Modulation of WNT signaling in gastrointestinal disorders

Abstract

To provide a method for treating gastrointestinal disorders using a modified WNT agonist and a modulator of the WNT signaling pathway. [Solution] In one embodiment, the present disclosure includes an engineered WNT agonist 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, wherein the polypeptide sequence has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOs: 1 to 18, or a polypeptide sequence disclosed in any one of SEQ ID NOs: 1 to 25, Figure 2, Figure 6, Table 1, or Table 3, or a functional fragment or variant thereof, for example, its binding fragment, for example, 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 on 10 March 2021, U.S. Provisional Patent Application No. 63 / 190,535 filed on 19 May 2021, and U.S. Provisional Patent Application No. 63 / 247,151 filed on 22 September 2021, which are incorporated herein by reference in their entirety.

[0002] Sequence List This application was filed electronically via EFS-Web and includes an electronically submitted sequence listing in .txt format. The .txt file contains a sequence listing titled SRZN_020_03WO_ST25.txt, was created on March 7, 2022, and is 80 kilobytes in size. The sequence listing contained in this .txt file is part of this specification and is incorporated herein by reference in its entirety.

[0003] Field of Invention This disclosure provides a WNT signal modulator as a treatment for gastrointestinal disorders, particularly inflammatory bowel disease. [Background technology]

[0004] background WNT proteins form a family of highly conserved secreted signaling molecules that regulate intercellular interactions during embryogenesis. WNT genes and WNT signaling are also linked to cancer. Insights into the mechanisms of WNT action have arisen from several systems: genetics in Drosophila and Caenorhabditis elegans; and biochemistry in cell culture and ectopic gene expression in African clawed frog embryos. Many WNT genes are mutated in mice, leading to highly specific developmental defects. As is currently understood, WNT proteins bind to Frizzled family receptors on the cell surface. The signal is transmitted to betacatenin through several intracytoplasmic relay components, which then enter the nucleus and form a complex with TCF to activate the transcription of WNT target genes. WNT protein expression 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 hampered by the need for functional redundancy and conditional inactivation strategies. Dickkopf-1 (Dkk1) was recently identified as a founding family member of secreted proteins that strongly antagonistize 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 co-receptors LRP5 and LRP6, as well as the transmembrane protein Kremen. The resulting ternary complex leads to rapid internalization of LRP6 and impaired WNT signaling due to the absence of a functional Frizzled / LRP6 WNT receptor complex (see, for example, 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 Tcf locus knockout exhibit loss of proliferative stem cell compartments in the small intestine during late embryogenesis. However, the knockout is lethal and therefore has not been tested in adults. In chimeric transgenic mice, which allow for adult analysis, expression of constitutively active NH2 truncated β-catenin stimulated proliferation in the small intestinal crypts, but increased crypt apoptosis was also induced by either NH2 truncated β-catenin or Lef-1 / β-catenin fusions. The cause of the intestinal stem cell defect remains unclear, as β-catenin / Lef / Tcf-dependent transcription is regulated by a variety of factors, including non-Frizzled GPCRs and PTEN / PI-3 kinases.

[0007] Adult intestinal epithelium is characterized by the continuous replacement of epithelial cells through a typical cycle of cell division, differentiation, migration, and shedding that occurs during the crypt-villous transition time of 5–7 days. While putative growth factors regulating proliferation within the adult intestinal stem cell niche have not yet been fully identified, several studies have suggested a cell-specific role for β-catenin / Lef / Tcf signaling within the proliferative crypt compartment.

[0008] Many pathological conditions affect the cells of the intestines. Inflammatory bowel disease (IBD) involves either the small intestine, the large intestine, or both. Crohn's disease and ulcerative colitis are the best-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 structural changes such as crypt distortion and scarring. Cryptal abscesses can occur in many forms of IBD.

[0009] Crohn's disease can involve any part of the gastrointestinal tract, but most frequently the distal small intestine and colon. Inflammation is typically transthick, ranging from small ulcers (aphthous ulcers) across lymphoid follicles to deep fissuring ulcers leading to transmural scarring and chronic inflammation. One-third of cases have granulomas, and granulomas can also be present in external colonic sites such as lymph nodes, the liver, and joints. Transthick inflammation leads to the development of fistulas between the intestinal loop and other structures. Inflammation is typically segmental, with the affected areas of the intestine separated by the uninvolved parts. 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 further parts of the colon may be involved, sometimes spreading in a continuous pattern from the rectum proximally. The etiology of UC is unknown. Patients with long-term UC are at high risk of developing colon cancer. Patients with UC are also at risk of developing liver diseases, including sclerosing cholangitis and cholangiocarcinoma. Currently, all and most of the drugs used in clinics to treat UC focus on reducing inflammation and do not directly induce epithelial healing, which highlights the unaddressed need for therapies 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 [Overview of the Initiative] [Means for solving the problem]

[0011] Developing pharmacological agents to regulate intestinal epithelial growth is of clinical interest. However, the search for WNT agonists as pharmacological agents is hampered in part by the fact that WNT agonists are not naturally soluble and are diffusible molecules. This 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 epithelial-specific transient Wnt signaling activation, thereby driving robust epithelial regeneration and barrier restoration, ultimately leading to reduced inflammation and improvement of colitis.

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

[0013] In one embodiment, the present disclosure includes an engineered WNT agonist 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, wherein the polypeptide sequence has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOs: 1 to 18, or a polypeptide sequence disclosed in any one of SEQ ID NOs: 1 to 25, Figure 2, Figure 6, Table 1, or Table 3, or a functional fragment or variant thereof, for example, its binding fragment, for example, a VHH domain, a heavy chain variable domain, or a light chain variable domain. In certain embodiments, one or more binding domains that bind to one or more FZDs bind to 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 manipulated 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 the sequences disclosed in SEQ ID NOs: 1-18 or 19-25 or Table 3. In a particular embodiment, the manipulated 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 polypeptide sequences (e.g., two) having at least 90% or at least 95% homology to the lipeptide sequence and SEQ ID NO: 8; (h) One or more polypeptide sequences (e.g., two) having at least 90% or at least 95% homology to SEQ ID NO: 11 and one or more polypeptide sequences (e.g., two) having at least 90% or at least 95% homology to SEQ ID NO: 12; (i) One or more polypeptide sequences (e.g., two) having at least 90% or at least 95% homology to SEQ ID NO: 15 and one or more polypeptide sequences (e.g., two) having at least 90% or at least 95% homology to SEQ ID NO: 16;or (j) comprising 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 a CDR 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 to 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 LALAPG or N297G modification. In certain embodiments, the WNT agonist has any of the structures or formats disclosed herein, including any of the structures or formats related to various antibodies. Examples of suitable formats include 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, as long as they exhibit the desired biological activity, e.g., WNT agonist activity. In certain embodiments, the WNT agonist is R2M13-h26. R2M13 is a humanized form of the parent R2M13-26, also containing an LALAPG substitution in the Fc domain. R2M13-h26 may also be referred to herein as R2M13-h26-LALAPG, R2M13-26 humanized LALAPG, or humanized LALPG.

[0014] In a relevant aspect, the Disclosure provides pharmaceutical compositions comprising an engineered WNT agonist disclosed herein and a pharmaceutically acceptable carrier, diluent, or excipient.

