissues and other cell types in the eye. For example, age-related macular degeneration (AMD), other retinal degenerative diseases, corneal diseases, Fuchs' dystrophy, vitreoretinopathy, genetic diseases, and the like, can benefit from the compositions of the present invention. AMD is characterized by a progressive decrease in central vision and visual acuity. Fuchs' dystrophy is characterized by a progressive loss of corneal endothelial cells. Wnt signals and enhanced Wnt signals can promote the regeneration of corneal endothelium, retinal epithelium, and the like in ocular tissues. In other embodiments, the compositions of the present invention can be used for retinal regeneration and treatment of macular degeneration, for example, in injections; in matrices or other depot systems; or in other topical applications to the eye.

[0234] A specific population of proliferative cells for homeostatic regeneration of hepatocytes, such as Axin 2 positive cells in the pericentral region, has been identified through lineage tracing studies. Lineage tracing studies have also identified additional potential hepatic progenitor cells, including but not limited to Lgr positive cells. Self-renewing hepatocytes and other populations of potential progenitor cells, including Lgr5 positive and Axin 2 positive cells, have been identified as capable of regenerating after injury in response to Wnt signaling and / or R-spondin. Numerous preclinical models of acute liver injury and liver failure as well as chronic liver disease have shown the recovery and regeneration of hepatocytes that benefit from enhancing Wnt signaling.

[0235] In certain embodiments, compositions comprising the engineered WNT agonist disclosed herein (or a polynucleotide encoding the engineered WNT agonist, or a vector or cell comprising a polynucleotide encoding the engineered WNT agonist) can be used to promote liver regeneration, reduce fibrosis, and / or improve liver function.In certain embodiments, the compositions and methods disclosed herein are used to increase liver weight, increase liver to body weight ratio, increase the number of PCNA and pH3 positive nuclei in the liver, increase Ki67 and / or cyclin D1 expression in the liver, increase hepatocyte proliferation and / or mitosis, reduce fibrosis after chronic liver injury, or increase hepatocyte function.

[0236] In certain embodiments, the compositions of the present invention can be used to treat acute liver failure, acute alcoholic liver injury, chronic liver disease caused by hepatitis C or B virus infection or after antiviral drug therapy, chronic alcoholic liver disease, alcoholic hepatitis, nonalcoholic fatty liver disease and nonalcoholic steatohepatitis (NASH), cirrhosis and severe chronic liver disease caused by all causes, and enhance the regeneration of liver cells.The method for regenerating liver tissue benefits from the administration of the compound of the present invention, which can be systemic or localized.These include but are not limited to the method of systemic administration and the method of localized administration, for example, by injection into liver tissue, by injection into veins or blood vessels leading to the liver, by implantation of sustained release formulations, etc.

[0237] In certain embodiments, compositions comprising an engineered WNT agonist (or a polynucleotide encoding an engineered WNT agonist, or a vector or cell comprising a polynucleotide encoding an engineered WNT agonist) disclosed herein are used to treat or prevent liver diseases or disorders, including, but not limited to, any of the following, or to treat or prevent liver injury or damage resulting from any of the following: acute liver failure (all causes), chronic liver failure (all causes), cirrhosis, liver fibrosis, liver disease (all causes), portal hypertension, alcoholic liver disease including alcoholic hepatitis, nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD) (fatty liver), alcoholic hepatitis, hepatitis C virus induced liver disease (HCV), hepatitis B virus induced liver disease (HBV), other viral hepatitis (e.g., hepatitis A virus induced liver disease (HAV) and hepatitis D virus induced liver disease (HDV)), primary biliary cirrhosis, autoimmune hepatitis, liver surgery, liver injury, liver transplantation, "small for size" syndrome in liver surgery and transplantation, congenital liver diseases and disorders, any other liver disorder or defect resulting from genetic disease, degeneration, aging, drugs, or injury.

[0238] Wnt signaling plays an important role in the regeneration of various epithelial tissues. A variety of epidermal conditions benefit from treatment with the compounds of the present invention. Mucositis occurs when there is a breakdown in the rapidly dividing epithelial cells lining the gastrointestinal tract, making the mucosal tissue susceptible to ulceration and infection. The part of the epithelial 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. Oral mucositis is perhaps the most common debilitating complication of cancer treatment, especially chemotherapy and radiation. In addition, the compositions of the present invention may also be beneficial in the treatment of short bowel syndrome, inflammatory bowel disease (IBD), or other gastrointestinal disorders. Other epidermal conditions include epidermal wound healing, diabetic foot ulcers, syndromes involving teeth, nails, or skin hypoplasia, and the like. The molecules of the present invention may be used for all of these conditions, in which case regenerative cells are contacted with the compounds of the present invention. Methods for regenerating epithelial tissue benefit from the administration of the compounds of the present invention, which may be systemic or localized. Contacting can be topical, including intradermal, subcutaneous, such as in a gel, lotion, cream, etc., applied to the targeted site.

[0239] In addition to skin and gastrointestinal tract, Wnt signaling and the enhancement and promotion of Wnt signaling also play an important role in the repair and regeneration of tissues, including pancreas, kidney, and lung in preclinical models.Engineered WNT agonists can be beneficial for various disease conditions involving the exocrine and endocrine pancreas, kidney, or lung.Engineered WNT agonists can be used to treat metabolic syndrome; treat diabetes, treat acute or chronic pancreatitis, treat exocrine pancreatic insufficiency, treat acute kidney injury, treat chronic kidney disease, treat lung diseases, including but not limited to chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, especially idiopathic pulmonary fibrosis (IPF), and other conditions that cause the loss of lung epithelial tissue.The methods for regenerating these tissues benefit from the administration of the compounds of the present invention, which can be systemic or localized.

[0240] Epidermal Wnt signaling, in cooperation with signaling through other developmental factors, is crucial for adult hair follicle regeneration.Alopecia is a common problem, and androgenetic alopecia, often referred to as male pattern baldness, is the most common form of alopecia in men.In some embodiments, hair follicle regeneration is enhanced by contacting responsive cell population with the molecules of the present invention.In some such embodiments, contacting is performed in vivo.In other such embodiments, contacting is performed ex vivo.Molecules can be localized to the site of action, for example, in topical lotions, gels, creams, etc.

[0241] Stroke, traumatic brain injury, Alzheimer's disease, multiple sclerosis, and other conditions that affect the blood-brain barrier (BBB) ​​can be treated with engineered WNT agonists. Angiogenesis is crucial to ensure the supply of oxygen and nutrients to many tissues throughout the body, and is particularly important to the CNS, because neural tissues are highly sensitive to hypoxia and ischemia. CNS endothelial cells that form the BBB are highly polarized cells held together by tight junctions and differ from endothelial cells in non-neural tissues in that they express specific transporters. Wnt signaling regulates angiogenesis and / or function in the CNS. Conditions in which the BBB is compromised can benefit from administration of the compounds of the invention, which can be systemic or localized, for example, by direct injection, intrathecal administration, implantation of sustained release formulations, and the like. In addition, Wnt signaling is actively involved in neurogenesis and plays a role in neuroprotection after injury. The compositions of the invention can also be used to treat spinal cord injury, other spinal cord diseases, stroke, traumatic brain injury, and the like.

[0242] Wnt signal also plays a role in angiogenesis.Engineered WNT agonist can be useful for treating conditions that angiogenesis is beneficial, such as myocardial infarction, coronary artery disease, heart failure, diabetic retinopathy, and conditions that result from genetic diseases.The method for regenerating these tissues benefits from the administration of the compound of the present invention, which can be systemic or localized.

[0243] In certain embodiments, the method of the present invention promotes tissue regeneration in tissues that have undergone, for example, injury or tissue or cell loss or loss. Loss or injury can be anything that causes a decrease in cell number, including disease or injury. For example, trauma can be constituted by accidents, autoimmune disorders, treatment side effects, or pathologies. Tissue regeneration increases the number of cells in the tissue, preferably allowing connections between cells of the tissue to be reestablished, and more preferably regaining the functionality of the tissue.

[0244] The terms "administering" or "introducing" or "providing" as used herein refer to the delivery of a composition to a cell, to a cell, tissue and / or organ of a subject, or to a subject. Such administering or introducing steps can occur in vivo, in vitro or ex vivo.

[0245] In certain embodiments, the pharmaceutical composition is administered parenterally, for example, intravenously, orally, rectally, or by injection. In some embodiments, the pharmaceutical composition is administered locally, for example, topically or intramuscularly. In some embodiments, the composition is administered to a target tissue, for example, to bone, joint, ear tissue, eye tissue, gastrointestinal tract, skin, wound site, or spinal cord. The method of the present invention can be performed in vivo or ex vivo. In some embodiments, the step of contacting the target cell or tissue with the WNT agonist is performed ex vivo, with subsequent implantation of the cell or tissue, for example, activated stem or progenitor cells, into the subject. Those skilled in the art can determine the appropriate site and route of administration based on the disease or disorder being treated.

[0246] The dose and dosage regimen may depend on a variety of factors, such as the nature of the disease or disorder, the characteristics of the subject, and the medical history of the subject, which are readily determined by a physician. In certain embodiments, the amount of engineered WNT agonist administered or provided to a subject ranges from about 0.01 mg to about 50 mg, about 0.1 mg to about 500 mg, or about 0.1 mg to about 50 mg per kg of the subject's body weight. In certain embodiments of any of the methods disclosed herein, the WNT agonist is administered to a subject, e.g., a mammal, intravenously, e.g., as a bolus injection, or subcutaneously. In certain embodiments, the WNT agonist is administered at least once per week. In certain embodiments, the subject is administered about 0.5 to about 100 mg of WNT agonist per kg of body weight, or about 2 to about 50 mg of WNT agonist per kg of body weight, for example, about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, or about 50 mg / kg of WNT agonist. In certain embodiments, the subject is administered about 25 mg, about 75 mg, about 250 mg, about 750 mg, about 1500 mg, or about 2250 mg of WNT agonist. In certain embodiments, a subject is administered about 3 to about 30 mg / kg of body weight of R2M13-h26 intravenously or subcutaneously at least once per week, where R2M13-h26 comprises two polypeptides of SEQ ID NO:9 and two polypeptides of SEQ ID NO:10 linked by disulfide bonds.

[0247] The terms "treatment", "treating" and the like are used herein to generally mean obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, in terms of completely or partially preventing a disease or its symptoms, e.g., reducing the likelihood that a disease or its symptoms will occur in a subject, and / or may be therapeutic, in terms of partially or completely curing a disease and / or adverse effects caused by the disease. "Treatment", as used herein, covers any treatment of a disease in a mammal, including: (a) preventing the disease from occurring in a subject who is 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. Therapeutic agents (e.g., engineered WNT agonists) may be administered before, during, or after the onset of the disease or injury. Treatment of ongoing disease, where the treatment stabilizes or reduces undesirable clinical symptoms in the patient, is particularly targeted. Such treatment is desirably performed before complete loss of function in the affected tissue. The subject therapy is desirably administered during, and in some cases after, the symptomatic stage of the disease. In some embodiments, the subject method provides a therapeutic benefit, such as preventing the onset of a disorder, halting the progression of a disorder, reversing the progression of a disorder, etc. In some embodiments, the subject method includes a step of detecting that a therapeutic benefit has been achieved. Those skilled in the art will understand that such measures of therapeutic efficacy are applicable to the particular disease being modified, and will recognize the appropriate detection method to use to measure therapeutic efficacy.

[0248] In certain embodiments, the methods disclosed herein result in one or more of the following PK / PD parameters following administration of an engineered WNT agonist disclosed herein to a subject: clearance (mL / day / kg), 10-50 or about 25; terminal t1 / 2, 2-5 days or about 4 days; Cmax (μg / mL), 50-300 or 100-200, or about 140, MRT, about 3-4 (days), or about 4, or AUC(day*μg / mL), about 100-1000, or about 100-500, or about 190.

[0249] Other embodiments relate in part to the use of engineered WNT agonists disclosed herein for promoting or enhancing the growth or proliferation of cells, tissues and organoids, for example, by contacting cells or tissues with one or more engineered WNT agonists, optionally in combination with Norrin or Rspondin polypeptide.In certain embodiments, cells or tissues are contacted ex vivo, in vitro or in vivo.Such methods can be used to generate cells, tissues or organoids for therapeutic use, for example, transplanted or grafted into subjects.They can also be used to generate cells, tissues or organoids for research use.Engineered WNT agonists are widely applied in non-therapeutic methods, for example, in vitro research methods.

[0250] In certain embodiments, engineered WNT agonists, including those disclosed herein, can be used to preserve cells, tissues, organs, or organoids, such as tissues or organs for transplantation. For example, cells, tissues, organs, or organoids can be contacted with engineered WNT agonists in vivo or ex vivo. In the context of preserving cells, tissues, or organs for transplantation, cells, tissues, organs, or organoids can be contacted with engineered WNT agonists while still in the donor (i.e., before being removed from the donor) and / or after being removed from the donor. The method can maintain or enhance the viability of cells, tissues, or organs, for example, during storage or before transplantation into a recipient. In certain embodiments, cells, tissues, or organs are perfused with a composition or solution that includes engineered WNT agonists. In certain embodiments, certain organ tissues are contacted with WNT superagonist molecules to maintain the viability of the tissue. In certain embodiments, the organ tissue is a donor organ tissue that is transplanted into a recipient in need of transplantation. In certain embodiments, donor organ tissue is perfused in vivo with a solution comprising the engineered WNT agonist disclosed herein, for example, before organ tissue is removed from donor.In certain embodiments, donor organ tissue is perfused ex vivo with a solution comprising the engineered WNT agonist disclosed herein, for example, during storage or transportation from donor to recipient.In certain embodiments, the organ tissue that is contacted with engineered WNT agonist remains viable for transplantation at least 10%, at least 20%, at least 50%, or at least 100% longer than when it is not contacted with engineered WNT agonist.In certain embodiments, the organ tissue is liver tissue.

[0251] In certain embodiments, engineered WNT agonists, including those disclosed herein, are used to expand and / or maintain ex vivo tissue, such as skin tissue. In certain embodiments, tissue is isolated from a donor or patient. Tissue can be contacted with engineered WNT agonists in vivo or ex vivo (e.g., maintained or cultured in the presence of engineered WNT agonists). In certain embodiments, tissue is contacted ex vivo, for example, by perfusion with a composition comprising engineered WNT agonists.

[0252] In another embodiment, engineered WNT agonist, including those disclosed herein, can be used to generate or maintain organoid or organoid culture.For example, organoid culture can be contacted with engineered WNT agonist, for example, by culturing organoid in medium containing engineered WNT agonist.In certain embodiments, organoid culture is generated, grown or maintained by contacting with one or more engineered WNT agonists disclosed herein.In certain embodiments, engineered WNT agonist is present in the culture medium used to grow or maintain organoid tissue.

[0253] The present invention provides a method for tissue regeneration of damaged tissue such as the above-mentioned tissue, comprising administering engineered WNT agonist to cells.Engineered WNT agonist can be administered directly to cells in vivo, can be administered to subjects orally, intravenously, or by other methods known in the art, or can be administered to cells ex vivo.In some embodiments, when engineered WNT agonist is administered to cells ex vivo, these cells can be transplanted to subjects before, after, or during administration of engineered WNT agonist.