[0015] In further relevant embodiments, the Disclosure provides a method for treating a disease or disorder suitable for treatment by enhanced WNT pathway signaling in a subject, the method comprising the step of 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 fistula formation, 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, subjects are administered approximately 0.5 to 100 mg of a WNT agonist per kg of body weight, or approximately 2 to 50 mg of a WNT agonist per kg of body weight, for example, approximately 2 mg / kg, approximately 3 mg / kg, approximately 4 mg / kg, approximately 5 mg / kg, approximately 10 mg / kg, approximately 15 mg / kg, approximately 20 mg / kg, approximately 25 mg / kg, approximately 30 mg / kg, approximately 35 mg / kg, approximately 40 mg / kg, approximately 45 mg / kg, or approximately 50 mg / kg of a WNT agonist. In certain embodiments, subjects are administered approximately 3 to 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 a disulfide bond.

[0016] In another related embodiment, the present disclosure provides a method for increasing WNT signaling in cells, comprising the step of contacting the cells with an engineered WNT agonist disclosed herein. In a particular embodiment, the WNT agonist is R2M13-h26.

[0017] In another related embodiment, the Disclosure provides a method for modulating the expression of WNT pathway molecules in one or more tissues and / or cells of a subject having a gastrointestinal disorder, comprising the step of 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 manipulated Wnt agonist, the expression of WNT pathway molecules (genes or proteins) in one or more tissues and / or cells of interest increases by at least 20%, at least 50%, at least 80%, at least 1.1 times, at least 1.2 times, at least 1.3 times, at least 1.4 times, at least 1.5 times, 2 times, at least 5 times, at least 10 times, or at least 20 times, or decreases by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%. In certain embodiments, the tissue is epithelial tissue. In certain embodiments, the cells are gastrointestinal epithelial cells, optionally stem cells, TA1, TA2, basal goblet cells, injury-induced alternative progenitor (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, subjects are administered approximately 0.5 to 100 mg of a WNT agonist per kg of body weight, or approximately 2 to 50 mg of a WNT agonist per kg of body weight, for example, approximately 2 mg / kg, approximately 3 mg / kg, approximately 4 mg / kg, approximately 5 mg / kg, approximately 10 mg / kg, approximately 15 mg / kg, approximately 20 mg / kg, approximately 25 mg / kg, approximately 30 mg / kg, approximately 35 mg / kg, approximately 40 mg / kg, approximately 45 mg / kg, or approximately 50 mg / kg of a WNT agonist. In certain embodiments, subjects are administered approximately 3 to 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 a disulfide bond.

[0018] In another related embodiment, the Disclosure provides a method for stimulating tissue repair in a subject having a gastrointestinal disorder, comprising the step of administering to the subject an engineered WNT agonist or pharmaceutical composition disclosed herein. In certain embodiments, tissue repair is stimulated (or the method brings about such modulation) by modulation of at least one WNT pathway molecule selected from the group consisting of genes related to the cell cycle, genes related to the regeneration and differentiation of stem cells and progenitor cells, genes related to the repair and barrier restoration of epithelial cells, and / or genes listed in any of Tables 4-8. In certain embodiments, the genes related to the cell cycle are those provided in Table 4, or selected from 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 genes related to the regeneration and differentiation of stem cells and progenitor cells are those provided in Table 8, as well as selected from Axin2, Id1, Hmga2, Nhp2, Foxq1, and Adh1. In certain embodiments, the genes associated with epithelial cell repair and barrier restoration are those provided in Table 6, or selected from Apex1, Agr2, B3gnt7, Fcgbp, Muc2, Muc3, Tff3, Zg16, and Sprr2a3. In certain embodiments, after administration of the manipulated Wnt agonist, the expression of the gene in one or more tissues and / or cells of interest is increased by at least 20%, at least 50%, at least 80%, at least 2x, at least 5x, at least 10x, or at least 20x, or decreased 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 a WNT agonist at about 0.5 to about 100 mg per kg of body weight, or at about 2 to about 50 mg per kg of body weight, such as a WNT agonist at 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. In certain embodiments, the subject is administered intravenously at least once per week with about 3 to about 30 mg of R2M13-h26 per kg of body weight, where 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 of reducing inflammation in a subject having a gastrointestinal disorder (or tissue or cells of the subject), the method comprising administering to the subject an engineered WNT agonist or pharmaceutical composition disclosed herein. In certain embodiments, the inflammation is reduced (or the method results in the modulation) by modulation of a gene provided in Table 5, or at least one WNT pathway molecule selected from the group consisting of Adamdec1, Atf3, Gpx2, Gsta3, Gstm1, Gstm3, Gdf15, Ihh, Il18, Lyz2, Nox1, Reg4, Sycn, Selenbp1, Tgfbr2, and Timp3. In certain embodiments, the inflammation is reduced in gastrointestinal tissue, optionally in epithelial tissue. In certain embodiments, the inflammation is in gastrointestinal epithelial cells, epithelial stem cells, TA1, TA2, basal goblet cells, injury-induced alternative progenitor cells (Alt progenitor), injury-induced alternative intestinal cells (Alt) It reduces WNT pathway molecules in Enterocytes, EnteroPrecur cells, Goblet cell 1, Goblet cell 2, or enteroendocrine cells or tuft cells. In certain embodiments, after administration of the engineered Wnt agonist, the expression of WNT pathway molecules in one or more tissues and / or cells of interest increases by at least 20%, at least 50%, at least 80%, at least 1.1 times, at least 1.2 times, at least 1.3 times, at least 1.4 times, at least 1.5 times, 2 times, at least 5 times, at least 10 times, or at least 20 times, or decreases 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, subjects are administered approximately 0.5 to 100 mg of a WNT agonist per kg of body weight, or approximately 2 to 50 mg of a WNT agonist per kg of body weight, for example, approximately 2 mg / kg, approximately 3 mg / kg, approximately 4 mg / kg, approximately 5 mg / kg, approximately 10 mg / kg, approximately 15 mg / kg, approximately 20 mg / kg, approximately 25 mg / kg, approximately 30 mg / kg, approximately 35 mg / kg, approximately 40 mg / kg, approximately 45 mg / kg, or approximately 50 mg / kg of a WNT agonist. In certain embodiments, subjects are administered approximately 3 to 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 a disulfide bond.

[0020] In any particular embodiment of the disclosed method, the manipulated Wnt agonist comprises R2M13-h26 or a functional variant or fragment thereof. In any particular embodiment of the disclosed method, the subject is a mammal, and optionally a human.

[0021] In another related embodiment, the Disclosure provides a method for restoring the gastrointestinal epithelial barrier in a subject having damaged epithelium, the method comprising the step of administering to the subject an engineered WNT agonist or pharmaceutical composition disclosed herein. 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, 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, subjects are administered R2M13-h26 intravenously at a dose of approximately 3 to 30 mg per kg of body weight 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 a disulfide bond. 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 related to the cell cycle, genes related to the regeneration and differentiation of stem cells and progenitor cells, genes related to the repair and barrier restoration of epithelial cells, and / or genes listed in any of Tables 4, 5, 6, 7, 8, and 11. Genes related to the cell cycle are provided in Table 4, or can be selected from 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 related to the regeneration and differentiation of stem cells and progenitor cells are provided in Table 8, as well as can be selected from Axin2, Id1, Hmga2, Nhp2, Foxq1, and Adh1.Genes associated with epithelial cell repair and barrier restoration can be those provided in Table 6 or can be selected from 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 after administration of the engineered Wnt agonist, the 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 decreased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%. In some embodiments, the gastrointestinal epithelial barrier is restored by modulation of at least one WNT pathway molecule, where within about 24 hours after administration of the engineered Wnt agonist, the expression of the WNT pathway molecule in one or more tissues and / or cells of the subject is increased. 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 the subject does not induce hyperproliferation of normal epithelium.

[0023] In another related embodiment, the Disclosure provides a method for inducing epithelial progenitor cell differentiation in a subject having a gastrointestinal disorder, comprising the step of administering to the subject an engineered WNT agonist or pharmaceutical composition disclosed herein. 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, 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, subjects are administered R2M13-h26 intravenously at a dose of approximately 3 to 30 mg per kg of body weight 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 a disulfide bond. In some embodiments, epithelial cell differentiation is induced by modulation of at least one WNT pathway molecule selected from the group consisting of genes related to the cell cycle, genes related to the regeneration and differentiation of stem cells and progenitor cells, genes related to the repair and barrier restoration of epithelial cells, and / or genes listed in any of Tables 4, 5, 6, 7, 8, and 11. Genes related to the cell cycle are provided in Table 4, or can be selected from 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 related to the regeneration and differentiation of stem cells and progenitor cells are provided in Table 8, as well as can be selected from Axin2, Id1, Hmga2, Nhp2, Foxq1, and Adh1.Genes associated with epithelial cell repair and barrier restoration are those provided in Table 6, or can be selected from 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, after administration of the engineered Wnt agonist, the expression of the WNT pathway molecule in one or more tissues and / or cells of the subject increases 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 decreases by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%. In some embodiments, epithelial cell differentiation is induced by modulation of at least one WNT pathway molecule, where, within approximately 24 hours after administration of the engineered Wnt agonist, the expression of the WNT pathway molecule in one or more tissues and / or cells of the subject increases.