[0254] Wnt signaling is an important component of stem cell culture.For example, WO2010 / 090513, WO2012 / 014076, Sato et al., 2011 (GASTROENTEROLOGY 201 1; 141: 1762-1772) and Sato et al., 2009 (Nature 459, 262-5) described stem cell culture medium.The engineered WNT agonist disclosed herein is suitable for use in these stem cell culture medium, or can be combined with Rspondin.

[0255] Therefore, in one embodiment, the present disclosure provides a method for enhancing the proliferation of stem cells, comprising contacting stem cells with one or more engineered WNT agonists disclosed herein.In one embodiment, the present disclosure provides a cell culture medium comprising one or more engineered WNT agonists disclosed herein.In some embodiments, the cell culture medium can be any cell culture medium already known in the art that normally comprises Wnt or Rspondin, but Wnt or Rspondin is replaced (fully or partially) by engineered WNT agonist(s) disclosed herein or is supplemented with engineered WNT agonist(s) disclosed herein. For example, the culture medium may be as described in WO2010 / 090513, WO2012 / 014076, Sato et al., 2011 (GASTROENTEROLOGY 201 1; 141: 1762-1772) and Sato et al., 2009 (Nature 459, 262-5), the entireties of which are incorporated herein by reference.

[0256] Stem cell culture media often contain additional growth factors. Thus, the method may further include a step of supplying the stem cells with growth factors. Growth factors commonly used in cell culture media 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), hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF, Peprotech, also known as FGF7). EGF is a potent mitogen for a variety of ectodermal and mesodermal cultured cells, and has been shown to be effective in in vivo and in vivo cell cultures. It has a significant effect on the differentiation of certain cells in vitro as well as some fibroblasts under cell culture. EGF precursor exists as a membrane-bound molecule and is proteolytically cleaved to generate a 53 amino acid peptide hormone that stimulates the cells. Thus, EGF or other mitogenic growth factors can be supplied to stem cells. During the culture of stem cells, mitogenic growth factors can be added to the culture medium every 2 days, and at the same time, the culture medium is renewed, preferably every 4 days. In general, the mitogenic factors are selected from the group 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-alpha and KGF; viii) TGF-alpha, and FGF7; ix) or TGF-alpha and a FGF. In certain embodiments, the present disclosure encompasses stem cell culture media comprising an engineered WNT agonist disclosed herein, for example, optionally in combination with one or more of the growth factors or combinations thereof described herein.

[0257] These methods of enhancing stem cell proliferation can be used to grow new organoids and tissues from stem cells, for example, as described in WO2010 / 090513, WO2012 / 014076, Sato et al., 201 1 (GASTROENTEROLOGY 2011; 141: 1762-1772) and Sato et al., 2009 (Nature 459, 262-5).

[0258] In some embodiments, engineered WNT agonist is used to enhance stem cell regeneration.Exemplary stem cells of interest include, but are not limited to, muscle satellite cells; hematopoietic stem cells and progenitor cells derived therefrom (US 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; hepatic stem cells; adipose tissue-derived stem cells, etc.

[0259] The present invention is based in part on the use of engineered WNT agonists to regulate gastrointestinal epithelial proliferation, particularly in inflammatory bowel disease.

[0260] In one embodiment, the present invention provides a method for treating a subject suffering from a gastrointestinal disorder, comprising administering to the subject an engineered WNT agonist as disclosed herein. In certain embodiments, the gastrointestinal disease is inflammatory bowel disease. In further embodiments, the inflammatory bowel disease is selected from the group consisting of Crohn's disease (CD), CD with fistulization, and ulcerative colitis (UC). In certain embodiments, the engineered WNT agonist reduces inflammatory cytokine expression in the intestine or colon and / or repairs the intestinal epithelium.

[0261] In a particular aspect, the present invention also provides a method for treating a subject suffering from a gastrointestinal disorder, comprising administering to the subject a tissue-specific WNT signal enhancer molecule. In a particular embodiment, the WNT signal enhancer molecule comprises a) a first domain that binds to one or more E3 ubiquitin ligases; and b) a second domain that binds to a tissue-specific receptor. In a further embodiment, the E3 ubiquitin ligase is selected from the group consisting of zinc and ring finger protein 3 (ZNRF3) and ring finger protein 43 (RNF43). In another embodiment, the first domain comprises an R-spondin (RSPO) polypeptide. In a further embodiment, the RSPO polypeptide is selected from the group consisting of RSPO-1, RSPO-2, RSPO-3, and RSPO-4. In a particular embodiment, the RSPO polypeptide comprises a first furin domain and a second furin domain. In certain embodiments, the second furin domain is wild type or mutated to reduce binding to leucine-rich repeat-containing G protein-coupled receptors 4-6 (LGR4-6). In certain embodiments, the engineered agonist or Wnt signal enhancing molecule incorporates a tissue targeting molecule. In further embodiments, the tissue targeting molecule is an antibody or a fragment thereof that binds to a tissue-specific cell surface antigen. In certain embodiments, the tissue targeting molecule is selected from the group consisting of GPA33, CDH17, and MUC-13 or a functional fragment or variant thereof. In some embodiments, the WNT agonist is administered with a binding domain that specifically binds to an inflammatory molecule. In certain embodiments, the binding domain specific for the inflammatory molecule is an antagonist of the inflammatory molecule. In further embodiments, the antagonist of the inflammatory molecule is an antagonist of TNFα, an antagonist of IL-12, an antagonist of IL-12 and IL-23, or an antagonist of IL-23. In some embodiments, the gastrointestinal disease is inflammatory bowel disease. In further embodiments, the inflammatory bowel disease is selected from the group consisting of Crohn's disease (CD), CD with fistulization, and ulcerative colitis (UC).

[0262] In another embodiment, the present invention provides a method for treating a subject suffering from gastrointestinal disorder, comprising administering to the subject an engineered WNT agonist and an engineered tissue-specific WNT signal enhancing molecule. The engineered WNT agonist and the engineered tissue-specific WNT signal enhancing molecule can be administered at the same time or at different times. In some embodiments, the subject comprises both an engineered WNT agonist and an engineered tissue-specific WNT signal enhancing molecule in effective amounts during an overlapping period. In certain embodiments, the engineered WNT agonist comprises one or more binding domains that bind to FZD5, FZD8, FZD1, FZD2, FZD7, FZD5 and 8, or FZD1, 2, and 7, and one or more binding domains that bind to LRP5, LRP6, or LRP5. In some embodiments, the engineered WNT agonist comprises a tissue targeting molecule. In certain embodiments, the tissue targeting molecule is an antibody or a fragment thereof that binds to a tissue-specific cell surface antigen. In further embodiments, the tissue targeting molecule is selected from the group consisting of GPA33, CDH17, and MUC-13 or functional fragments or variants thereof. In certain embodiments, the engineered WNT signal enhancing molecule comprises a first domain that binds to one or more E3 ubiquitin ligases and a second domain that binds to a tissue-specific receptor. In further embodiments, the E3 ubiquitin ligase is selected from the group consisting of zinc and ring finger protein 3 (ZNRF3) and ring finger protein 43 (RNF43). In some embodiments, the first domain comprises an R-spondin (RSPO) polypeptide. In other embodiments, the RSPO polypeptide is selected from the group consisting of RSPO-1, RSPO-2, RSPO-3, and RSPO-4. In further embodiments, the RSPO polypeptide comprises a first furin domain and a second furin domain. In yet further embodiments, the second furin domain is wild-type or mutated to reduce binding to leucine-rich repeat-containing G protein-coupled receptors 4-6 (LGR4-6). In further embodiments, the engineered WNT agonist is disclosed in Table 3.In some embodiments, the engineered WNT agonist and engineered tissue-specific WNT signal enhancing molecule are administered with a binding domain that specifically binds to an inflammatory molecule. In a further embodiment, the binding domain that specifically binds to an inflammatory molecule is an antagonist of the inflammatory molecule. In yet a further embodiment, the antagonist of the inflammatory molecule is an antagonist of TNFα, an antagonist of IL-12, an antagonist of IL-12 and IL-23, or an antagonist of IL-23. In certain embodiments, the gastrointestinal disease is an inflammatory bowel disease. In a further embodiment, the inflammatory bowel disease is selected from the group consisting of Crohn's disease (CD), CD with fistulization, and ulcerative colitis (UC).

[0263] In another embodiment, the present invention provides a method for treating a subject suffering from gastrointestinal disorder, comprising administering to the subject an engineered WNT agonist and an engineered tissue-specific WNT signal enhancement combination molecule.In certain embodiments, the combination molecule comprises: a) an engineered WNT agonist comprising one or more binding domains that bind to FZD5, FZD8, FZD1, FZD2, FZD7, FZD5 and 8, or FZD1, 2, and 7, and one or more binding domains that bind to LRP5, LRP6, or LRP5; and b) an engineered WNT signal enhancement molecule comprising a first domain that binds to one or more E3 ubiquitin ligases, and a second domain that binds to a tissue-specific receptor.In further embodiments, the E3 ubiquitin ligase is selected from the group consisting of zinc and ring finger protein 3 (ZNRF3) and ring finger protein 43 (RNF43).In some embodiments, the first domain comprises an R-spondin (RSPO) polypeptide. In other embodiments, the RSPO polypeptide is selected from the group consisting of RSPO-1, RSPO-2, RSPO-3, and RSPO-4. In further embodiments, the RSPO polypeptide comprises a first furin domain and a second furin domain. In yet further embodiments, the second furin domain is wild-type or mutated to reduce binding to leucine-rich repeat-containing G protein-coupled receptors 4-6 (LGR4-6). In some embodiments, the combination molecule incorporates a tissue targeting molecule. In certain embodiments, the tissue targeting molecule is an antibody or fragment thereof that binds to a tissue-specific cell surface antigen. In further embodiments, the tissue targeting molecule is selected from the group consisting of GPA33, CDH17, and MUC-13, or a functional fragment or variant thereof. In some embodiments, the combination molecule is administered with a binding domain that specifically binds to an inflammatory molecule. In further embodiments, the binding domain specific for the inflammatory molecule is an antagonist of the inflammatory molecule.In yet further embodiments, the antagonist of the inflammatory molecule is an antagonist of TNFα, an antagonist of IL-12, an antagonist of IL-12 and IL-23, or an antagonist of IL-23. In certain embodiments, the gastrointestinal disease is inflammatory bowel disease. In further embodiments, the inflammatory bowel disease is selected from the group consisting of Crohn's disease (CD), CD with fistulization, and ulcerative colitis (UC).

[0264] In certain embodiments of any of the methods disclosed herein, the engineered WNT agonist is selected from those disclosed in any of the following: PCT Application Publication No. WO2016 / 040895; US Application Publication No. US2017-0306029; US Application Publication No. US2017-0349659; PCT Application Publication No. WO2019 / 126398; or PCT Application Publication No. WO2020 / 01030. In certain embodiments of any of the methods disclosed herein, the tissue-specific WNT signal enhancing molecule is selected from those disclosed in any of the following: PCT Application Publication No. WO2018 / 140821; US ​​Application Publication No. US2020-0048324; or PCT Application Publication No. WO2020 / 14271. All of which are incorporated herein by reference in their entirety.

[0265] In a related embodiment, the present disclosure provides a method for treating a subject suffering from a gastrointestinal disorder, comprising administering to the subject an engineered WNT agonist, an engineered WNT signal enhancing molecule, and / or a combination molecule disclosed herein, or a pharmaceutical composition comprising an engineered WNT agonist or a combination molecule disclosed herein. In some embodiments, the gastrointestinal disorder is an inflammatory bowel disease, optionally selected from the group consisting of Crohn's disease (CD), CD with fistulization, and ulcerative colitis (UC). Any of the methods disclosed herein can be carried out using any of the engineered WNT agonists, engineered WNT signal enhancing molecules, and / or combination molecules disclosed herein.

[0266] In certain embodiments of any of the methods disclosed herein, the WNT agonist is administered intravenously to a subject, e.g., a mammal, e.g., as a bolus injection. In certain embodiments, the WNT agonist is administered at least once per week. In certain embodiments, the subject is administered about 0.5 to about 100 mg of WNT agonist per kg of body weight, or about 2 to about 50 mg of WNT agonist per kg of body weight, e.g., about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, or about 50 mg / kg of WNT agonist. In certain embodiments, a subject is administered about 3 to about 30 mg of R2M13-h26 per kg of body weight intravenously at least once per week, where R2M13-h26 comprises two polypeptides of SEQ ID NO: 9 and two polypeptides of SEQ ID NO: 10 linked by disulfide bonds. In certain embodiments, a method is used to treat IBD, e.g., moderate to severe IBD, with a WNT agonist disclosed herein, e.g., R2M13-h26. In certain embodiments, the IBD is selected from the group consisting of Crohn's disease, Crohn's disease with fistulization, or ulcerative colitis.

[0267] Any of the methods disclosed herein can also be carried out using a combination of WNT agonist molecules and tissue-specific WNT signal enhancing molecules, or a combination molecule that includes, for example, both the WNT agonist molecules and tissue-specific WNT signal enhancing (combined molecules) described herein. In one embodiment, the WNT agonist molecules and / or tissue-specific WNT signal enhancing molecules, or combination molecules, are provided to a subject having a disease involving inappropriate or deregulated WNT signaling. In certain embodiments, the methods disclosed herein include providing the WNT agonist molecules and / or tissue-specific WNT signal enhancing molecules, alone or in combination, or combination molecules, to a subject in need thereof. In certain embodiments, the WNT agonist molecules and tissue-specific WNT signal enhancing molecules are provided to a subject in the same or different pharmaceutical compositions. In some embodiments, the WNT agonist molecules and tissue-specific WNT signal enhancing molecules are provided to a subject at the same time or at different times, for example, one before or after the other. In some embodiments, the method comprises providing an effective amount of WNT agonist molecule and / or tissue-specific WNT signal enhancing molecule to the subject.In some embodiments, the effective amount of WNT agonist molecule and tissue-specific WNT signal enhancing molecule are present in the subject during an overlapping period, for example, 1 day, 2 days, or 1 week.In other embodiments, the method disclosed herein comprises providing a combination molecule comprising WNT agonist molecule and tissue-specific WNT signal enhancing molecule (combination molecule) to the subject in need thereof.

[0268] In certain embodiments, any of the methods disclosed herein may be performed to reduce inflammation (e.g., inflammation associated with IBD or in tissue affected by IBD, e.g., gastrointestinal tissue, e.g., the small intestine, large intestine, or colon), increase WNT signaling, reduce any of the histological symptoms of IBD (e.g., those disclosed herein), reduce cytokine levels in inflamed tissue (e.g., gastrointestinal tissue), or reduce a disease activity index as disclosed herein.

[0269] In certain embodiments, WNT agonist molecule or tissue-specific WNT signal enhancing molecule or combination molecule can be used to enhance WNT signaling pathway in tissue or cell. Agonizing WNT signaling pathway can include, for example, increasing WNT signaling or enhancing WNT signaling in tissue or cell. Thus, in some aspects, the present disclosure provides a method for agonizing WNT signaling pathway in cell, comprising contacting tissue or cell with an effective amount of WNT agonist molecule and / or tissue-specific WNT signal enhancing molecule or combination molecule disclosed herein, or a pharmaceutically acceptable salt thereof, wherein the WNT agonist molecule and / or tissue-specific WNT signal enhancing molecule or combination molecule is a WNT signaling pathway agonist. In certain embodiments, the present disclosure provides a method for increasing WNT signaling in cell, comprising contacting cell with engineered WNT agonist disclosed herein. In certain embodiments, the WNT agonist is R2M13-h26. In some embodiments, the contacting step occurs in vitro, ex vivo, or in vivo. In certain embodiments, the cells are cultured cells and the contacting step occurs in vitro.