[0025] In some embodiments, administration of a manipulated Wnt agonist induces progenitor cell differentiation into intestinal cells, goblet cells, enteroendocrine cells, or tuft cells in the subject. In some embodiments, substantial progenitor cell differentiation is induced in the subject within approximately 48 hours of administration of the manipulated Wnt agonist. In some embodiments, administration of a manipulated Wnt agonist does not induce excessive proliferation of normal epithelium in the subject. In embodiments of the present invention, for example, the following items are provided. (Item 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 A modified WNT agonist containing, The manipulated WNT agonist comprises a polypeptide sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with any of SEQ ID NOs: 1 to 25, or a polypeptide sequence disclosed in Figure 2, Figure 6, Table 1, or Table 3, or a conjugated fragment thereof. If necessary, one or more binding domains that bind to one or more FZDs 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 A manipulated WNT agonist that binds to it. (Item 2) (a) A polypeptide sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with any one of Sequence IDs 1 to 25, or a sequence disclosed in Table 3; or (b) A polypeptide sequence containing two or three CDR sequences located in either the VHH domain, VH domain, or VL domain disclosed in Figure 2. Includes, If necessary, the manipulated WNT agonist according to item 1, comprising a CDR in which the polypeptide sequence is present in any one of sequence numbers 1 to 25. (Item 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) polypeptide sequences having at least 90% or at least 95% homology to SEQ ID NO: 3 and polypeptide sequences having at least 90% or at least 95% homology to SEQ ID NO: 4; (c) polypeptide sequences having at least 80%, at least 90%, or at least 95% homology to SEQ ID NO: 5 and polypeptide sequences having at least 80%, at least 90%, or at least 95% homology to SEQ ID NO: 6; (d) polypeptide sequences having at least 90% or at least 95% homology to SEQ ID NO: 7 and polypeptide sequences having at least 90% or at least 95% homology to SEQ ID NO: 8; (e) polypeptide sequences having at least 90% or at least 95% homology to SEQ ID NO: 9 and polypeptide sequences 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 Includes, If necessary, the manipulated WNT agonist described in item 2, wherein the polypeptide contains a CDR present in any one of sequence numbers 1 to 18. (Item 4) An engineered WNT agonist according to any one of items 1 to 3, wherein one or more binding domains that bind to LRP5, LRP6, or both LRP5 and LRP6 are humanized. (Item 5) An operated WNT agonist according to any one of items 1 to 4, comprising a modified Fc domain, wherein the modified Fc domain comprises a LALAPG or N297G modification. (Item 6) A pharmaceutical composition comprising an engineered WNT agonist as described in any one of items 1 to 5, and a pharmaceutically acceptable carrier, diluent, or excipient. (Item 7) A method for treating a disease or disorder suitable for treatment by increased WNT pathway signaling in a subject, comprising the step of administering to the subject an engineered WNT agonist according to any one of items 1 to 5 or a pharmaceutical composition according to item 6. (Item 8) The method according to item 7, wherein the disease or disorder is a gastrointestinal disorder. (Item 9) The method according to item 8, wherein the gastrointestinal disorder is inflammatory bowel disease. (Item 10) The method according to item 9, wherein the inflammatory bowel disease is selected from the group consisting of Crohn's disease (CD), CD with fistula formation, and ulcerative colitis (UC). (Item 11) The method according to any one of items 7 to 10, wherein the manipulated WNT agonist is administered orally or parenterally. (Item 12) The method according to item 11, wherein the manipulated WNT agonist is administered intravenously, intraperitoneally, or subcutaneously. (Item 13) A method for increasing WNT signaling in cells, comprising the step of contacting the cells with an engineered WNT agonist described in any one of items 1 to 5. (Item 14) A method for modulating the expression of WNT pathway molecules in one or more tissues or cells in a subject having a gastrointestinal disorder, comprising the step of administering to the subject an engineered WNT agonist according to any one of items 1 to 5 or a pharmaceutical composition according to item 6. (Item 15) The method according to item 14, wherein the WNT pathway molecule is a gene or protein listed in any one of Tables 4, 5, 6, 7, 8, and 11. (Item 16) The method according to item 14, wherein the WNT pathway molecule is selected from the group consisting of glutathione peroxidase 2 (Gpx2), interferon regulator 8 (Irf8), Rel, RelA, RelB, RNAse4, angiogenin, Gsta3, Rnf43, Axin2, Ki67, occurdin, or any of the genes or proteins listed in Table 7. (Item 17) After administration, the expression of the WNT pathway molecule in one or more tissues and / or cells of the subject increases by at least 20%, at least 50%, at least 80%, at least 2x, at least 5x, at least 10x, or at least 20x, or by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or A method described in any one of items 14 to 16, which reduces by 90%. (Item 18) The method according to any one of items 14 to 17, wherein the tissue is epithelial tissue and / or the cells are gastrointestinal epithelial cells, optionally stem cells, TA1, TA2, goblet cell progenitor, injury-induced alternative progenitor (Alt progenitor), injury-induced alternative enterocyte (Alt Enterocyte), intestinal progenitor (EnteroPrecur), goblet cell progenitor (goblet_PC), goblet cell 1, goblet cell 2, or enteroendocrine cells. (Item 19) A method for stimulating tissue repair in a subject with a gastrointestinal disorder, comprising the step of administering to the subject an engineered WNT agonist according to any one of items 1 to 5 or a pharmaceutical composition according to item 6. (Item 20) The method according to item 19, wherein the tissue repair is stimulated by the modulation of at least one WNT pathway molecule selected from the group consisting of genes related to the cell cycle, genes related to the regeneration and differentiation of stem cells and progenitor cells, genes related to the repair and barrier restoration of epithelial cells, and / or genes listed in any of Tables 4, 5, 6, 7, 8, and 11. (Item 21) The method according to item 20, wherein the cell cycle-related genes are those provided in Table 4, or selected from 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. (Item 22) The method according to item 20, wherein the genes related to the regeneration and differentiation of the aforementioned stem cells and progenitor cells are those provided in Table 8, and selected from Axin2, Id1, Hmga2, Nhp2, Foxq1, Hes6, and Adh1. (Item 23) The method according to item 20, wherein the genes related to the repair and restoration of the epithelial cells are those provided in Table 6, or selected from Apex1, Agr2, B3gnt7, Fcgbp, Muc2, Muc3, Tff3, Zg16, and Sprr2a3. (Item 24) The method according to any one of items 20 to 23, wherein, after administration of the manipulated Wnt agonist, the 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 decreased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%. (Item 25) A method for reducing inflammation in a subject having a gastrointestinal disorder, comprising the step of administering to the subject an engineered WNT agonist according to any one of items 1 to 5 or a pharmaceutical composition according to item 6. (Item 26) The method according to item 19, wherein the inflammation is reduced by the modulation of a gene provided in Table 5, or at least one molecule selected from the group consisting of Adamdec1, Atf3, Gpx2, Gsta3, Gstm1, Gstm3, Gdf15, Ihh, Il18, Lyz2, Nox1, Reg4, Sycn, Selenbp1, Tgfbr2, and Timp3. (Item 27) The method according to item 25 or item 26, wherein the inflammation is reduced in gastrointestinal tissue, and epithelial tissue if necessary. (Item 28) The method according to item 27, wherein the gastrointestinal tissue comprises gastrointestinal epithelial cells, optionally stem cells, TA1, TA2, goblet cell progenitor, injury-induced alternative progenitor (Alt progenitor), injury-induced alternative enterocyte (Alt Enterocyte), intestinal progenitor (EnteroPrecur), goblet cell progenitor (goblet_PC), goblet cell 1, goblet cell 2, or enteroendocrine cells. (Item 29) The method according to any one of items 25 to 28, wherein, after administration, the expression of the WNT pathway molecule in one or more tissues and / or cells of the subject increases 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 decreases by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%. (Item 30) The method according to any one of items 7 to 29, wherein the manipulated Wnt agonist is R2M13-h26, or comprises a functional variant or fragment thereof. (Item 31) A method for generating, culturing, or maintaining organs, tissues, cells, or organoid cultures, wherein the organs, tissues, cells, or organoid cultures a) A modified WNT agonist as described in any one of items 1 through 5; or b) Pharmaceutical compositions described in item 6 A method including the step of bringing into contact with. (Item 32) a) A step of ex vivo contacting an organ or tissue obtained from a donor with a composition containing the manipulated WNT agonist or the pharmaceutical composition, by perfusion as necessary; or b) The method of item 31 for maintaining the viability of a donor organ or tissue ex vivo, comprising the step of contacting the donor organ or tissue in vivo with a composition comprising the manipulated WNT agonist or the pharmaceutical composition. (Item 33) The method according to item 31 for generating or maintaining the organoid culture, comprising, if necessary, the step of contacting the organoid culture by culturing it in a medium containing the manipulated WNT agonist. (Item 34) A method for restoring the gastrointestinal epithelial barrier in a subject having damaged epithelium, comprising the step of administering to the subject an engineered WNT agonist according to any one of items 1 to 5 or a pharmaceutical composition according to item 6. (Item 35) The method according to item 34, wherein the gastrointestinal epithelial barrier is restored by modulation of at least one WNT pathway molecule selected from the group consisting of genes related to the cell cycle, genes related to the regeneration and differentiation of stem cells and progenitor cells, genes related to the repair and barrier restoration of epithelial cells, and / or genes listed in any of Tables 4, 5, 6, 7, 8, and 11. (Item 36) The method according to item 35, wherein the cell cycle-related genes are those provided in Table 4, or selected from 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. (Item 37) The method according to item 35, wherein the genes related to the regeneration and differentiation of the aforementioned stem cells and progenitor cells are those provided in Table 8, and selected from Axin2, Id1, Hmga2, Nhp2, Foxq1, Hes6, and Adh1. (Item 38) The method according to item 35, wherein the genes related to the repair and restoration of the epithelial cells are those provided in Table 6, or selected from Apex1, Agr2, B3gnt7, Fcgbp, Muc2, Muc3, Tff3, Zg16, and Sprr2a3. (Item 39) The method according to any one of items 35 to 38, wherein, after administration of the manipulated Wnt agonist, the 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 decreased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%. (Item 40) The method according to item 39, wherein the expression of the WNT pathway molecule in one or more tissues and / or cells of the subject is increased within approximately 24 hours after administration of the manipulated Wnt agonist. (Item 41) The method according to any one of items 34 to 40, wherein the damaged epithelium of the subject is substantially restored within approximately 6 days after administration of the manipulated Wnt agonist. (Item 42) The method according to any one of items 34 to 41, wherein administering the manipulated Wnt agonist to the subject does not induce excessive proliferation of normal epithelium. (Item 43) A method for inducing epithelial progenitor cell differentiation in a subject having a gastrointestinal disorder, comprising the step of administering to the subject an engineered WNT agonist according to any one of items 1 to 5 or a pharmaceutical composition according to item 6. (Item 44) The method according to item 43, wherein the epithelial cell differentiation is induced by modulation of at least one WNT pathway molecule selected from the group consisting of genes related to the cell cycle, genes related to the regeneration and differentiation of stem cells and progenitor cells, genes related to the repair and barrier restoration of epithelial cells, and / or genes listed in any of Tables 4, 5, 6, 7, 8, and 11. (Item 45) The method according to item 44, wherein the cell cycle-related genes are those provided in Table 4, or selected from 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. (Item 46) The method according to item 44, wherein the genes related to the regeneration and differentiation of the aforementioned stem cells and progenitor cells are those provided in Table 8, and selected from Axin2, Id1, Hmga2, Nhp2, Foxq1, Hes6, and Adh1. (Item 47) The method according to item 44, wherein the genes related to the repair and restoration of the epithelial cells are those provided in Table 6, or selected from Apex1, Agr2, B3gnt7, Fcgbp, Muc2, Muc3, Tff3, Zg16, and Sprr2a3. (Item 48) The method according to any one of items 44 to 47, wherein, after administration of the manipulated Wnt agonist, the 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 decreased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%. (Item 49) The method according to item 48, wherein the expression of the WNT pathway molecule in one or more tissues and / or cells of the subject is increased within approximately 24 hours after administration of the manipulated Wnt agonist. (Item 50) The method according to any one of items 43 to 49, wherein administration of the manipulated Wnt agonist induces precursor cell differentiation into intestinal cells, goblet cells, enteroendocrine cells, or tuft cells in the subject. (Item 51) The method according to any one of items 43 to 50, wherein substantial progenitor cell differentiation is induced in the subject within approximately 48 hours after administration of the manipulated Wnt agonist. (Item 52) The method according to any one of items 43 to 51, wherein administering the manipulated Wnt agonist to the subject does not induce excessive proliferation of normal epithelium. [Brief explanation of the drawing]

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

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

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

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

[0030] [Figure 5] Figures 5A-5D show the in vitro activity of the Fzd5,8 subfamily-specific Wnt mimetic R2M13-26. Figure 5A is a graph showing the binding affinity of the R2M13-26 Fzd5,8 binding factor IgG to its target Fzd5 CRD, as measured by Octet. Figure 5B is a graph showing the binding affinity of the R2M13-26 Fzd5,8 binding factor IgG to its target Fzd8 CRD, as measured by Octet. Figure 5C is a graph showing the binding specificity of the R2M13-26 Fzd5,8 binding factor IgG to each of the 10 Fzd CRDs, as investigated by Octet. Figure 5D is a graph showing the dose-dependent STF activity of R2M13-26, Fzd1,2,7-specific mimetic 1RC07-26, and Fzd1,2,5,7,8-generate specific mimetic R2M3-26 in the presence of 20 nM RSPO2, as measured in Huh-7 cells.

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

[0032] [Figure 7] Figure 7 shows a schematic diagram of the DSS model for acute colitis, as well as serum antibody exposures resulting after treatment with various dehumanized and humanized versions, including R2M13-03-LALAPG (dehumanized), R2M13-26-LALAPG (dehumanized), R2M13-36-LALAPG (dehumanized), R2M13-humanized-03-LALAPG, R2M13-humanized-26-LALAPG, R2M13-humanized-36-LALAPG, R2M13-humanized-03-N297G, and R2M13-humanized-36-N297G.

[0033] [Figure 8] Figure 8 provides a graph showing disease activity indices in animals treated with various dehumanized and humanized versions, including R2M13-03-LALAPG (dehumanized), R2M13-26-LALAPG (dehumanized), R2M13-36-LALAPG (dehumanized), R2M13-humanized-03-LALAPG, R2M13-humanized-26-LALAPG, R2M13-humanized-36-LALAPG, R2M13-humanized-03-N297G, and R2M13-humanized-36-N297G. As of day 10, the lines on the graph from top to bottom correspond to the following: 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]Figure 9 provides a graph showing cytokine levels in animals treated with various controls, including DSS-free, 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, as well as non-humanized and humanized versions, from left to right.

[0035] [Figure 10] Figure 10 provides a graph showing the levels of lipocalin 2 in animals treated with various controls and dehumanized and humanized versions, including DSS-free, anti-GFP, parental R2M13-03-LALAPG (dehumanized), parental R2M13-26-LALAPG (dehumanized), parental R2M13-36-LALAPG (dehumanized), 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 a micrograph showing the restoration of ZO-1, an epithelial tight junction marker, in vivo after treatment with a modified WNT agonist in a DSS model of acute colitis. The brightly stained area represents ZO-1.

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

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

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

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

[0041] [Figure 16] Figure 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 10 days, the lines in the graph, from top to bottom, correspond to: anti-GFP, cyclosporine A, R2M13-h26 (2 mpk × 1), R2M13-h26 (20 mpk × 1), R2M13-h26 (1 mpk × 2), R2M13-h26 (6 mpk × 1), R2M13-26 (3 mpk × 2), R2M13-h26 (10 mpk × 2), R2M13-26 (10 mpk × 2), and no DSS.

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

[0043] [Figure 18] Figure 18 provides a diagram of an animal model of chronic DSS colitis.

[0044] [Figure 19] Figure 19 shows a micrograph of a transverse colon section after the procedure described.

[0045] [Figure 20] Figure 20 provides a graph showing the histological score and overall disease activity index after the indicated treatment.

[0046] [Figure 21] Figure 21 provides a graph showing the expression of lipocalin-2 and IL-6 after the treatments shown.

[0047] [Figure 22] Figure 22 shows a diagram of an animal model of chronic DSS colitis.

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

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

[0050] [Figure 25] Figure 25 provides a graph showing the expression of Axin2 and Ki67 after treatment with R2M13-26-LALAPG (R2M13-26).

[0051] [Figure 26-1]Figures 26A–26C show the different cell types detected in the colon by scRNA-seq for uninjured mice and DSS-treated mice: Figure 26A is a schematic diagram showing the experimental design of the scRNA-seq experiment. Figure 26B is a plot of the first two principal components: lineage / tissue layers are shown, with three groups radiating from the center. Figure 26C provides a graph showing the strong effect of DSS injury on numerous differentially expressed genes 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 uninjured mice on days 5 and 6; the graph on the right shows the number of differentially expressed genes from each tissue layer treated with R2M13-26 compared to anti-GFP on days 5 and 6. The tissues / lineages correspond to epithelium, immune tissue, and stroma from top to bottom on each rod, and on day 5, almost all are epithelium treated with R2M13-26-LALAPG (R2M13-26). [Figure 26-2] Same as above.

[0052] [Figure 27-1]Figures 27A-27C show that the effects of DSS extend to all tissue layers by day 5, while the primary effect of R2M13-26-LALAPG (R2M13-26) at day 5, 24 hours after 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 post-injury epithelium. Figure 27A is a table listing selected top gene sets (from GSEA) enriched in DSS-injury epithelium treated with R2M13-26 compared to DSS-injury epithelium treated with anti-GFP. Figures 27B and 27C show validation of scRNA-seq analysis in tissue. 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 the proliferative cell marker MKI67 in colon samples (day 6) treated with non-injured, DSS / anti-GFP, and DSS / R2M13-26; nuclei labeled with DAPI. Scale bar represents 100 microns. [Figure 27-2] Same as above.

[0053] [Figure 28-1]Figures 28A–28E show that in the DSS model, R2M13-26-LALAPG (R2M13-216) treatment induced accelerated and appropriate differentiation: Figures 28A–D provide graphs showing homogeneous manifold approximation projection (UMAP) plots of epithelial cells. Figure 28A is a graph showing the UMAP of epithelial cells color-coded by cluster / cell type. Figure 28B is a graph showing the UMAP color-coded by experimental conditions of cells. Figure 28C is a graph showing the minimum spanning tree of cluster medoids, where clusters are connected based on similarity. Only cell types in which damaged cells were almost exclusively absent were included. Stem cells and TA2 cell types were integrated and set as the starting cluster. Figure 28D is a graph showing the completed slingshot predicted slingshot trajectory, illustrating the transition from stem cells / TA cells to EnteroPrecur cells (upward) during the transition to immature and mature intestinal cells; as well as the branching from stem cells / TA cells downward towards either brush cells or goblet cells and enteroendocrine cells, with a second branching from the goblet progenitor cell type between them. Figure 28E provides a histogram of the number of cells from the indicated treatment group at the indicated positions along the pseudo-time series or slingshot trajectory axis derived from the intestinal cell lineage shown in Figure 28D, at 48 hours / day 6. The vertical red dashed lines indicate the same positions 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. Figure 28E shows that progression toward the intestinal cell lineage was increased with the 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 differential expression patterns of Frizzled family receptors in the small intestinal epithelium. Figures 29A–29L provide graphs showing the expression of 10 Fzd receptors (Fzd1–10), Axin2, and Lgr5 in a normal duodenum, as determined by RNAscope in situ hybridization, respectively. Figures 29A'–29L' provide enlarged graphs of photographs showing Fzd expression in the small intestinal crypts. The arrows in panel E' indicate intestinal stem cells.