[0270] WNT agonist and / or tissue-specific WNT signal enhancing molecule, or combination molecule, can be used to treat gastrointestinal disorders, including but not limited to inflammatory bowel disease, including but not limited to Crohn's disease, Crohn's disease with fistulization, and ulcerative colitis. In certain embodiments, WNT agonist can be used to treat gastrointestinal disorders, including but not limited to inflammatory bowel disease, including but not limited to Crohn's disease with or without fistulization, including but not limited to ulcerative colitis, including but not limited to acute intestinal GVHD (graft-versus-host disease), including but not limited to short bowel syndrome, and any other gastrointestinal disease in which epithelial barrier is compromised or intestine is shortened. In particular, the present invention provides a WNT / β-catenin signaling WNT / β-catenin agonist that enhances the regeneration of intestinal epithelium as a result of the injury caused by these disorders. In certain embodiments, the WNT agonist is R2M13-h26.

[0271] The engineered WNT agonist can also be used to modulate various tissue and / or cellular processes, as well as to modulate gene expression in tissues and / or cells. In certain embodiments, the present disclosure provides a method of modulating gene expression, comprising contacting a subject, organ, tissue, or cell with an engineered WNT agonist disclosed herein, for example, in Table 3. The engineered WNT agonist can be administered to the subject, and the organ, tissue, or cell can be contacted with the engineered WNT agonist in vivo, ex vivo, or in vitro. In certain embodiments, the method results in the upregulation or downregulation of one or more genes in the WNT signaling pathway, including, but not limited to, any of the genes disclosed in Tables 4-8. The upregulation or downregulation of gene expression may be measured at the RNA or protein level and may result in at least a 2-fold, at least a 5-fold, at least a 10-fold, or at least a 20-fold increase or at least a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% decrease in one or more tissues and / or cells of the subject after administration. In certain embodiments, the increase or decrease may be determined based on a comparison with a predetermined control level or a level determined for a corresponding cell or tissue that has not been contacted with the engineered WNT agonist.

[0272] In some embodiments, the disclosure provides a method of modulating expression of a WNT pathway molecule in one or more tissues and / or cells of a subject having a gastrointestinal disorder, comprising administering to the subject an engineered WNT agonist or pharmaceutical composition disclosed herein. In certain embodiments, the WNT pathway molecule is a gene or protein listed in any one of Tables 4-7. In certain embodiments, the WNT pathway molecule is selected from the group consisting of RNAse4, angiogenin, Gsta3, Rnf43, Axin2, Ccnb1, or any of the genes or proteins listed in Table 7. In certain embodiments, after administration of the engineered Wnt agonist, the expression of WNT pathway molecules (genes or proteins) in one or more tissues and / or cells of the subject is increased by at least 1.1-fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 2-fold, at least 5-fold, at least 10-fold, or at least 20-fold, or reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%. In certain embodiments, the tissue is an epithelial tissue. In certain embodiments, the cell is a gastrointestinal epithelial cell, optionally a stem cell, TA1, TA2, basal goblet cell, injury-induced alternative progenitor cell (AltEnteroPC), injury-induced alternative enterocyte (AltEntero), enteroprogenitor cell (EnteroPrecur), goblet cell 1, goblet cell 2, enteroendocrine cell, or tuft cell. In a particular embodiment, the WNT agonist is R2M13-h26.

[0273] In another embodiment, the disclosure provides a method of stimulating tissue repair in a subject having a gastrointestinal disorder, comprising administering to the subject an engineered WNT agonist or pharmaceutical composition disclosed herein. In certain embodiments, tissue repair is stimulated by (or the method results in modulation of) at least one WNT pathway molecule selected from the group consisting of genes associated with cell cycle, genes associated with stem and progenitor cell renewal and differentiation, genes associated with epithelial cell repair and barrier restoration, and / or any of the genes listed in any of Tables 4-8. In certain embodiments, the cell cycle-related genes are selected from those provided in Table 4, or Aurka, Aurkb, Ccna2, Ccnb1, Ccnb2, Ccnd2, Ccne1, Cdc45, Cdk1, Cdkn3, Cenpm, Cenpp, Cenpq, Cenpu, Hells, Mcm4, Mcm5, Mcm6, Mcm7, Myc, Pbk, Plk1, Rrm1, and Rrm2. In certain embodiments, the stem and progenitor cell renewal and differentiation-related genes are selected from those provided in Table 8, and Axin2, Id1, Hmga2, Nhp2, Foxq1, and Adh1. In certain embodiments, the genes related to epithelial cell repair and barrier restoration are selected from those provided in Table 6, or Apex1, Agr2, B3gnt7, Fcgbp, Muc2, Muc3, Tff3, Zg16, and Sprr2a3. In certain embodiments, after administration of the engineered Wnt agonist, expression of the genes in one or more tissues and / or cells of the subject is increased by at least 2-fold, at least 5-fold, at least 10-fold, or at least 20-fold, or reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%. In certain embodiments, the WNT agonist is R2M13-h26.

[0274] In another embodiment, the present disclosure provides a method for reducing inflammation in a subject (or tissue or cell of the subject) having a gastrointestinal disorder, comprising administering to the subject an engineered WNT agonist or pharmaceutical composition disclosed herein.In certain embodiments, inflammation is reduced (or modulated by the method) by modulation of at least one WNT pathway molecule selected from the group consisting of the genes provided in Table 5, or Adamdec1, Atf3, Gpx2, Gsta3, Gstm1, Gdf15, Il18, Nox1, Reg4, Sycn, Selenbp1, Tgfbr2, and Timp3.In certain embodiments, inflammation is reduced in gastrointestinal tissue, optionally epithelial tissue. In certain embodiments, inflammation is reduced in gastrointestinal epithelial cells, epithelial stem cells, TA1, TA2, basal goblet cells, injury-induced alternative progenitor cells (AltEnteroPC), injury-induced alternative enterocytes (AltEntero), enteric progenitor cells (EnteroPrecur), goblet cells 1, goblet cells 2, or enteroendocrine cells. In certain embodiments, after administration of the engineered Wnt agonist, expression of WNT pathway molecules in one or more tissues and / or cells of the subject is increased by at least 1.1-fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, 2-fold, at least 5-fold, at least 10-fold, or at least 20-fold, or reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%. In certain embodiments, the WNT agonist is R2M13-h26.

[0275] In certain embodiments of any of the methods disclosed herein, the WNT agonist molecule may also incorporate a tissue targeting moiety, such as an antibody or fragment thereof that recognizes a lung tissue-specific receptor or cell surface molecule.

[0276] The present invention also provides combination treatment with known and new treatments for gastrointestinal disorders, particularly inflammatory bowel disease (IBD).For example, WNT agonist can be combined with some known treatments for IBD, including but not limited to 5-aminosalicylate (5-ASA); immunosuppressants, such as corticosteroids, azathioprine or 6-mercaptopurine, methotrexate, and cyclosporine-A or tacrolimus; TNFα inhibitors, such as infliximab, adalimumab, and golimumab; anti-integrins, such as vedolizumab; inflammatory cytokine antagonists, such as ustekinumab; Janus kinase (JAK) inhibitors, such as tofacitinib; SMAD7 inhibitors, such as mongersen; and S1P modulators, such as ozanimod and etrasimod, as well as any new drugs that may be marketed for the above-mentioned disorders. The above therapeutic agents may be administered sequentially or simultaneously with the molecules of the invention.

[0277] The therapeutic agent (e.g., engineered WNT agonist and / or tissue-specific WNT signal enhancing molecule or combination molecule) can be administered before, during or after the onset of disease or injury. Of particular interest is the treatment of ongoing disease, where treatment stabilizes or reduces the patient's undesirable clinical symptoms. Such treatment is desirably performed before complete loss of function in the affected tissue. The subject therapy is desirably administered during, and in some cases after, the symptomatic stage of the disease. In some embodiments, the subject method provides a therapeutic benefit, such as preventing the development of a disorder, halting the progression of a disorder, reversing the progression of a disorder, etc. In some embodiments, the subject method includes a step of detecting that a therapeutic benefit has been achieved. Those skilled in the art will understand that such measures of therapeutic efficacy are applicable to the particular disease to be altered, and will recognize the appropriate detection method to be used to measure therapeutic efficacy.

[0278] All of the above U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification and / or listed in the Application Data Sheets are hereby incorporated by reference in their entireties.

[0279] From the foregoing, it will be understood that, although specific embodiments of the present disclosure have been described herein for purposes of illustration, various modifications may be made without departing from the spirit and scope of the disclosure. Accordingly, the disclosure is not limited, except as by the appended claims.

[0280] The scope of the present invention is best understood with reference to the following examples, which are not intended to limit the invention to the specific embodiments. EXAMPLES

[0281] Example 1 Common methods Standard methods in molecular biology were used, for example those described in: Maniatis et al. (1982) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Sambrook and Russell (2001) Molecular Cloning, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Wu (1993) Recombinant DNA, Vol. 217, Academic Press, San Diego, Calif. Standard methods can also be found in Ausbel et al. (2001) Current Protocols in Molecular Biology, Vols. 1-4, John Wiley and Sons, Inc. New York, NY, which describes cloning and DNA mutagenesis in bacterial cells (Vol. 1), cloning in mammalian cells and yeast (Vol. 2), glycoconjugates and protein expression (Vol. 3), and bioinformatics (Vol. 4).

[0282] Methods for purifying proteins, including immunoprecipitation, chromatography, electrophoresis, centrifugation, and crystallization, are described, for example, in Coligan et al. (2000) Current Protocols in Protein Science, Vol. 1, John Wiley and Sons, Inc., New York. Chemical analysis, chemical modification, post-translational modification, production of fusion proteins, and glycosylation of proteins have been described; see, e.g., Coligan et al. (2000) Current Protocols in Protein Science, Vol. 2, John Wiley and Sons, Inc., New York; Ausubel et al. (2001) Current Protocols in Molecular Biology, Vol. 3, John Wiley and Sons, Inc., NY, NY, pp. 16.0.5-16.22.17; Sigma-Aldrich, Co. (2001) Products for Life Science Research, St. Louis, Mo.; pp. 45-89; Amersham Pharmacia Biotech (2001) BioDirectory, Piscataway, NJ, pp. 384-391. The production, purification, and fragmentation of polyclonal and monoclonal antibodies are described, for example, in Coligan et al. (2001) Current Protocols in Immunology, Vol. 1, John Wiley and Sons, Inc., New York; Harlow and Lane (1999) Using Antibodies, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Harlow and Lane, supra. Standard techniques are available for characterizing ligand / receptor interactions.See, e.g., Coligan et al. (2001) Current Protocols in Immunology, Vol. 4, John Wiley, Inc., New York.

[0283] Methods of flow cytometry are available, including fluorescence-activated cell sorting detection systems (FACS®); see, e.g., Owens et al. (1994) Flow Cytometry Principles for Clinical Laboratory Practice, John Wiley and Sons, Hoboken, NJ; Givan (2001) Flow Cytometry, 2nd ed.; Wiley-Liss, Hoboken, NJ; Shapiro (2003) Practical Flow Cytometry, John Wiley and Sons, Hoboken, NJ. Fluorescent reagents suitable for modifying nucleic acids, including nucleic acid primers and probes, polypeptides, and antibodies, for use, e.g., as diagnostic reagents, are available. Molecular Probes (2003) Catalogue, Molecular Probes, Inc., Eugene, Oreg.; Sigma-Aldrich (2003) Catalogue, St. Louis, Mo.

[0284] Standard methods for immune system histology have been described, see, e.g., Muller-Harmelink (ed.) (1986) Human Thymus: Histopathology and Pathology, Springer Verlag, New York, NY; Hiatt, et al. (2000) Color Atlas of Histology, Lippincott, Williams, and Wilkins, Phila, Pa.; Louis, et al. (2002) Basic Histology: Text and Atlas, McGraw-Hill, New York, NY.

[0285] For example, software packages and databases are available for determining antigenic fragments, leader sequences, protein folding, functional domains, glycosylation sites, and sequence alignments. For example, GenBank, Vector NTI® Suite (Informax, Inc., Bethesda, Md.); GCG Wisconsin Package (Accelrys, Inc., San Diego, Calif.); DeCypher® (TimeLogic Corp., Crystal Bay, Nev.); Menne et al. (2000) Bioinformatics 16: 741-742; Menne et al. (2000) Bioinformatics Applications Note 16: 741-742;Wren et al. (2002) Comput. Methods Programs Biomed. 68: 177-181;von Heijne (1983) Eur. J. Biochem. 133: 17-21;von Heijne (1986) Nucleic Acids Res. 14: See 4683-4690.

[0286] Exemplary methods and materials used in this disclosure are presented below.

[0287] RNA in situ hybridization: mRNA expression was detected by RNAscope in situ hybridization (ACD Bio). The RNAscope probes used are listed below. For colorimetric visualization, standard RNAscope® 2.5 HD Assay-Red protocols (www.acdbio.com) were followed and images were acquired with a Leica DMi8 microscope equipped with a DFC7000T color camera. For fluorescent RNAscope in situ hybridization, standard RNAscope Multiplex Fluorescent Reagent Kit v2 Assay protocols were followed (ACD Bio Document#323100-USM) in conjunction with the TSA Plus Cyanine 3 System and the TSA Plus Cyanine 5 System. Fluorescent images were acquired using a Leica Thunder imaging system.

[0288] RNA isolation and RT-qPCR: For RNA isolation, the MagMAX™ mirVana (Thermofisher, A27828) Total RNA Isolation Kit was used with the KingFisher (Thermofisher) sample purification system. Reverse transcription was performed using the Applied Biosystems High-Capacity cDNA Reverse Transcription Kit (Thermofisher, 4368814), and Applied Biosystems TaqMan Fast Advanced Master Mix (Thermofisher, 4444557) was used for qPCR.

[0289] Affinity measurement: The binding kinetics of the Fzd-binding moieties of R2M13, R2M13-26, and Fab to the CRDs of Fzd5 and 8 were determined by biolayer interferometry (BLI) using an Octet Red 96 (PALL ForteBio, Fremont, CA) instrument at 30°C and 1000 rpm with a streptavidin (SA) biosensor. Biotinylated CRDs of Fzds diluted to 25 nM in running buffer (PBS, 0.05% Tween®-20, 0.5% BSA, pH 7.2) were captured by the SA biosensor and then immersed in wells containing various concentrations of R2M13 Fab protein in running buffer or with only running buffer as a reference channel. The KD for each binder was calculated by the Octet System software based on fitting to a 1:1 binding model. The binding specificity of R2M13 IgG to the 10 Fzds was also investigated by BLI assay. Biotinylated Fzd CRD (H. Chen, Lu, Lee, & Li, 2020) diluted to 50 nM in running buffer was captured by the SA biosensor and then immersed in wells containing 200 nM R2M13 IgG in running buffer.