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

[0056] [Figure 31] Figure 31 shows the reduction in inflammation due to a reduction in neutrophil infiltration. S100A9 is a marker of neutrophil infiltration, and CD45 is a marker of activated inflammatory cells.

[0057] [Figure 32] Figure 32 provides a graph showing the increase in serum ALP after administration of the indicated dose of R2M13-h26.

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

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

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

[0061] [Figure 36] Figures 36A and 36B provide pairs of graphs showing the increase in ALP from days 0 to 7 (Figure 36A) and from days 28 to 42 (Figure 36B) for different dosage groups during R2M13-h26.

[0062] [Figure 37] Figure 37 provides a graph showing the mean serum R2M13-h26 concentration after a single dose of R2M13-h26.

[0063] [Figure 38] Figure 38 provides a table showing the PK parameters for R2M13-h26 after a single dose. [Modes for carrying out the invention]

[0064] Detailed explanation As used herein, including in the attached claims, singular words such as “a,” “an,” and “the” encompass multiple corresponding references unless the context clearly indicates otherwise.

[0065] All references cited herein are incorporated by reference to the same extent as individual publications, patent applications, or patents are incorporated by reference specifically and individually as indicated in those publications.

[0066] I. Definition The “activity” of a molecule may describe or refer to its ability to bind to a ligand or receptor, for example, for catalytic activity, for its ability to stimulate gene expression, for antigenic activity, or for modulating the activity of other molecules. The “activity” of a molecule may also refer to its activity in intercellular interactions, such as activity in modulating or maintaining adhesion, or in maintaining cellular structures, such as the cell membrane or cytoskeleton. “Activity” may also mean specific activity, such as [catalytic activity] / [protein 1 mg] or [immunological activity] / [protein 1 mg].

[0067] The terms “administer,” “introduce,” or “deliver,” as used herein, mean delivering a composition to one cell, multiple cells, tissue, tissue organoid, and / or organ, or subject. Such administration or introduction may be carried out in vivo, in vitro, or ex vivo.

[0068] As used herein, the term “antibody” means an isolated or recombinant conjugate containing a variable region sequence necessary for specific binding to an antigen epitope. Therefore, an antibody is any form of antibody or fragment thereof that exhibits a desired biological activity, e.g., binding to a specific target antigen. Hence, the term “antibody” is used in its broadest sense, specifically covering monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, VHH antibodies, camel antibodies, nanobodies, diabodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, including but not limited to scFv, Fab, and Fab2, if they exhibit the desired biological activity.

[0069] An "antibody fragment" is a portion of an intact antibody, such as the antigen-binding region or variable region of an 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 polyspecific antibodies formed from antibody fragments. Papain digestion of an antibody produces two identical antigen-binding fragments, called "Fab" fragments, each having a single antigen-binding site, and the remaining "Fc" fragment (a designation reflecting its ability to easily crystallize). Pepsin treatment produces an F(ab')2 fragment, which has two antigen-binding sites and can still crosslink antigens.

[0070] The term "antigen" refers to a molecule or part of a molecule that can be used in an animal to produce an antibody that can be bound to a selective binder, such as an antibody, and that can further bind to the epitope of that antigen. In certain embodiments, a binder (e.g., an engineered WNT agonist or its binding region, or a WNT antagonist) can be said to bind specifically 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 immunoglobulin heavy and / or light chains, or Nanobody® (Nab), that binds to the antigen of interest, in particular one or more FZD receptors, or LRP5 and / or LRP6. In this regard, the antigen-binding fragments of antibodies described herein may include all one, two, three, four, five, or six CDRs of VH and VL derived from the antibody that binds to one or more FZD receptors or LRP5 and / or LRP6.

[0072] As used herein, the terms “bioactive” and “biologically active” refer to activity attributable to a particular biological element within a cell. For example, the “bioactive” of a WNT agonist, or a fragment or variant thereof, refers to its ability to mimic or enhance WNT signaling. As another example, the bioactive 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 or enzymatic activity. In some embodiments, the 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 bioactive of a gene regulatory element, such as a promoter, enhancer, or Kozak sequence, refers to the ability of the regulatory element or a functional fragment or variant thereof to regulate the expression of the gene to which it is operably linked, i.e., to promote, enhance, or activate the translation of that gene, respectively.

[0073] As used herein, the term "bifunctional antibody" refers to an antibody comprising a first arm having specificity for one antigen site and a second arm having specificity for a different antigen site, i.e., a bifunctional antibody having bispecificity.

[0074] "Bispecific antibodies" are, in this specification, obtained 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 introducing mutations into the Fc region, thereby resulting in multiple different immunoglobulin species, one of which is a functional bispecific antibody, using knob-into-hole or similar methods (see Holliger et al., The term is used to refer to full-length antibodies produced by (see Proc. Natl. Acad. Sci. USA, 90 (14): 6444-6448 (1993)). A bispecific antibody binds to one antigen (or epitope) on one of its two binding arms (one HC / LC pair) and to a different antigen (or epitope) on the second arm (a different HC / LC pair). According to this definition, a bispecific antibody has two distinct antigen-binding arms (in terms of both specificity and CDR sequence) and is monovalent for each antigen it binds to.

[0075] "Includes" means that the listed elements are required for, for example, a composition, method, kit, etc., but other elements may be included, for example, to form a composition, method, kit, etc. within the scope of the claims. For example, an expression cassette "including" 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, such as polyadenylated sequences, enhancer elements, other genes, linker domains, etc.

[0076] "Essentially derived from" means that the scope of the described, for example, composition, method, kit, etc., is limited to specified materials or steps that do not substantially affect the basic and novel features(s) of the composition, method, kit, etc. For example, an expression cassette "essentially derived from" a gene encoding a therapeutic polypeptide operably linked to a promoter and a polyadenylation sequence may include further sequences, such as linker sequences, provided that they do not substantially affect the transcription or translation of the gene. As another example, a variant, or mutant, polypeptide fragment "essentially derived from" an enumerated sequence has approximately 10 amino acid residues plus or minus the amino acid sequence of the enumerated sequence at the boundary of the sequence based on the full-length naive polypeptide from which it is derived, for example, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residue less than the enumerated binding amino acid residues, or 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 residues more than the enumerated binding amino acid residues.

[0077] "Consists of" means that any element, step, or component not explicitly stated in the claim is excluded from the composition, method, or kit. For example, a polypeptide or polypeptide domain "consists of" the listed sequences contains only the listed sequences.

[0078] A “regulatory element” or “regulatory sequence” is a nucleotide sequence involved in molecular interactions that contribute to the regulation of polynucleotide function, including replication, duplication, transcription, splicing, translation, or degradation. This regulation may affect the frequency, rate, or specificity of a process and may be either enhancing or inhibiting. Regulatory 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 to RNA polymerase and initiate transcription of a coding region typically located downstream (3' direction) of the promoter.

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

[0080] The portion of an antibody or antibody fragment that recognizes an epitope is called the "epitope-binding domain" or "antigen-binding domain." The epitope-binding domain of an antibody or antibody fragment is located in the Fab fragment, while the effector function is located in the Fc fragment. Six segments known as complementarity-determining regions (CDRs) within the variable regions (VH and VL) of the heavy and light chains form loops and interact with the remaining framework (FR region) globular structure of the antibody 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, but sometimes fewer CDRs can provide the primary binding motif.