[0290] Super TopFlash (STF) Assay: The signaling activity of Wnt mimetics was measured using Huh7 human hepatocytes containing a luciferase gene controlled by a WNT-responsive promoter (Super TopFlash reporter assay, STF) according to established protocols (H. Chen, Lu, Lee, & Li, 2020 ).

[0291] Organoid culture and proliferation assay: Mouse small intestinal organoids were maintained in Mouse IntestiCult™ Organoid Growth Medium (STEMCELL technologies) and passaged once a week until the day of assay for Wnt mimetic activity (H. Chen, Lu, Lee, & Li, 2020). To assay for organoid proliferation, organoids were dissociated with Gentle Cell Dissociation Reagent (STEMCELL technologies) by shaking for 10 min, washed twice with cold PBS (Gibco), and resuspended 1:1 in Matrigel (Corning) on ​​ice. 25 μl of cell resuspension in Matrigel was seeded in the center of each well of a pre-warmed 48-well tissue culture plate and placed at 37°C for 5 min to solidify. 300 μl of basal medium (Table 10), basal medium + IWP2 + anti-βGal or basal medium + IWP2 + Wnt mimetic was applied to the wells. Five to six replicates were included for each condition. Medium and treatments were changed once on day 4 after plating. On day 7, images of 3D cultured organoids were acquired.

[0292] Animal Care: Seven-week-old C57Bl / 6J female mice were obtained from Jackson Laboratories (Bar Harbor, ME, USA) and housed 4–5 mice per cage. All animal experiments conformed to the standards of the Guide for the Care and Use of Laboratory Animals, prepared by the National Academy of Sciences. Animal experimental protocols were approved by the Surrozen Institutional Animal Care and Use Committee. Mice were allowed to acclimate for a minimum of 2 days before starting the experiments. Mice were housed on a 12 / 12-h light / dark cycle in a 30%–70% humidity environment and at room temperatures ranging from 20°C to 26°C.

[0293] DSS-induced acute colitis: 7-8 week old female C57BL6 / J mice were fed 4% (wt / vol) dextran sodium sulfate (DSS, MP Biomedicals, molecular weight 36-50 kDa, Ref#160110) in drinking water from day 1 to day 7 to induce colitis and switched to 1% DSS from day 8. Protein treatment was administered either once on day 7 or twice on days 4 and 7. Animals were sacrificed on day 10 to allow for a 6 day course of protein treatment. Colons were harvested for histology and RT-qPCR. In one of the studies, DSS-induced mouse weight loss in animals treated with anti-GFP was nearly 25% on day 9, therefore, animals were switched to drinking water without DSS for IACUC compliance. A disease activity index (DAI) was calculated based on the mean scores of weight loss, stool consistency and the extent of intestinal bleeding (Wirtz Stefan et al., 2017). A grading scoring system was developed on a scale of 0–4 using the following parameters: weight loss (0, 0–1%; 1, 1–6%; 2, 6–12%; 3, 12–18%; 4, >18%), stool consistency (0, normal; 1, soft but formed; 2, soft; 3, very soft and wet; 4, watery diarrhea) and intestinal bleeding (0–1, hemoccult negative; 2, hemoccult positive; 3, visible traces of blood in stool; 4, gross rectal bleeding).

[0294] Histology: The small intestine and colon were removed and weighed and measured for length after removal of fecal contents. The desired small intestinal segments (duodenum, jejunum, ileum) and colonic segments (ascending, transverse, and descending colon) were excised and directly fixed overnight in 10% neutral buffered formalin (NBF). The tissues were then transferred to 70% ethanol before being embedded in paraffin. The paraffin tissue blocks were then sectioned at 5 μM thickness and stained with hematoxylin and eosin (H&E) for histological analysis. Pathology read-out was performed by an independent pathologist.

[0295] Immunohistochemistry and indirect immunofluorescence: Briefly, 5-micron-thick formalin-fixed paraffin-embedded tissue sections on slides were deparaffinized and then subjected to antigen retrieval with citrate buffer (pH 6) in a steamer. Slides were then washed thoroughly with tap water, followed by one wash with PBST. Tissue sections were subsequently blocked with serum-free protein block (Agilent, X090930-2) for 1 h at room temperature, and then incubated in primary antibodies. After incubation with primary antibodies, tissue sections were washed at least three times with 0.1% TX-100 in PBS (PBST), followed by incubation in secondary antibodies. Tissue sections were then washed with PBST and mounted with coverslips using Vectashield Vibrance antifade mounting medium with DAPI (Vector Laboratories, H-1800).

[0296] Fluorescence-activated cell sorting (FACS) Mouse colons were dissociated as follows and resuspended in FACS buffer (HBSS, 2% FBS, 10 mM HEPES, 1 mM sodium pyruvate, and 1% Pen-strep or antibiotic / antimycotic solution). Prior to FACS, cells were passed through a 40 micron filter and DAPI was added to distinguish live / dead cells. Prior to target antibody incubation, FcR blocking reagent (Miltenyi Biotec, 130-092-575) was added to the samples and incubated for 10 minutes.

[0297] Single-cell RNA sequencing (scRNA-seq): tissue dissociation, cell isolation, library preparation, and sequencing For the acute DSS model, mice were treated with 4% DSS in drinking water throughout the duration of the experiment. DSS-treated animals were administered 10 mpk of R2M13-26 or anti-GFP antibody on the fourth day of DSS treatment. Cells were collected from two non-injured naive mice (no DSS) on the fifth and sixth days, as well as from anti-GFP-treated and R2M13-26-treated DSS animals at each time point, with three replicates each. Each animal was considered as one replicate.

[0298] The transverse colon was isolated from each animal and cleared of feces. After a brief wash with cold PBS, the colon was cut longitudinally to open the tube into a flat sheet, and the tissue was cut into 3-4 mm long fragments. The tissue fragments were incubated with 5 mM EDTA in pre-warmed (37°C) PBS at 37°C, 150 rpm in a shaker for 15 min. After 15 min, the tubes containing the samples were shaken vigorously for 10 s to release more epithelial cells. The epithelial cells floating in suspension were removed and placed in a new tube and centrifuged at 200 rcf for 2 min.

[0299] The remaining tissue, containing the remaining epithelium and stroma / lamina propria, was then incubated in 8-12.5 mL of lamina propria dissociation buffer (AdvDMEM / F12 with 10 mM HEPES, 0.2% FBS, DNAse1 (80 U / mL), Liberase™ (0.2 mg / mL), and 1% antibiotic / antimycotic) for 30 min at 37°C with horizontal shaking at 150 rpm. After pelleting, the epithelial cells were resuspended in 1 mL of TrypLE with DNase1, which was incubated at 37°C for 5 min and triturated with a P1000 pipette for 30 s. After trituration, 10 mL of PBS + 50 U / mL DNAse1 was added to the epithelial cells, which were centrifuged at 4°C, 500 rcf, and the supernatant was removed. The epithelial cells were then washed once with FACS buffer (HBSS, 2% FBS, 10 mM HEPES, 1 mM sodium pyruvate, and 1% Pen-strep or antibiotic / antimycotic solution) before another round of centrifugation and final resuspension in 0.5 mL of FACS buffer. After dissociation in LP dissociation buffer for 30 minutes, the remaining tissue fragments and suspension were centrifuged at 500 rcf for 5 minutes. The supernatant was removed and reduced to 1 mL, and the sample was ground with a P1000 until the solution was homogenous and all tissue fragments were dissociated. After grinding, the sample was centrifuged at 500 rcf for 5 minutes at 4°C, washed with FACS buffer, and then resuspended in 1 mL of FACS buffer in preparation for FACS.

[0300] All cells were passed through a 40 micron filter prior to FACS. Viability was assessed by FACS using DAPI, and only viable (DAPI negative) cells were collected. A negative control without DAPI was used to ensure proper DAPI gating. Cells were collected from the epithelial fraction and then from the epithelial / lamina propria fraction, combined (1:5 ratio), and counted on a hemocytometer prior to cell capture. Standard 10x Genomics Chromium 3'v3 scRNA-seq reagents (PN1000075) were used. Approximately 4000-4500 cells were loaded per channel. Cells from one individual animal replicate were captured per channel. Standard 10x Genomics Chromium 3'v3 scRNA-seq RT, cDNA amplification, and sequencing library preparation protocols were followed. The multiplexed sequencing libraries were sequenced on an Illumina Nova Seq 6000 S1 lane, with an average of approximately 50,000 reads per cell.

[0301] scRNA-seq analysis: Illumina read data were processed using the 10x Genomics Cellranger (version 3.0.2) pipeline running STAR aligner against the mm10-3.0.0 version of the mouse transcriptome. The demultiplexed UMI counting data were then evaluated and after exploratory data analysis, low-quality cells and low-expressing genes were partially removed by using the R package scone (version 1.14.0) and filtering cutoffs specific to the dataset: only cells with UMIs greater than 1000, 500 to 6500 genes, and UMIs less than or equal to 60000 were kept to remove likely empty droplets and qualify doublets. Cells with mitochondrial gene percentages greater than one standard deviation above the mean were filtered. Only genes expressed in the top quartile of at least three cells were obtained, yielding 16039 genes. UMI counting data were normalized using deconvolution scaling in the R package scran (version 1.18.5; (Lun, Bach, & Marioni, 2016)). After normalization, we did not observe batch-specific cell populations when evaluated in reduced dimensional space, nor did we observe strong correlations between QC metrics and observed gene expression principal components.

[0302] A shared nearest neighbor (SNN) graph-based clustering method (Xu & Su, 2015) was applied to the complete, filtered dataset by using a wrapper function (buildSNNGraph) from the R package scran (version 1.18.5) in conjunction with the cluster_louvain function from the R package igraph (1.2.6) on the first 10 principal components derived from the top 2000 most variable genes across the dataset, with k equal to 40. This allowed for a rough grouping of cells within the three tissue layers / lineages (immune, stromal, epithelial) and identification of cell types. Based on this initial clustering, the data were subset into these three smaller data sets and cells within each layer / lineage were clustered using an SNN graph-based method and the walktrap algorithm implemented using the cluster_walktrap function in the igraph package, applied to the first 15 principal components derived from the top 2000 most variable genes within the subsetted layers / lineages (immune, stromal, epithelial). Cell type / subtype identity was determined using established marker genes and published literature.

[0303] Differential gene expression analysis between experimental conditions was performed using the R package edgeR (version 3.32.1) (Y. Chen, Lun, & Smyth, 2016; Robinson, McCarthy, & Smyth, 2010) on pseudo-bulk samples after aggregation of single cells within biological replicates. This type of DE analysis was implemented at the lineage level and at the cell type / cluster level. For each time point (24 or 48 h), differential expression comparisons were performed within each of the three layers / lineages (epithelial, stromal, immune) and within individual clusters / cell types within each lineage, between experimental conditions (DSS-injured vs. non-injured, and within DSS-injured samples for R2M13-26-treated vs. anti-GFP-treated). Gene set enrichment analysis (GSEA), also known as pathway analysis, was applied by implementing the fry function (Y. Chen, Lun, & Smyth, 2016) in the R package edgeR (version 3.32.1). Gene sets were obtained from the Broad Institute's Molecular Signature Database (MSigDB), which included KEGG, Biocarta, PID, Reactome, ST, SIG, Hallmark and curated (C2) gene sets of SA type. We also implemented the kegga function in the edgeR package using only KEGG pathways, and similar results were observed (data not shown). To identify pathways differentially enriched in one experimental condition compared to another, GSEA was applied both pairwise and with more specific contrasts on pseudo-bulk samples aggregated per replicate.

[0304] Lineage trajectory inference was performed using the R package slingshot (version 1.8.0; ( Street et al., 2018 ).

[0305] To confirm the ability of R2M13-26 to affect Wnt target gene expression, additional genes with literature support were added to the Wnt signaling target gene list (Gougelet et al., 2014) and differentially expressed genes by tissue layer are shown in Table 7. Table 7 shows the Wnt target genes that were differentially expressed within the epithelial lineage when R2M13-26 treatment was compared to anti-GFP treatment at either 24 or 48 hours. Differential expression was filtered based on a corrected p-value (false discovery rate (FDR)) < 0.05.

[0306] Example 2 Engineered Wnt agonists Engineered Wnt agonists in IgG1 format were synthesized, including Wnt agonists fused to the N-terminus of each light chain of a Fzd-binding antibody with a humanized Lrp5 / 6 binding domain. Exemplary structures are shown in FIG. 1. The Lrp5 / 6 binding domains were derived from different camelid single chain antibody (VHH) binding domains selected from VHH03, VHH26, or VHH36. The VHH03 domain binds Lrp5, the VHH26 domain binds Lrp6, and the VHH36 domain binds Lrp5 and Lrp6. The camelid single chain antibodies were humanized by retaining the CDR sequences but replacing other sequences with the human antibody framework. The resulting LRP5 / 6 binding domains were modified to remove potential disadvantages.

[0307] Humanization of VHH26 was performed as follows: Camelid VHH domains are considered difficult to humanize because they are derived from single-chain homodimeric antibodies that lack the VL:CL or VH:CH interactions present in heterotetrameric human IgG1 antibodies. The surface properties of camelid VHHs (Muyldermans (2013) Annu. Rev. Biochem. 82: 775-797; Vincke et al (2009) J. Biol. Chem. 284: 3273-3284) have been evolutionarily re-engineered to optimize the stability of the homodimeric nature of single-chain antibodies. Humanization of camelid VHH26 was first performed by CDR grafting (for review: Safdari et al., (2013) Biotechnol. Genet. Eng. Rev. 27: 175-186) to the human germline sequence with the highest sequence identity. In a next step, several different humanized VHH26 constructs with back mutations to the camelid sequence were generated to identify engineered VHHs with optimal expression, homogeneity and biophysical properties such as binding affinity to the Lrp6 receptor. An alignment of VHH26 (Table 1) with its closest human germline sequence IGHV3-23*01 is shown in Figure 2A. The sequences of the six different humanized VHH26s (H1-H6) are listed in Table 1 and their alignment to the parental VHH26 is shown in Figure 2B.

[0308] These six humanized VHH26 variants H1-H6 and the parental VHH26 were transiently expressed with a C-terminal hexa-histidine tag in Expi293 cells (80 mL scale). Proteins were purified using His-Complete resin (Roche, USA) according to standard procedures. Expression levels and homogeneity of VHH26 and its humanized variants were analyzed by SDS-PAGE and SEC (size exclusion chromatography). For Lrp6:VHH26 affinity determination, binding kinetics of VHH26-H1, VHH26-H2, VHH26-H3, VHH26-H4, VHH26-H5, VHH26-H6, and VHH26_His to biotinylated LRP6E3E4 (Chen et al., (2020) Cell Chemical Biol. 27, 1-12) was determined by biolayer interferometry (BLI) using an Octet Red 96 (PALL ForteBio, Fremont, CA) instrument at 30 °C, 1000 rpm, and a streptavidin (SA) biosensor. Biotinylated LRP6E3E4 diluted to 50 nM in running buffer (PBS, 0.05% Tween®-20, 0.5% BSA, pH 7.2) was captured by the SA biosensor and then immersed in wells containing various concentrations of the indicated VHH26 proteins in running buffer or wells with running buffer alone as a reference channel. The KD for each binder was calculated by the Octet System software based on fitting to a 1:1 binding model. Kinetic values ​​(Kon, Koff, KD) from each experiment were calculated using Octet Data Analysis 9.0 (PALL ForteBio, Fremont, CA) from seven technical replicates with different concentrations of the tested molecules.

[0309] SDS-PAGE, SEC, and Octet-BLI profiles for VHH26 and its humanized variants are shown in Figure 3A-3B. SDS-PAGE analysis on Ni-pulldown samples revealed that among the six VHH26 human variants, VHH26-H2, VHH26-H4, and VHH26-H5 exhibited higher levels of expression compared to VHH26-H1, VHH26-H3, and VHH26-H6 (Figure 3A). SEC analysis of all six humanized VHH26 constructs revealed two peaks. The results are summarized in Figure 3B. The central fractions of each of these peaks were investigated by Octect-BLI for their ability to interact with Lrp6. The kinetic parameters such as kon, koff, and KD for the interaction between VHH26 constructs and Lrp6 E3E4 domain are listed in Table 2. Analysis of these parameters revealed that the VHH26-H5 humanized variant had little effect on the binding affinity for Lrp6 compared to the parental VHH26 (Table 2; Figure 3B). For comparison, an alignment of the parental VHH26 and VHH26-H5 is shown in Figure 2B.

[0310] Based on the above, VHH26-H5 was used as a humanized LRP binding domain (e.g., tetravalent bispecific WNT agonist) fused with an Fzd binding domain in further experiments. The Fzd binding domain was derived from the R2M13 antibody that binds to Fzd5 and Fzd8, and contained an effector-free Fc region that retains FcRn binding, such as LALAPG or N297G (Wang X et al., Protein Cell 2018, 9: 63-73). N297G is a non-glycosylated form of IgG1 antibody in which Asn is replaced by Gly. In the case of R2M13-26 humanized N297, N297 corresponds to amino acid N302, and therefore the N297G mutation may alternatively be referred to as N302G. LALAPG represents three mutations in the Fc domain of IgG1. Using standard IgG1 sequence numbering, Leu234 and Leu235 are mutated to Ala; similarly, Pro329 is mutated to Gly. Thus, this triple mutant in the Fc domain is called "LALAPG". In the case of R2M13-h26, these mutations are at positions 239, 240, and 334 of the sequence, respectively. VHH26-H5 was fused to the N-terminus of the light chain of the R2M13 antibody via a 5 amino acid linker, thus generating an IgG-like molecule that contains the R2M13 antibody with VHHs at both N-termini of the antibody light chain.

[0311] The sequences of the R2M13 heavy chain IgG and R2M13 light chain fused by amino acid linkers to the various LRP5 / 6 VHH binding domains present in the various Wnt agonists are provided in Table 3. The sequences of the heavy and light chains present in the parent R2M13-03, R2M13-26, R2M13-36 Wnt agonists, which do not have the LALPG or N297G modifications, are provided as SEQ ID NOs: 136-138 (light chain, respectively) and SEQ ID NO: 153 (heavy chain) in PCT Publication No. WO2019 / 126398, which is incorporated herein by reference in its entirety. The Wnt agonists shown contain two heavy chains and two light chains in an antibody-type format, with the chains connected by disulfide bonds.

Table 1

Table 2-1

Table 2-2

Table 3-1

Table 3-2

Table 3-3

Table 3-4

[0312] The activity of Wnt agonists in the context of a fully engineered Wnt agonist format, in which the Fzd binding factor R2M13 was paired with various humanized Lrp binding domains, was determined using a Super TOPFlash luciferase reporter (STF) assay that measures activation of canonical Wnt signaling in a Wnt-responsive Huh-7 reporter cell line (Huh-7STF). The results are shown in FIG. 4. The R2M13-humanized_26-LALAPG construct ("R2M13-26 humanized LALAPG"; also referred to herein as R2M13-h26, R2M13-h26-LALAPG, or humanized LALPG) exhibited the most active humanized Lrp binding domain. The R2M13 humanized_26-N297G construct (R2M13-26 humanized N297G; humanized N297G) was not stable. Humanization of VHH03 and VHH36 significantly reduced potency in vitro when paired with R2M13, but their absolute EC50 values ​​were comparable to VHH26 paired with R2M13. The heavy and light chain sequences of the R2M13-humanized_26-LALAPG construct (R2M13-h26) are shown in Figure 6. This construct consisted of two heavy and two light chains linked by disulfide bonds. Effector function was removed by LALAPG mutations in the Fc domain (see, e.g., Wang, et al. (2018) Protein Cell. 9:63-73). The various domains of the R2M13-h26 construct are shown and the domains of other constructs can be easily determined based on these.

[0313] Example 3 Dose-response of engineered Wnt agonists in an animal model of DSS acute colitis The objectives of this study were to investigate the efficacy and dose response of R2M13-26, a Fzd5,8-specific Wnt mimetic disclosed in U.S. Patent Application Publication No. 2020-0308287, in an acute DSS colitis mouse model; to characterize the in vivo activity of R2M13-26 at various doses and frequencies in an acute DSS colitis mouse model; and to evaluate the effects of R2M13-26 on 1) body weight, fecal scores and occult blood, 2) epithelial / barrier repair by histology, and 3) inflammatory cytokines in serum and colon.

[0314] Six- to eight-week-old C57Bl / 6J female mice (86 in total) were obtained from Jackson Laboratories (Bar Harbor, ME, USA) and housed five per cage. All animal experiments conformed to the standards of the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences. Animal experimental protocols were approved by the Surrozen Institutional Animal Care and Use Committee.

[0315] To induce acute colitis, 7-8 week-old female mice were given drinking water containing 4.0% (w / v) dextran sulfate sodium (DSS, MP Biomedicals, MFCD00081551) ad libitum for 7 days, followed by drinking water containing 1.0% (w / v) DSS for 3 days. Groups of mice were either untreated, treated once on day 4 or twice on days 4 and 7 with an isotype control antibody (anti-GFP), or treated with the indicated engineered Wnt agonists.

[0316] R2M13-26 treatment at 1 mg / kg, 3 mg / kg, 10 mg / kg, 30 mg / kg once weekly and at 0.3 mg / kg, 1 mg / kg, 3 mg / kg, 10 mg / kg twice weekly reduced the disease activity index (DAI) in acute DSS mouse model. A single dose or twice weekly doses starting with 1 mg / kg R2M13-26 were able to repair the damaged colonic epithelium and improve histology scores. A single dose or twice weekly doses starting with 1 mg / kg R2M13-26 were able to reduce serum inflammatory and colonic cytokine levels.

[0317] This study confirmed that Fzd5,8-specific Wnt mimetic (R2M13-26) alone can improve disease activity index, repair damaged colonic epithelium, and reduce inflammatory cytokine levels in colon and serum in an acute DSS mouse model. Overall, in an acute mouse IBD model (acute DSS), R2M13-26 with a wide dose range of treatment improved fecal scores and body weight, repaired damaged colonic epithelium, and reduced inflammatory cytokine levels in colon and serum.

[0318] Example 4 Engineered Wnt agonists repair damaged colonic epithelium in an animal model of DSS-induced acute colitis Various engineered humanized Wnt agonists were tested in the DSS model of acute colitis, as outlined in Figure 7. Constructs tested included non-humanized and humanized versions, including R2M13-03-LALAPG, R2M13-26-LALAPG, R2M13-36-LALAPG, R2M13-humanized-03-LALAPG, R2M13-humanized-26-LALAPG, R2M13-humanized-36-LALAPG, R2M13-humanized-03-N297G, and R2M13-humanized-36-N297G.

[0319] Six-week-old C57Bl / 6J female mice (96 in total) were obtained from Jackson Laboratories (Bar Harbor, ME, USA) and housed five per cage. All animal experiments conformed to the standards of the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences. Animal experimental protocols were approved by the Surrozen Institutional Animal Care and Use Committee. Mice were allowed to acclimate for a minimum of 2 days before starting the experiments. Mice were housed in a 30%–70% humidity environment and at room temperatures ranging from 20°C to 26°C with a 12 / 12-h light / dark cycle.

[0320] To induce acute colitis, 7-8 week old female mice were given free access to drinking water containing 4.0% (w / v) dextran sulfate sodium (DSS, MP Biomedicals, MFCD00081551) for 7 days, followed by drinking water containing 1.0% (w / v) DSS for an additional 3 days (Figure 7). Groups of mice were either untreated, treated with an isotype control antibody (anti-GFP) or treated with 1 mg per kg of the indicated engineered Wnt agonist on days 4 and 7. All protein treatments showed comparable serum antibody exposure at the end point (Figure 7).

[0321] Control treated animals subjected to DSS developed severe colitis characterized by significant and persistent weight loss and bloody diarrhea, resulting in an increase in the disease activity index represented by the fecal score. Treatment with humanized R2M13-26 and humanized R2M13-36 in either the LALAPG or N297G form significantly improved body weight in DSS mice. Significant improvement in body weight was observed with humanized R2M13-36-LALAPG compared to the parent construct. These constructs also significantly reduced the disease activity index (DAI) in DSS mice (Figure 8), reduced fecal score in DSS mice, increased colon length and feces in DSS mice, and increased colon length and weight in DSS mice. Furthermore, humanized R2M13-26 (H-LALAPG26) and humanized R2M13-36 (H-LALAPG36) reduced serum levels of inflammatory cytokines, tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6) and interleukin-8 (IL-8) (Figure 9) and lipocalin-2, which were elevated in the DSS-treated group (Figure 10). Humanized R2M13-36-LALAPG showed significant improvement in body weight compared to the parent construct. Furthermore, humanized R2M13-26-LALAPG (R2M13-h26-LALAPG) was demonstrated to restore epithelial tight junction marker ZO-1 in vivo (Figure 11), repair damaged colonic epithelium (Figure 12), and restore epithelial cell lineages including colonocytes, goblet cells, and tuft cells (Figure 13). Thus, both humanized R2M13-26 and humanized R2M13-36 showed good efficacy in DSS mice.

[0322] Example 5 Pharmacokinetics (PK) of engineered Wnt agonists The pharmacokinetics (PK) of parental R2M13-26 (R2M13-26-LALAPG) and humanized R2M13-26 (R2M13-h26-LALAPG) following intravenous injection was determined by measuring the amount of antibody in serum at various time points following administration to rats and comparing with the data obtained from mice (Figure 14). The Cmax for humanized R2M13-26 (R2M13-h26) is higher than the Cmax for parental R2M13-26 (R2M13-26), thus maintaining the difference over time. However, the fold difference increased over time. The clearance of humanized R2M13-26 (25.3 mL / day / kg) was lower than that of the parent R2M13-26 (40.0 mL / day / kg), and the half-life of humanized R2M13-26 (3.75 days) was longer than that of the parent R2M13-26 (2.47 days).

[0323] Example 6 Evaluation of engineered Wnt agonists in a DSS chronic colitis model Because R2M13-26 treatment ameliorated acute colitis in the DSS model (Example 3), the engineered Wnt agonist, R2M13-26, was tested at different time points of repeated cycles of DSS-washout in the DSS model of chronic colitis to demonstrate the efficacy of the engineered Wnt agonist in the chronic colitis model.

[0324] Six- to eight-week-old C57Bl / 6J female mice were obtained from Jackson Laboratories (Bar Harbor, ME, USA) and housed 4–5 mice per cage. All animal experiments conformed to the standards of the Guide for the Care and Use of Laboratory Animals, published by the National Academy of Sciences. Animal experimental protocols were approved by the Surrozen Institutional Animal Care and Use Committee.

[0325] To induce chronic colitis, female mice were given free access to drinking water containing 3.0% (w / v) dextran sulfate sodium (DSS, MP Biomedicals, MFCD00081551) for 5 days followed by 3 cycles of pure drinking water for 7 days. Groups of mice were treated with an isotype control antibody (anti-GFP) or with 4 doses of R2M13-26-LALAPG (R2M13-26) at 10 mg / kg on days 16, 19, 28 and 31. Animals were sacrificed on day 33.

[0326] In the chronic DSS model, R2M13-26 treatment improved body weight and disease activity index. R2M13-26 also improved colon histology. Furthermore, R2M13-26 reduced serum inflammatory mediators IL-6 and lipocalin-2 at the end of the study on day 33 (data not shown).

[0327] Example 7 Effects of engineered Wnt agonists on DSS-induced acute colitis model In Examples 3 and 4, it was demonstrated that R2M13-26 and R2M13-h26 Wnt agonists specific for Fzd5,8 are effective in treating acute mouse colitis (acute DSS) model. The aim of this study was to use a similar model system and develop a more comprehensive understanding of the mechanism of action by which R2M13-26 affects colonic cells throughout the repair process.

[0328] Six- to seven-week-old C57Bl / 6J female mice were obtained from Jackson Laboratories (Bar Harbor, ME, USA) and housed 4–5 mice per cage. All animal experiments conformed to the standards of the Guide for the Care and Use of Laboratory Animals, published by the National Academy of Sciences. Animal experimental protocols were approved by the Surrozen Institutional Animal Care and Use Committee.

[0329] To induce acute colitis, female mice were given drinking water containing 4.0% (w / v) dextran sulfate sodium (DSS, MP Biomedicals, MFCD00081551) ad libitum for 7 days and drinking water containing 1.0% (w / v) DSS for 3 days. On day 4, groups of mice were either untreated, treated with a control antibody (anti-GFP), or treated with a single ip injection of R2M13-26-LALAPG (R2M13-26). A total of 123 mice were grouped (day 3 = 13, day 4 = 13, day 5 = 26, day 6 = 24, day 7 = 26, day 10 = 21), 91 were used for histology endpoints and 21 were used for scRNA-seq (group excluded due to machine). Daily food intake, BW, fecal scores, and occult blood were measured. At termination, mice were treated as follows: Groups A-E: Collect transverse colon for qPCR and histology (Group A terminated on days 3, 4, Group C terminated on days 3, 4, 5, 6, 7, Groups B, D, and E terminated on days 5, 6, 7, and 10). Assays / endpoints included RT-qPCR, histology, scRNA-seq, fecal scores for stool consistency and occult blood, Disease Activity Index (DAI)=(BW loss + stool consistency + blood) / 3, serum inflammatory cytokines (TNF-a, IL-6, lipocalin 2), and anatomical pathology: ascending, transverse, and descending colon, H&E. Histopathological scoring criteria included inflammation severity, spread of inflammation, mucosal erosion, crypt proliferation, and goblet cell loss.

[0330] No differences were observed between PBS and anti-GFP treatments on days 3-7 with DSS (data not shown). However, treatment with R2M13-26 showed healthier colon tissue at days 5-10, with notable histological improvement by day 7 in R2M13-26 treated animals (data not shown). R2M13-26 improved fecal scores and BW loss in DSS mice (data not shown), thus reversing experimental colitis in mice.

[0331] To evaluate gene expression changes, RT-qPCR analysis was performed on bulk colon samples. Wnt induction was examined, showing a significant decrease in Axin2 by DSS. R2M13-26 induced Axin2 expression in the absence of DSS. Proliferation markers were examined, showing a significant decrease in Ki67 by DSS at day 4 and rescue by R2M13-26. R2M13-26 rescued Cdk1 downregulation in the presence of DSS. Analysis of stem cell markers showed a significant decrease in Lrig1 by DSS at day 4 and rescued by R2M13-h26. Regarding clinical markers for IBD, a significant upregulation of Gpx2 was observed at days 5 and 6.