[0081] An "expression vector" is a vector containing a region encoding a gene product of interest, such as a plasmid, minicircle, viral vector, liposome, etc., as discussed herein or known in the art, used to induce the expression of the gene product in intended target cells. Expression vectors also include regulatory elements operably ligated to the coding region to facilitate the expression of the gene product in the target, such as promoters, enhancers, UTRs, and miRNA targeting sequences. The combination of regulatory elements and the gene(s) operably ligated for expression is sometimes called an "expression cassette," many of which are known, available, or readily constructed from components available in the art.

[0082] As used herein, the term “FR set” refers to the four adjacent amino acid sequences that frame the CDR of a CDR set in a heavy or light chain V region. Some FR residues can come into contact with the bound antigen; however, FRs, particularly those directly adjacent to the CDR, are primarily responsible for folding the V region into the antigen-binding site. Within the FR, 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 CDR is displayed as a protruding loop motif that forms the antigen-binding surface. It is generally understood that there are conserved structural regions in the FR that influence the folding shape of the CDR loop to be a certain “standard” structure, regardless of the exact CDR amino acid sequence. Furthermore, certain FR residues are known to be involved in non-covalent interdomain contacts that stabilize the interaction between the heavy and light chains of the antibody.

[0083] A "humanized" antibody or fragment refers to an antibody or fragment of a non-human species whose protein sequence has been modified to increase its similarity to antibody variants naturally produced in humans. The "humanization" process 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 human and non-human primates, including monkeys and humans; mammalian sports 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 antibodies" refer to a homogeneous population of antibodies composed of amino acids (naturally occurring and non-naturally occurring) involved in the selective binding of epitopes. Monoclonal antibodies are highly specific and target a single epitope. The term "monoclonal antibody" encompasses not only intact and full-length monoclonal antibodies, but also their fragments (e.g., Fab, Fab', F(ab')2, Fv), single chains (scFv), Nanobodies®, their variants, fusion proteins containing antigen-binding fragments of monoclonal antibodies, humanized monoclonal antibodies, chimeric monoclonal antibodies, and any other modified configurations of immunoglobulin molecules containing antigen-binding fragments (epitope-recognition sites) with the required specificity and ability to bind to epitopes, including engineered WNT agonists disclosed herein. It is not intended to be limited in terms of the source of the antibody or the method of antibody production (e.g., by hybridoma, phage selection, recombinant expression, transgenic animals, etc.). This term encompasses immunoglobulins as a whole, as well as fragments that fall under the definition of "antibody" as described herein.

[0086] The terms “native” or “wild-type,” as used herein, refer to a nucleotide sequence, e.g., a gene or gene product, e.g., RNA or protein, present in wild-type cells, tissues, organs, or organisms. 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 the reference polynucleotide or polypeptide sequence. In other words, a variant contains at least one amino acid difference (e.g., an amino acid substitution, an amino acid insertion, or an amino acid deletion) compared to the reference polynucleotide sequence, e.g., a native polynucleotide or polypeptide sequence. For example, a variant may be a polynucleotide having 50% or more, 60% or more, or 70% or more sequence identity with the full-length native polynucleotide sequence, e.g., 75% or 80% or more, e.g., 85%, 90% or 95% or more identity with the full-length native polynucleotide sequence, e.g., 98% or 99% identity. In another example, a variant may be a polypeptide having 70% or more sequence identity with the full-length native polypeptide sequence, e.g., 75% or 80% or more identity with the full-length native polypeptide sequence, e.g., 85%, 90%, or 95% or more identity, e.g., 98% or 99% identity. A variant may also include a variant fragment of a reference, e.g., the native sequence, that shares 70% or more sequence identity with a reference, e.g., the native sequence fragment, e.g., 75% or 80% or more identity with the native sequence, e.g., 85%, 90%, or 95% or more identity, e.g., 98% or 99% identity.

[0087] "Operationally linked" or "operationally linked" refers to the juxtaposition of gene elements, where these elements are related in a way that allows them to function in the expected manner. For example, if a promoter helps initiate transcription of a coding sequence, the promoter is operationally linked to the coding region. There may be intervening residues between the promoter and the coding region, as long as 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, for example, to involve disulfide bond formation, glycosylation, lipidation, phosphorylation, or conjugation with a labeling component.

[0089] The term “polynucleotide” refers to a nucleotide in a multimeric form of any length, including either a deoxyribonucleotide or a ribonucleotide, or an analog thereof. Polynucleotides may include modified nucleotides, such as methylated nucleotides and nucleotide analogs, and non-nucleotide components may be interposed in the polynucleotide. Where present, modifications to the nucleotide structure may be made before or after the assembly of the polymer. As used herein, the term polynucleotide refers to mutually interchangeable 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 two complementary single-stranded forms that are known to or are expected to constitute the double-stranded form.

[0090] A polynucleotide or polypeptide has a certain percentage of “sequence identity” with respect to another polynucleotide or polypeptide, where “sequence identity” means that when the two sequences are aligned and then compared, the percentage of bases or amino acids are the same. As used herein, the terms “identity” and “identity” refer to the percentage of residues that perfectly match in the alignment of the target polypeptide or polynucleotide sequence with its reference sequence, such as the alignment produced by the BLAST algorithm. Identity is calculated over the entire length of the reference sequence unless otherwise specified. Thus, the target sequence “shares at least x% identity” with the reference sequence if, when the reference sequence (as the query sequence) is aligned with the target sequence (as the subject sequence), at least x% (rounded down) of residues in the subject sequence align as an exact match to the corresponding residues in the query sequence, where the denominator is the entire length of the reference sequence plus the length of any gaps inserted into the reference sequence by the alignment of the reference sequence and the target sequence. If the subject sequence has a variable position (e.g., a residue indicated by X), any alignment with any 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, which is available via 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 yields the same results. Unless otherwise instructed, sequence identity is determined using the BLAST algorithm (e.g., bl2seq) with default parameters.

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

[0093] The goal is to provide a BestFit program that uses the local homology algorithm of Smith and Waterman (Advances in Applied Mathematics 2: 482-489 (1981)) to determine sequence identity. The gap generation penalty is generally in the range of 1 to 5, usually 2 to 4, but in many embodiments it is 3. The gap expansion penalty is generally in the range of about 0.01 to 0.20, but in many examples it is 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] When used herein, “promoter” encompasses a DNA sequence, i.e., the smallest sequence sufficient to direct transcription, that directs the binding of RNA polymerase and thereby promotes RNA synthesis. Promoter and corresponding protein or polypeptide expression may be universal, meaning strongly active in a wide range of cells, tissues, and species, or the promoter and expression may be cell type-specific, tissue-specific, or species-specific. Promoter may be “constitutive,” meaning continuously active, or promoter may be “inducible,” meaning that the promoter can be activated or inactivated by the presence or absence of biofactors or non-biological factors. Enhancer sequences, which may or may not be contiguous with promoter sequences, are also included in the nucleic acid constructs or vectors of the present invention. Enhancer sequences may affect promoter-dependent gene expression and may be located in the 5' or 3' region of the native gene.