[0332] For scRNA-seq experiments investigating gene expression in the DSS model, mice were treated with 4% DSS in drinking water throughout the duration of the experiment. DSS-treated animals were administered 10mpk of R2M13-26 or anti-GFP antibody on day 4 of DSS treatment. Three animals each treated with Wnt agonist and anti-GFP were collected on days 5 and 6, 24 and 48 hours after administration, respectively. Samples from two naive non-injured animals were also collected on days 5 and 6. Colon, small intestine, spleen and liver tissues were collected at the end and examined or frozen for mRNA analysis. Single-cell RNA sequencing (scRNA-seq) was performed on fresh transverse colon samples to isolate single cells, and RT-qPCR was performed on fresh transverse colon to isolate only the epithelium.

[0333] The transverse colon was isolated from each animal and cleared of feces. After a brief wash with cold PBS, the colon was cut longitudinally to open the tube into a flat sheet, and the tissue was cut into 3-4 mm long fragments. The tissue fragments were incubated in pre-warmed (37°C) PBS with 5 mM EDTA in a shaker at 37°C, 150 rpm for 15 min. After 15 min, the tube containing the sample was shaken vigorously for 10 s to release more epithelial cells. The epithelial cells floating in suspension were removed and placed in a new tube and centrifuged at 200 rcf for 2 min to pellet the epithelial cells dissociated from the tissue. The remaining tissue containing the remaining epithelium and lamina propria was then incubated in 8-12.5 mL of lamina propria dissociation buffer at 37°C, 150 rpm with horizontal shaking for 30 min. After pelleting, the epithelial cells were resuspended in 1 mL of TrypLE with DNase1, the epithelial cells were incubated at 37°C for approximately 8 minutes, and triturated with a P1000 pipette approximately 25 times. After trituration, the epithelial cells were centrifuged at 500 rcf at 4°C, and the supernatant was removed. The epithelial cells were then washed once with FACS buffer before another round of centrifugation and a final resuspension in 0.5 mL of FACS buffer. After dissociation in LP dissociation buffer for 30 minutes, the remaining tissue fragments and suspension were centrifuged at 500 rcf for 5 minutes. The supernatant was removed and reduced to 1 mL, and the sample was triturated with a P1000 until the solution was homogenous and all tissue fragments were dissociated. After trituration, the sample was centrifuged at 500 rcf at 4°C for 5 minutes, washed with FACS buffer, and then resuspended in 1 mL of FACS buffer in preparation for FACS.

[0334] All cells were passed through a 40 micron filter prior to FACS. Viability was assessed by FACS using DAPI, and only viable (DAPI negative) cells were collected. Cells were collected from the epithelial fraction and then from the epithelial / lamina propria fraction, combined and counted on a hemocytometer prior to cell capture. Approximately 4500-5000 cells were loaded per channel following the standard 10x Genomics 3'v3 scRNA-seq protocol. Samples from individual animals were captured per channel. Standard 10x Genomics 3'v3 scRNA-seq RT, cDNA amplification and sequencing library preparation followed. Sequencing of the multiplexed sequencing libraries was performed on an Illumina Nova Seq 6000 S1 lane.

[0335] Illumina read data was processed using the 10x Genomics Cellranger pipeline. Demultiplexed UMI counting data was then evaluated to remove low quality cells and low expressed genes. UMI counting data was normalized using deconvolution scaling in the R package scran, and cells were clustered using an SNN graph-based clustering method using the R package scran. Cell type identity was determined using established cell type markers. Differential gene expression comparisons at the single cell level for each cluster were performed using one versus all and pairwise comparisons within each series by running EdgeR using the R package clusterExperiment. Differential gene expression analysis between experimental conditions was performed with the R package edgeR on pseudo-bulk samples after aggregating biological replicate samples based on series or cell type / cluster level. Differential expression comparisons between experimental conditions (DSS-injured vs. non-injured, then within DSS-injured samples for R2M13-26-treated vs. anti-GFP-treated) were performed at each time point (24 or 48 h), along the epithelial lineage, and within individual clusters representing cell types within the epithelial lineage.

[0336] R2M13-26 exerts its effect primarily by directly affecting colonic epithelial cells, due to FZD5 being highly expressed in intestinal epithelial cells and enriched in stem and progenitor cell populations. Comparing expression across epithelial lineages and all cell types included between R2M13-26 and control treatments, the following Wnt target genes were increased (Table 7). Molecules that showed at least a two-fold increase between treatment and control across epithelial lineages and have been shown in the literature to be direct Wnt targets were selected. The majority of Wnt target genes were obtained from genetic manipulations and chromatin immunoprecipitation experiments published in Gougelet et al. (2014). Further scRNA-seq data are shown in Tables 4-6, and 8.

[0337] In addition to investigating molecules that show significant changes across the epithelial lineage, scRNA-seq data was used to investigate specific cell types and compare gene expression between R2M13-26 treated cells and control treated cells to identify Wnt target genes that were increased or decreased in each relevant cell type within the epithelial lineage. This type of differential expression analysis was performed for the following relevant epithelial cell types: stem cells, TA1, TA2, basal goblet cells, injury-induced alternative progenitor cells (AltEnteroPC), injury-induced alternative enterocytes (AltEntero), enteroprogenitor cells (EnteroPrecur), goblet cells 1, goblet cells 2, enteroendocrine, and tuft cells. A summary of Wnt target genes modulated across epithelial lineages and / or in specific epithelial subtypes, along with examples of log2 fold changes, is shown in Table 7. A heatmap of epithelial cells detected in the scRNA-seq experiment is shown in Figure 26B.

[0338] A number of molecules were identified that were significantly increased or decreased when comparing expression in any of the aggregated epithelial lineages and / or cell types included between R2M13-26 and control treatments. Molecules that showed at least a two-fold change between treatment and control across epithelial lineages or within at least one epithelial cell type in the acute DSS mouse model of IBD were selected. These molecules are shown in Tables 4-8.

[0339] Genes increased by treatment with R2M13-26 were crossed with a list of established cell cycle genes (Giotti et al., 2019) to identify genes involved in cell cycle progression and regulation that were increased by treatment with R2M13-26. The identified genes are listed in Table 4. One of the established roles of Wnt signaling is the maintenance of stem and progenitor cells, and cell cycle regulation is a key aspect of its function (Davidson, 2010; Hirata 2013). R2M13-26 promoted the expansion of stem and progenitor cells in injured colonic epithelium, which are essential for their ability to regenerate the epithelium. These data indicate that some of these genes are also direct Wnt targets (Table 8).

[0340] In addition to promoting the expansion of stem and progenitor cells to facilitate epithelial regeneration, Wnt signaling is crucial for the maintenance and renewal of stem and progenitor cell pools and the regulation of their differentiation (Pinto et al., 2003; Ma et al., 2016). R2M13-26 promotes epithelial repair and regeneration by maintaining stem and progenitor cells, as evidenced by increased expression of several key genes involved in this process, including Id1 (Hollnagel 1999; Meteoglu 2008; Ruzinova 2003), Nhp2 (Fong 2014; McCann 2020) and Hmga2 (Nishino 2008; Parisi 2020), Foxq1 (Tu 2018; Zhang 2018), and Aldh1 (Tomita 2016) (Table 8). Additionally, effects were observed on the expression of Areg (Fujii 2008; Mahtouk 2005; Suzuki 2010; Takahashi 2020), a ligand of EGFR signaling that is important for intestinal stem cell niche maintenance. Yet another interesting molecule that was induced in several stem and progenitor cells after R2M13-26 treatment and showed a significant increase in expression was glucagon (Gcg). Glucagon can be processed into multiple small peptides, including GLP-1 and GLP-2, which play a role in reducing inflammation in IBD. GLP-2 also acts as a growth factor that promotes proliferation of stem and progenitor cells and epithelial crypt regeneration (Drucker 1999; Markovic 2019; Zatorski 2019). These data indicate that Wnt signaling activation increases glucagon expression, which leads to increased levels of GLP-2 and contributes to the expansion of stem and progenitor cells.

[0341] In addition to regulating stem and progenitor cell self-renewal and differentiation, one of the key aspects for tissue repair and epithelial regeneration is the repair of intracellular and extracellular damage and the reconstruction of the epithelial barrier. To this end, some of the genes induced and / or increased upon treatment with R2M13-26 are associated with these processes (Table 6). For example, Apex1 is crucial for DNA repair (Park 2014). Mucus production and mucus barrier dysfunction are important aspects of IBD (Antoni 2014; Dorofeyev 2013; Kim, Ho 2010). Some of the genes increased upon treatment with R2M13-26 promote mucus secretion and establishment of the mucus barrier (B3gnt7, Agr2, Muc2, Muc3, Tff3, Fcgbp, and Zg16). These genes play important roles in mucus production, processing, and secretion of mucus (Agr2: Bergstrom 2014; Park 2009; B3gnt7: Arike 2017; Fcgbp: van der Post 2019; Muc2, Muc3: Arike 2017; Svensson 2018; Kim 2010; Ho 2006; Tff3: Aihara 2017; Zg16: Bergstrom 2016). Additionally, Sprr2a3, a member of small proline-rich repeat proteins involved in epithelial barrier formation (Gibbs 1993), was enriched.

[0342] Importantly, reduced or lost expression of many of these genes is associated with increased colitis severity and / or IBD onset and progression in mouse models (Dorofeyev 2013; van der Post 2019). For example, expression of MUC2, MUC3, and TFF3 is reduced in severe CD and UC (Dorofeyev 2012). In mouse colitis models, reduction of MUC2 renders mice more susceptible to DSS-induced colitis (Kim, Ho 2010). Furthermore, GWAS studies have identified risk alleles in Agr2 that appear to reduce its expression in promoted IBD (Zheng 2006).

[0343] In addition to affecting epithelial repair and regeneration by regulating stem and progenitor cell proliferation and differentiation, cellular repair, and barrier formation, R2M13-26 promoted the expression of many genes and pathways associated with reducing inflammatory responses in injury and IBD (Table 5). These molecules have anti-inflammatory effects and / or their reduction is associated with increased inflammation or exacerbation of IBD.

[0344] In the R2M13-26 treated group, a dose response was shown for serum antibody concentrations at 24 and 48 hours post-injection, with R2M13-26 showing linearity at doses of 1 mpk, 3 mpk, and 10 mpk. R2M13-26 increased Axin2 and Ki67 expression 2 days after a single IP injection (Figure 25), and R2M13-26 increased LGR5 expression 2 days after injection. R2M13-26 treatment increased occludin expression 2 days after injection.

[0345] Example 8 Evaluation of engineered Wnt agonists in comparison with other agents in the DSS colitis model In Examples 3 and 4, it was demonstrated that R2M13-26 and R2M13-h26 specific for Fzd5,8 are effective in treating the acute mouse colitis (acute DSS) model, and in Example 6, it was demonstrated that R2M13-26 is effective in treating the chronic mouse colitis (chronic DSS) model. The purpose of this study was to compare the efficacy of R2M13-h26 with that of other drugs, including cyclosporine A, anti-TNF antibody, and anti-IL-12 / 23 antibody, in treating the colitis model. 6-7 week old C57Bl / 6J female mice were obtained from Jackson Laboratories (Bar Harbor, ME, USA) and housed 4-5 per cage. All animal experiments were in accordance with the standards of the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences. Animal experimental protocols were approved by the Surrozen Institutional Animal Care and Use Committee.

[0346] The study with cyclosporine A is outlined in Figure 15. To induce acute colitis, female mice were given drinking water containing 4.0% (w / v) dextran sulfate sodium (DSS, MP Biomedicals, MFCD00081551) ad libitum for 7 days and drinking water containing 1.0% (w / v) DSS for 3 days. Groups of mice were either untreated, treated with an isotype control antibody (anti-GFP), or treated with R2M13-h26 at the indicated doses by a single IP injection on day 4 or two injections on days 4 and 7, or with cyclosporine A as indicated.

[0347] R2M13-h26 treatment improved body weight, reduced fecal scores, and reduced disease activity index (DAI) more than cyclosporine A (Figure 16). Furthermore, R2M13-h26 repaired colonic epithelium in vivo more effectively than cyclosporine A (Figure 17), improved colon histology scores (data not shown), and reduced serum levels of inflammatory cytokines more than cyclosporine A (data not shown). Overall, R2M13-h26 showed efficacy in repairing colonic epithelium, improving histology and disease activity index (DAI), and reducing inflammatory cytokines at doses as low as 1 mg / kg twice weekly or a single dose of 2 mg / kg. Cyclosporine A showed a mild effect on reducing DAI and lipocalin-2, and in general, its effect was much less than that of R2M13-h26.

[0348] A study comparing R2M13-h26 with anti-TNF in the chronic DSS model is outlined in Figure 18. Mice were administered 3% DSS for three 7-day cycles separated by 7 days without DSS, followed by a 3-day washout period of 1% DSS, thereby producing chronic intestinal epithelial injury. R2M13-h26 treatment was administered with 2, 4, or 6 injections at 1 mpk, 3 mpk, or 10 mpk. Anti-TNF was administered with 4 or 7 injections at 5 mpk or 25 mpk. Readings were taken on day 38.

[0349] R2M13-h26 restored colonic epithelium more effectively than anti-TNF in a chronic DSS colitis model (Figure 19). R2M13-h26 reduced colon histology scores, improved body weight, reduced fecal scores, and reduced DAI, whereas anti-TNF had no effect on these disease parameters (Figure 20 and data not shown). R2M13-h26 also reduced serum inflammatory cytokine levels, lipocalin-2 and IL-6, more than anti-TNF in a chronic in vivo model (Figure 21). In a chronic mouse IBD model (chronic DSS with three repeated cycles of DSS injury over 38 days), various dosing regimens (four doses of 1 mg / kg to two, four, or six doses of 10 mg / kg) of R2M13-h26 specific for Fzd5,8 were able to reach significant effects on repairing colonic epithelium, improving histology and disease activity indexes, and reducing inflammatory cytokines. In contrast, anti-TNF Abs were unable to reverse either epithelial damage or DAI in chronic DSS mice.

[0350] The efficacy of anti-IL12 / 23p40 compared to R2M13-h26 was also investigated in the chronic DSS colitis mouse model with respect to: 1) body weight, fecal score and occult blood, 2) epithelial / barrier repair by histology, and 3) serum inflammatory cytokines. Female C57BL / 6 mice aged 6-8 weeks were treated with three cycles of 3.0% dextran sulfate sodium (DSS) to induce chronic colonic colitis as outlined in Figure 22. The first two cycles consisted of 7 days of DSS and 7 days of water without DSS, and the third cycle consisted of 7 days of 3% DSS and 3 days of 1% DSS. R2M13-h26 treatment was administered by four injections at 0.1mpk and 1mpk. Anti-IL12 / 23 was administered by four or eight injections at 3mpk or 10mpk. Anti-IL12 / 23p40 was clone C17.8 from Invivoplus Bioxcell. Readout was performed on day 38.

[0351] R2M13-h26 treatment reduced the disease activity index (DAI) in the chronic DSS mouse model, whereas anti-IL12 / 23p40 treatment did not (Figure 23). Furthermore, R2M13-h26 treatment reduced serum cytokine levels more effectively than anti-IL12 / 23 (Figure 24). This study confirmed that R2M13-h26 could repair damaged colonic epithelium and reduce serum inflammatory cytokine levels in the chronic DSS mouse model, whereas BioXcell's anti-IL12 / 23p40 monoclonal antibody could not.

[0352] Example 9 Effect of engineered Wnt agonists on reducing Wnt pathway activation and inflammation Selective Wnt pathway activation Fzd5 has been shown to be highly expressed in the colon of a mouse model of colitis induced by dextran sulfate sodium, or DSS. In this model, DSS exposure leads to the disruption of the intestinal barrier, resulting in an inflammatory response similar to that seen in IBD patients. R2M13-h26 was observed to bind to DSS-injured intestinal cells, stimulate Wnt signaling, and restore tissue architecture, epithelial cell type composition, and epithelial barrier function, as measured by the expression of Axin2, a downstream target gene in the Wnt pathway. Exposure of mice to DSS for 7 days led to a breakdown of the intestinal barrier that could be easily visualized in stained colon cross sections. In the absence of DSS, an intact intestinal wall was present, with crypts tightly packed together to form a continuous structure. Following exposure to DSS, treatment with a negative control antibody, anti-GFP, led to several effects by day 10: disruption of the intestinal wall; shrinkage of colonic crypts; and creation of multiple discontinuous segments. However, in DSS-exposed mice treated with R2M13-h26 administered on days 4 and 7, doses of 1 mg / kg or higher led to a dose-dependent repair of this damage, reversing most of the damage visible by histology. Similar results were observed in a chronic model of DSS. The extent of epithelial repair measured by histology with R2M13-h26 was greater than that obtained in further experiments with cyclosporine A, anti-TNF antibodies, or anti-IL12 / 23 antibodies.

[0353] Histological staining showed that treatment with R2M13-26 and R2M13-h26 administration led to the restoration of markers of tight junctions, intercellular structures that contribute to the intestinal barrier preventing free exchange of materials between the intestinal tract and the peritoneal cavity. In healthy intestinal tissue, zonula occludens 1 protein, or ZO-1, a component of tight junctions, was found as a continuous layer along the intestinal brush border. In DSS-injured intestinal tissue, the continuous expression pattern of ZO-1 was perturbed. Treatment with R2M13-h26-LALAPG restored the localization of ZO-1 as a continuous layer along the intestinal brush border (Figure 11).

[0354] Reduce inflammation In the mouse DSS model, treatment with administration of R2M13-h26 led to a significant dose-dependent decrease in a number of inflammatory cytokines, such as TNFα, interleukin-6 or IL-6, and interleukin-8 or IL-8. Reductions in cytokine levels were observed in both colonic tissue and serum (data not shown). These results suggest that R2M13-h26 not only has the potential to repair the epithelium directly, but also as a result of reducing inflammation.

[0355] Example 10 Treatment with Wnt mimetics rapidly repaired DSS-damaged colonic epithelium Various dextran sodium sulfite (DSS)-induced colonic colitis models have been widely used as preclinical models to test the efficacy of therapeutic compounds and biological agents intended to treat ulcerative colitis. To test the effect of Wnt signal activation on epithelial repair, an acute severe DSS mouse model was established (see WO2020 / 185960A1, the entirety of which is incorporated herein by reference). In this model, a high percentage of DSS (4%) was used for the first 7 days to induce damage to the colonic epithelium. Animals were then maintained on 1% DSS until takedown on day 10 to maintain the established damage and minimize spontaneous epithelial repair. Consistent with a previously reported DSS study (Cooper, HS, Murthy, SN, Shah, RS, & Sedergran, DJ (1993). Clinicopathologic study of dextran sulfate sodium experimental murine colitis. Laboratory Investigation, 69(2), 238-249), damage to the colonic epithelium was visible by hematoxylin and eosin (H&E) staining on day 4 and continued to progress through day 7 (see WO2020 / 185960A1). RNAscope in situ hybridization analysis showed reduced mRNA expression of the Wnt target genes Axin2, Lgr5, Rnf43 and the Wnt ligands Wnt2b and Wnt5a in the colonic epithelium and surrounding mesenchymal cell layers, respectively. mRNA expression of Rspo3, a mouse intestinal R-spondin that predominates in mesenchymal cells beneath the colonic crypts, was not affected by DSS (see WO2020 / 185960A1).

[0356] In an established DSS model, mice were injected with two doses of R2M3-26 or a Wnt mimetic targeting FZD1, 2, 5, 7, 8 and LRP6, called FA-L6 in Fowler et al. (Fowler, T. W et al., (2021). Development of selective bispecific Wnt mimetics for bone loss and repair. Nature Communications, 12 (1). https: / / doi.org / 10.1038 / s41467-021-23374-8), starting on day 4, when DSS damage to the epithelium is already visible, followed by another dose on day 7 and evaluating the effect on epithelial repair on day 10 (6 days of treatment). Colons treated with R2M3-26 were similar to those not treated with DSS, with restored crypt architecture and less tissue inflammation compared to PBS or anti-GFP control treatments (see WO2020 / 185960A1). Colon tissues were examined by a pathologist blinded to treatment and scored for general colitis pathology (see methods described in Example 1). Histology scores consistently showed that R2M3-26 effectively repaired colon tissue damaged by DSS, reducing colitis scores from 4.75 to 2.0 (see WO2020 / 185960A1).

[0357] Since RSPO has previously been reported to reverse DSS-induced colitis in mice (Zhao et al., 2007), we also investigated the effect of RSPO2 in the DSS mouse model described herein. RSPO2 was IP injected either twice weekly or daily, starting on the fourth day of DSS treatment. Repair of damaged colonic epithelium was observed with RSPO2 treatment, but the effect was not significant compared to the effect of R2M3-26. Similarly, combined treatment of R2M3-26 and RSPO2, both twice weekly and daily, restored colonic crypt architecture and improved colonic histology, but to a lesser extent than R2M3-26 alone (see WO2020 / 185960A1).

[0358] It has previously been shown that RSPO alone or combined treatment with the Wnt mimetic 18R5-DKK1c stimulates hyperproliferation of intestinal stem cells and transient amplifying (TA) cells in normal mice, leading to growth in intestinal crypt and villus length (Yan Kelley S. et al., 2017). In the DSS model described herein, we also observed an expansion of Ki67 expression (data not shown) in the duodenum and colon by RSPO2 treatment or combined treatment with R2M3-26 and RSPO2 at day 10. However, R2M3-26 alone did not lead to an expansion of Ki67 in either the duodenal or colonic epithelium of the DSS model at day 10, which was consistent with previous studies expressing Wnt agonists in non-injured animals (Yan Kelley S. et al., 2017). The results indicate that Wnt agonist treatment alone was able to repair colonic epithelium damaged by DSS without causing hyperproliferation in normal colon or small intestine.

[0359] Example 11 R2M13-26, a Wnt mimetic targeted to Fzd5, 8, stimulated the growth of mouse intestinal organoids RNAscope in situ hybridization analysis showed that Fzd5 was expressed at the highest level in mouse small intestinal epithelium (Fig. 29E), followed by Fzd1 (Fig. 29A) and Fzd7 (Fig. 29G). Fzd1 and Fzd7 were mostly expressed near the crypt base where Lgr5-positive stem cells reside (Fig. 29L). Fzd5 expression was concentrated in columnar stem cells near the crypt-villus boundary and at the crypt base in the duodenum, overlapping with a strongly Axin2-positive domain that was also positive for the stem cell marker Lgr5 (Fig. 29K).

[0360] We then tested whether stimulation of Wnt signaling with a Wnt agonist specific for both the Fzd5 and Fzd8 subfamilies (R2M13-26) or a Wnt agonist specific for the Fzd1, 2, and 7 subfamilies (1RC07-26) was sufficient to stimulate epithelial cell proliferation in mouse small intestinal organoid cultures. The subfamily-specific Wnt mimetics were active in vitro in the Super TopFlash (STF) assay (Figure 5). Mouse small intestinal organoids were treated with the Porcupine inhibitor IWP2 to inhibit endogenous Wnt ligand secretion in the cultured organoids. When these organoids were not treated with the protein or treated with control anti-β-gal IgG, the organoids were not maintained and rapidly degenerated. In contrast, treatment with R2M3-26, a pan-specific Fzd1,2,5,7,8 Wnt mimetic, over a wide dose range, was able to stimulate cell proliferation and resulted in growing transparent spherical organoids. Both the Fzd5,8 specific Wnt mimetic ("R2M13-26") and the Fzd1,2,7 specific Wnt mimetic ("1RC07-26", also called FB-L6 in (Fowler et al., 2021)) were able to stimulate organoid proliferation and growth (see WO2020 / 185960A1). The effects of the subfamily-specific Wnt mimetics were comparable to those of the pan-specific agonists.

[0361] Example 12 R2M13-26, an Fzd5,8-specific Wnt mimetic, was effective in repairing colonic epithelium damaged by DSS In situ analysis demonstrated that colonic epithelium exhibited a similar Fzd expression pattern as the small intestine (Figure 30), and that Fzd5 was also expressed at the highest level among all Fzds in colonic epithelium. Under DSS conditions, this differential expression of Fzd was maintained, even though the expression of all Fzds was reduced by DSS (Figures 30K-30T).

[0362] We next investigated whether Fzd subfamily-specific mimetics could repair colonic epithelium damaged by DSS. In the DSS model, two doses of control anti-GFP IgG treatment or protein treatment were IP injected on days 4 and 7, and animals were sacrificed on day 10 for histology and serum analysis. In contrast to the severe tissue damage and inflammation observed in colons not treated with protein or treated with anti-GFP, both R2M13-26 (Fzd5,8) and 1RC07-26 (Fzd1,2,7) treatments led to repair of colonic epithelium. The effect of two Fzd subfamily-specific Wnt mimetics (R2M13-26 and 1RC07-26) on colonic histology was comparable to that of Fzd1,2,5,7,8 pan-specific mimetic R2M3-26) (see WO2020 / 185960A1).

[0363] Similar to R2M3-26 treatment, R2M13-26 and 1RC07-26 treatments also improved fecal scores and disease activity index (DAI) (see WO2020 / 185960A1). Improvements in fecal scores and DAI were more pronounced with R2M13-26 compared to R2M3-26 or 1RC07-26. To further understand the extent of tissue repair with the various Wnt mimetics, colon tissues were analyzed again by a pathologist blinded to the treatment group (see WO2020 / 185960A1). Consistent with the DAI, the overall histology score of R2M13-26-treated DSS colons was significantly improved and better than colons treated with 1RC07-26. This suggests that the Fzd5,8-specific Wnt mimetic R2M13-26 is more effective at reducing colitis and repairing epithelium than the Fzd1,2,7-specific Wnt mimetic 1RC07-26.

[0364] We then determined whether the reduction in colitis observed with the Wnt mimetics was accompanied by a reduction in serum cytokine levels. Treatment with each of the three Wnt mimetics reduced serum levels of the pro-inflammatory cytokines TNF-α, IL6 and IL-8 induced by DSS (see WO2020 / 185960A1).

[0365] The efficacy of R2M13-26 in the DSS model was further tested in a dose-ranging study in which R2M13-26 was IP injected once on day 4 at 1mpk, 3mpk, 10mpk and 30mpk, or twice on days 4 and 7 at 0.3mpk, 1mpk, 3mpk and 10mpk. Significant improvements in tissue histology, DAI and histology scores were observed for all dose groups (data not shown). All dose groups also showed significant reductions in serum and tissue levels of the pro-inflammatory cytokines TNF-α, IL-6 and IL-8 (see WO2020 / 185960A1).

[0366] (Example 13) Although DSS insult induced a robust inflammatory response in all tissue layers, the primary direct effect of R2M13-26 was on epithelial cells. Using scRNA-seq, we determined which cells were the first to respond to treatment with R2M13-26, how R2M13-26 affected epithelial cell differentiation, and whether the effect on inflammatory cytokine reduction occurred directly on immune cells or indirectly through epithelial restoration. To test these questions, we applied scRNA-seq to investigate the early transcriptomic response of colons treated with R2M13-26 in an acute DSS mouse model. As in the above examples, 4% (w / v) DSS was administered in water and mice were IP injected with either 10 mg / kg anti-GFP control protein or 10 mg / kg R2M13-26 on day 4, with endpoints on days 5 and 6, 24 and 48 hours after injection, respectively (Figure 26A). After filtering, the dataset contained 22,717 total cells. Normalization and cluster analysis were applied to the full dataset to identify each lineage / group, followed by each sub-lineage / group. Dimensionality reduction and cluster analysis were applied to each cell subset (Figure 26B). Three major cell groups were present: immune (4835 cells), mesenchymal / stromal (7509), and epithelial (10373) (Figure 26B).

[0367] DSS insult had a strong effect on all three lineages at each time point, resulting in differential gene expression ranging from 500 to over 1400 genes in each tissue layer, with the immune lineage showing the greatest number of changes (Figure 26C).

[0368] To understand the impact of R2M13-26 in the DSS model, we first assessed the impact of DSS injury by comparing DSS, anti-GFP conditions with non-injury conditions. DSS induced distinct cell types in each tissue layer or lineage, which accounted for the majority of differential gene expression at the lineage level. In the immune lineage, no cell types were lost upon injury. Rather, by day 5 of DSS treatment, several cell types appeared in injured colon samples, including activated neutrophils (ActNeutropil), two populations of pro-inflammatory monocytes (InjuryMono1, 2), stimulated dendritic cells (ActDendritic), and two groups of B cells enriched for IgM heavy chain gene expression and Ighd (Bcell1_IgM, Bcell2_IgM). In stromal cells, DSS injury resulted in the appearance of new populations of fibroblasts expressing inflammatory cytokines and chemokines, consistent with recent reports of proinflammatory fibroblasts in UC patients and DSS mouse models (Kinchen et al., 2018; Smillie et al., 2019). The two groups of fibroblasts consisted almost entirely of injured cells (Inj...

Claims

1. (a) one or more binding domains that bind to one or more FZDs; and (b) one or more binding domains that bind to LRP5, LRP6, or both LRP5 and LRP6.

1. An engineered WNT agonist comprising: The engineered WNT agonist comprises a polypeptide sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOs: 1-25, or a polypeptide sequence disclosed in FIG. 2, FIG. 6, Table 1, or Table 3, or a binding fragment thereof; Optionally, the one or more binding domains that bind to the one or more FZDs are i) FZD5; ii) FZD8; iii) FZD1; iv) FZD2; vi) FZD7; vi) FZD5 and FZD8; vii) FZD1, FZD2, and FZD7; viii) FZD1, FZD2, FZD7, FZD5 and FZD8; ix) FZD4; x) FZD9; or xi) FZD10 An engineered WNT agonist that binds to

2. (a) a polypeptide sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 1-25, or a sequence disclosed in Table 3; or (b) a polypeptide sequence comprising two or three of the CDR sequences present in any of the VHH domains, VH domains, or VL domains disclosed in Figure 2; Including, Optionally, the polypeptide sequence comprises CDRs present in any one of SEQ ID NOs: 1-25.

3. (a) a polypeptide sequence having at least 90%, or at least 95%, homology to SEQ ID NO:1 and a polypeptide sequence having at least 90%, or at least 95%, homology to SEQ ID NO:2; (b) a polypeptide sequence having at least 90%, or at least 95%, homology to SEQ ID NO:3 and a polypeptide sequence having at least 90%, or at least 95%, homology to SEQ ID NO:4; (c) a polypeptide sequence having at least 80%, at least 90%, or at least 95% homology to SEQ ID NO:5 and a polypeptide sequence having at least 80%, at least 90%, or at least 95% homology to SEQ ID NO:6; (d) a polypeptide sequence having at least 90%, or at least 95%, homology to SEQ ID NO:7 and a polypeptide sequence having at least 90%, or at least 95%, homology to SEQ ID NO:8; (e) a polypeptide sequence having at least 90%, or at least 95%, homology to SEQ ID NO:9 and a polypeptide sequence having at least 90%, or at least 95%, homology to SEQ ID NO:10; (f) a polypeptide sequence having at least 90%, or at least 95%, homology to SEQ ID NO:7 and a polypeptide sequence having at least 90%, or at least 95%, homology to SEQ ID NO:

8. (g) a polypeptide sequence having at least 90%, or at least 95%, homology to SEQ ID NO:11 and a polypeptide sequence having at least 90%, or at least 95%, homology to SEQ ID NO:12; (h) a polypeptide sequence having at least 90%, or at least 95%, homology to SEQ ID NO: 13 and a polypeptide sequence having at least 90%, or at least 95%, homology to SEQ ID NO: 14; (i) a polypeptide sequence having at least 90%, or at least 95%, homology to SEQ ID NO: 15 and a polypeptide sequence having at least 90%, or at least 95%, homology to SEQ ID NO: 16; or (j) a polypeptide sequence having at least 90%, or at least 95%, homology to SEQ ID NO: 17 and a polypeptide sequence having at least 90%, or at least 95%, homology to SEQ ID NO:

18. Including, 3. The engineered WNT agonist of claim 2, optionally wherein the polypeptide comprises CDRs present in any one of SEQ ID NOs: 1-18.

4. The engineered WNT agonist of any one of claims 1 to 3, wherein one or more binding domains that bind to LRP5, LRP6, or both LRP5 and LRP6 are humanized.

5. 5. The engineered WNT agonist of any one of claims 1 to 4, comprising a modified Fc domain, said modified Fc domain comprising a LALAPG or N297G modification.

6. 10. A pharmaceutical composition comprising an engineered WNT agonist according to any one of claims 1 to 5 and a pharma- ceutically acceptable carrier, diluent, or excipient.

7. A composition comprising an engineered WNT agonist according to any one of claims 1 to 5 or a pharmaceutical composition according to claim 6 for treating a disease or disorder suitable for treatment by increased WNT pathway signaling in a subject.

8. The composition or pharmaceutical composition according to claim 7, wherein the disease or disorder is a gastrointestinal disorder.

9. 9. The composition or pharmaceutical composition of claim 8, wherein the gastrointestinal disorder is inflammatory bowel disease.

10. 10. The composition or pharmaceutical composition of claim 9, wherein the inflammatory bowel disease is selected from the group consisting of Crohn's disease (CD), CD with fistulizing formation, and ulcerative colitis (UC).

11. A composition or pharmaceutical composition according to any one of claims 7 to 10, characterized in that the composition or pharmaceutical composition is administered orally or parenterally.

12. The composition or pharmaceutical composition according to claim 11, characterized in that the composition or pharmaceutical composition is administered intravenously, intraperitoneally or subcutaneously.

13. A composition for use in a method for increasing WNT signaling in a cell, comprising an engineered WNT agonist as described in any one of claims 1 to 5, said method comprising the step of contacting said cell with said engineered WNT agonist.

14. A composition comprising an engineered WNT agonist according to any one of claims 1 to 5 or a pharmaceutical composition according to claim 6 for modulating expression of a WNT pathway molecule in one or more tissues or cells in a subject with a gastrointestinal disorder.

15. 15. The composition or pharmaceutical composition of claim 14, wherein the WNT pathway molecule is a gene or protein listed in any one of Tables 4, 5, 6, 7, 8, and 11.

16. 15. The composition or pharmaceutical composition of claim 14, wherein the WNT pathway molecule is selected from the group consisting of glutathione peroxidase 2 (Gpx2), interferon regulatory factor 8 (Irf8), Rel, RelA, RelB, RNAse4, angiogenin, Gsta3, Rnf43, Axin2, Ki67, occludin, or any of the genes or proteins listed in Table 7.

17. 17. The composition or pharmaceutical composition of any one of claims 14 to 16, wherein after administration, expression of the WNT pathway molecule in the one or more tissues and / or cells of the subject is increased by at least 20%, at least 50%, at least 80%, at least 2-fold, at least 5-fold, at least 10-fold, or at least 20-fold, or is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%.

18. 18. The composition or pharmaceutical composition of any one of claims 14 to 17, wherein the tissue is an epithelial tissue and / or the cell is a gastrointestinal epithelial cell, optionally a stem cell, TA1, TA2, goblet cell progenitor cell, injury-induced alternative progenitor cell (Alt progenitor), injury-induced alternative enterocyte cell (Alt Enterocyte), intestinal progenitor cell (EnteroPrecur), goblet cell precursor cell (goblet_PC), goblet cell 1, goblet cell 2, or an enteroendocrine cell.

19. A composition comprising an engineered WNT agonist according to any one of claims 1 to 5 or a pharmaceutical composition according to claim 6 for stimulating tissue repair in a subject with a gastrointestinal disorder.

20. 20. The composition or pharmaceutical composition of claim 19, wherein the tissue repair is stimulated by modulation of at least one WNT pathway molecule selected from the group consisting of genes associated with cell cycle, genes associated with stem and progenitor cell renewal and differentiation, genes associated with epithelial cell repair and barrier restoration, and / or any of the genes listed in any of Tables 4, 5, 6, 7, 8, and 11.

21. 21. The composition or pharmaceutical composition of claim 20, wherein the cell cycle related genes are selected from those provided in Table 4 or Aurka, Aurkb, Ccna2, Ccnb1, Ccnb2, Ccnd2, Ccne1, Cdc45, Cdk1, Cdkn3, Cenpm, Cenpp, Cenpq, Cenpu, Hells, Mcm4, Mcm5, Mcm6, Mcm7, Myc, Pbk, Plk1, Rrm1, and Rrm2.

22. 21. The composition or pharmaceutical composition of claim 20, wherein the genes associated with stem and progenitor cell renewal and differentiation are selected from those provided in Table 8, and Axin2, Id1, Hmga2, Nhp2, Foxq1, Hes6, and Adh1.

23. 21. The composition or pharmaceutical composition of claim 20, wherein the genes associated with epithelial cell repair and barrier restoration are selected from those provided in Table 6, or Apex1, Agr2, B3gnt7, Fcgbp, Muc2, Muc3, Tff3, Zg16, and Sprr2a3.

24. 24. The composition or pharmaceutical composition of any one of claims 20 to 23, wherein after administration of the composition or pharmaceutical composition, expression of the WNT pathway molecule in one or more tissues and / or cells of the subject is increased by at least 20%, at least 50%, at least 80%, at least 2-fold, at least 5-fold, at least 10-fold, or at least 20-fold, or is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%.

25. 10. A composition comprising an engineered WNT agonist according to any one of claims 1 to 5 or a pharmaceutical composition according to claim 6 for reducing inflammation in a subject with a gastrointestinal disorder.

26. 20. The composition or pharmaceutical composition of claim 19, wherein the inflammation is reduced by modulation of at least one molecule selected from the group consisting of a gene provided in Table 5 or Adamdec1, Atf3, Gpx2, Gsta3, Gstm1, Gstm3, Gdf15, Ihh, Il18, Lyz2, Nox1, Reg4, Sycn, Selenbp1, Tgfbr2, and Timp3.

27. 27. The composition or pharmaceutical composition according to claim 25 or claim 26, wherein the inflammation is reduced in gastrointestinal tissue, optionally epithelial tissue.

28. 28. The composition or pharmaceutical composition of claim 27, wherein the gastrointestinal tissue comprises gastrointestinal epithelial cells, optionally stem cells, TA1, TA2, goblet cell progenitor cells, injury-induced alternative progenitor cells (Alt progenitor), injury-induced alternative enterocytes (Alt Enterocyte), enteroprogenitor cells (EnteroPrecur), goblet cell progenitor cells (goblet_PC), goblet cell 1, goblet cell 2, or enteroendocrine cells.

29. 29. The composition or pharmaceutical composition of any one of claims 25 to 28, wherein after administration, expression of the WNT pathway molecule in one or more tissues and / or cells of the subject is increased by at least 20%, at least 50%, at least 80%, at least 2-fold, at least 5-fold, at least 10-fold, or at least 20-fold, or is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%.

30. 30. The composition or pharmaceutical composition of any one of claims 7 to 29, wherein the engineered Wnt agonist is R2M13-h26 or comprises a functional variant or fragment thereof.

31. 10. A composition comprising the engineered WNT agonist of any one of claims 1 to 5 or the pharmaceutical composition of claim 6 for use in a method of generating, culturing or maintaining an organ, tissue, cell or organoid culture, said method comprising: a) the engineered WNT agonist; or b) The pharmaceutical composition A composition or pharmaceutical composition comprising the step of contacting with.

32. A composition or pharmaceutical composition according to claim 31 for maintaining viability of said organ or tissue ex vivo, said method comprising: a) contacting an organ or tissue obtained from a donor ex vivo with said composition or said pharmaceutical composition, optionally by perfusion; or b) contacting a donor organ or tissue in vivo with said composition or pharmaceutical composition.

33. A composition or pharmaceutical composition as described in claim 31 for generating or maintaining the organoid culture, said method comprising a step of contacting the organoid culture by culturing the organoid culture in a medium optionally containing the engineered WNT agonist.

34. A composition comprising an engineered WNT agonist according to any one of claims 1 to 5 or a pharmaceutical composition according to claim 6 for restoring gastrointestinal epithelial barrier in a subject with damaged epithelium.

35. 35. The composition or pharmaceutical composition of claim 34, wherein the gastrointestinal epithelial barrier is restored by modulation of at least one WNT pathway molecule selected from the group consisting of genes associated with cell cycle, genes associated with stem and progenitor cell renewal and differentiation, genes associated with epithelial cell repair and barrier restoration, and / or any of the genes listed in any of Tables 4, 5, 6, 7, 8, and 11.

36. 36. The composition or pharmaceutical composition of claim 35, wherein the cell cycle related genes are selected from those provided in Table 4 or Aurka, Aurkb, Ccna2, Ccnb1, Ccnb2, Ccnd2, Ccne1, Cdc45, Cdk1, Cdkn3, Cenpm, Cenpp, Cenpq, Cenpu, Hells, Mcm4, Mcm5, Mcm6, Mcm7, Myc, Pbk, Plk1, Rrm1, and Rrm2.

37. 36. The composition or pharmaceutical composition of claim 35, wherein the genes associated with stem and progenitor cell renewal and differentiation are selected from those provided in Table 8, and Axin2, Id1, Hmga2, Nhp2, Foxq1, Hes6, and Adh1.

38. 36. The composition or pharmaceutical composition of claim 35, wherein the genes associated with epithelial cell repair and barrier restoration are selected from those provided in Table 6, or Apex1, Agr2, B3gnt7, Fcgbp, Muc2, Muc3, Tff3, Zg16, and Sprr2a3.

39. 39. The composition or pharmaceutical composition of any one of claims 35 to 38, wherein after administration of the composition or pharmaceutical composition, expression of the WNT pathway molecule in one or more tissues and / or cells of the subject is increased by at least 20%, at least 50%, at least 80%, at least 2-fold, at least 5-fold, at least 10-fold, or at least 20-fold, or is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%.

40. 40. The composition or pharmaceutical composition of claim 39, wherein expression of the WNT pathway molecule is increased in one or more tissues and / or cells of the subject within about 24 hours of administering the composition or pharmaceutical composition.

41. 41. The composition or pharmaceutical composition of any one of claims 34 to 40, wherein the subject's damaged epithelium is substantially repaired within about 6 days of administering the composition or pharmaceutical composition.

42. 42. The composition or pharmaceutical composition of any one of claims 34 to 41, wherein administration of said composition or pharmaceutical composition to said subject does not induce hyperproliferation of normal epithelium.

43. A composition comprising an engineered WNT agonist according to any one of claims 1 to 5 or a pharmaceutical composition according to claim 6 for inducing epithelial progenitor cell differentiation in a subject with a gastrointestinal disorder.

44. 44. The composition or pharmaceutical composition of claim 43, wherein the epithelial cell differentiation is induced by modulation of at least one WNT pathway molecule selected from the group consisting of genes associated with cell cycle, genes associated with stem and progenitor cell renewal and differentiation, genes associated with epithelial cell repair and barrier restoration, and / or any of the genes listed in any of Tables 4, 5, 6, 7, 8, and 11.

45. 45. The composition or pharmaceutical composition of claim 44, wherein the cell cycle related genes are selected from those provided in Table 4 or Aurka, Aurkb, Ccna2, Ccnb1, Ccnb2, Ccnd2, Ccne1, Cdc45, Cdk1, Cdkn3, Cenpm, Cenpp, Cenpq, Cenpu, Hells, Mcm4, Mcm5, Mcm6, Mcm7, Myc, Pbk, Plk1, Rrm1, and Rrm2.

46. 45. The composition or pharmaceutical composition of claim 44, wherein the genes associated with stem and progenitor cell renewal and differentiation are selected from those provided in Table 8, and Axin2, Id1, Hmga2, Nhp2, Foxq1, Hes6, and Adh1.

47. 45. The composition or pharmaceutical composition of claim 44, wherein the genes associated with epithelial cell repair and barrier restoration are selected from those provided in Table 6, or Apex1, Agr2, B3gnt7, Fcgbp, Muc2, Muc3, Tff3, Zg16, and Sprr2a3.

48. 48. The composition or pharmaceutical composition of any one of claims 44 to 47, wherein after administration of the composition or pharmaceutical composition, expression of the WNT pathway molecule in one or more tissues and / or cells of the subject is increased by at least 20%, at least 50%, at least 80%, at least 2-fold, at least 5-fold, at least 10-fold, or at least 20-fold, or is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%.

49. 49. The composition or pharmaceutical composition of claim 48, wherein expression of the WNT pathway molecule is increased in one or more tissues and / or cells of the subject within about 24 hours of administering the composition or pharmaceutical composition.

50. 50. The composition or pharmaceutical composition of any one of claims 43 to 49, wherein administration of the composition or pharmaceutical composition induces progenitor cell differentiation into enterocytes, goblet cells, enteroendocrine, or tuft cells in the subject.

51. 51. The composition or pharmaceutical composition of any one of claims 43 to 50, wherein substantial progenitor cell differentiation is induced in the subject within about 48 hours of administering the composition or pharmaceutical composition.

52. 52. The composition or pharmaceutical composition of any one of claims 43 to 51, wherein administration of said composition or pharmaceutical composition to said subject does not induce hyperproliferation of normal epithelium.

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