[0096] When applied to polynucleotides, "recombinant" means that the polynucleotide is the product of a diverse combination of cloning, restriction, or ligation steps and other procedures that result in a construct distinct from naturally occurring polynucleotides.

[0097] The terms “treatment,” “to treat,” etc., are used herein to generally mean obtaining a desired pharmacological and / or physiological effect. This effect may be prophylactic in that it completely or partially prevents a disease or its symptoms, for example, by reducing the likelihood that the disease or its symptoms will occur in a subject, and / or therapeutic in that it partially or completely cures the disease and / or adverse effects resulting from the disease. When used herein, “treatment” covers any treatment of a disease in a mammal and includes: (a) preventing the disease from occurring in a subject that is susceptible to the disease but has not yet been diagnosed with it; (b) inhibiting the disease, i.e., halting its development; or (c) reducing the disease, i.e., causing regression of the disease. Therapeutic agents may be administered before, during, or after the onset of a disease or injury. Treatment of an ongoing disease is particularly targeted, where the treatment stabilizes or reduces undesirable clinical symptoms in the patient. Such treatment is preferably performed before complete loss of function in the affected tissue. Targeted therapy is preferably administered during the symptomatic phase of the disease, and in some cases, after the symptomatic phase.

[0098] The implementation of this invention will, unless otherwise indicated, utilize conventional techniques of cell biology, molecular biology, microbiology, biochemistry, and immunology, which are within the scope of those skilled in the art. Such techniques are described in “Molecular Cloning: A Laboratory Manual,” second edition (Sambrook et al.). 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 “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., This is adequately explained in literature such as 1991, and each of these documents is explicitly incorporated herein by reference.

[0099] Some aspects of the present invention are described below in relation to examples of applications for illustrative purposes. It should be understood that a number of specific details, relationships, and methods are shown to give a full understanding of the present invention. However, it will be readily apparent to those skilled in the art that the present invention can be carried out without using one or more of these specific details, or by other methods. The present invention is not limited by the order of the exemplary actions or events, for some actions may be carried out in a different order and / or in combination with other actions or events. Furthermore, not all exemplary actions or events are necessarily required to carry out the methodology according to the present invention.

[0100] The terminology used herein is intended solely to describe specific embodiments and is not intended to limit the invention. Where used herein, the singular forms “a,” “an,” and “the” are intended to include the plural unless otherwise explicitly indicated by the context. Furthermore, where the terms “including,” “includes,” “having,” “has,” “accompany,” or their variants are used in either the detailed description and / or the claims, such terms are intended to be inclusive, as is the term “comprising.”

[0101] The terms “about” or “approximately” mean within an acceptable margin of error for a particular value, as determined by those skilled in the art, and this acceptable margin of error depends in part on the constraints of the method or determination of the value, i.e., the measurement system. For example, “about” may mean within a range of 1 or a standard deviation greater than 1, according to practices in the art. Alternatively, “about” may mean 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. Or, particularly with respect to biological systems or processes, the term may mean within one order of magnitude of the value, preferably up to five times, and more preferably up to two times. Where a particular value is described in this application and claims, unless otherwise stated, the term “about” should be assumed to mean within an acceptable margin of error for that particular value.

[0102] All publications referenced herein are incorporated herein by reference to disclose and describe methods and / or materials relating to those publications cited. In the event of any conflict, this disclosure shall prevail over any disclosure in the incorporated publications.

[0103] It should be further noted that claims may be conceived to exclude elements as needed. Therefore, this statement is intended to serve as a basis prior to the use of exclusive terminology such as “exclusively,” “simply,” or “negative” limitations in relation to the description of elements of the claims.

[0104] Unless otherwise indicated, all terms used herein have the same meaning as those to the art, and the implementation of the present invention will utilize conventional techniques of microbiology and recombinant DNA technology that are within the scope of knowledge of those skilled in the art.

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

[0106] WNT ("Wingless-associated integration site," "Wingless and Int-1," or "Wingless-Int") ligands and their signaling play crucial roles in regulating development, homeostasis, and regeneration in many vital organs and tissues, including bone, liver, skin, stomach, intestines, kidneys, central nervous system, mammary glands, taste buds, ovaries, cochlea, lungs, and many other tissues (as outlined, e.g., by Clevers, Loh, and Nusse, 2014; 346:1248012). Modulation of the Wnt signaling pathway has potential for treating degenerative diseases and tissue injuries.

[0107] One of the challenges for modulating WNT signaling as a therapeutic agent is the presence of multiple WNT ligands and receptors, Frizzled 1-10 (FZD1-10), and many tissues express multiple overlapping FZDs. In addition to FZDs, low-density lipoprotein (LDL) receptor-related protein 5 (LRP5) and / or low-density lipoprotein (LDL) receptor-related protein 6 (LRP6) are also involved in canonical WNT signaling as co-receptors widely expressed in various tissues. LRP5 and LRP6 are collectively called LRP5 / 6, and references such as "LRP5 / 6 binding" indicate binding to LRP5 and / or LRP6.

[0108] R-spongins 1-4 (RSPO1-4) are a family of ligands that amplify WNT signaling. Each R-spongin functions via a receptor complex containing zinc and ring finger 3 (ZNRF3) or ring finger protein 43 (RNF43) at one end and leucine-rich repeat-containing G protein-coupled receptors 4-6 (LGR4-6) at the other (as outlined, e.g., by Knight and Hankenson 2014, Matrix Biology; 37: 157-161). R-spongins may also function through further 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. The binding of R-spondin to ZNRF3 / RNF43 and LGR4-6 induces clearance or sequestration of the ternary complex, thereby removing E3 ligase from the WNT receptor, stabilizing the WNT receptor, and consequently enhancing 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. Fragments of R-spondin containing furin domains 1 and 2 are sufficient to amplify WNT signaling. Although the R-spondin effect is dependent on WNT signaling, the effect of R-spondin is not tissue-specific because 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 a variety of diseases and disorders, including gastrointestinal disorders. Similarly, amplification of WNT signaling by RSPO or RSPO mimetic can be used to treat a variety of diseases and disorders, including gastrointestinal disorders. Previous studies in the literature have suggested that RSPO can be used to treat experimental 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 may play an important role in regulating the regeneration of intestinal epithelium in both homeostatic and injured states.

[0110] The two main functions of the digestive tract organs are determined by the two lineages of intestinal epithelium: absorptive and secretory. Secretory cells secrete hormones and, primarily through the secretion of mucus and antimicrobial peptides, provide an important barrier against foodborne microorganisms, toxins, and antigens. In contrast, absorptive cells are mainly located at the tips of the villi or in the upper part of the colonic crypts in the small intestine, and thus constitute the majority of luminal cells across the intestinal surface area, thus performing 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 regenerate within 3 to 10 days.

[0111] Different niche factors maintain intestinal stem cell (ISC) activity, and distinct non-epithelial and / or epithelial cells generate various signals that create the cellular niche. Such niche factors include not only standard signals such as WNT, R-spongin, Notch, and bone morphogenetic protein (BMP), but also the effects of inflammation and diet. Upon injury, the ISC niche transcends its homeostatic state and adapts to interpret pathogenic stimuli and translate them into epithelial regeneration. This regeneration is mediated by either surviving Lgr5+ ISCs, or other mature cell types such as intestinal cells, enteroendocrine cells, or Paneth cells that can be converted back into Lgr5+ ISCs to assist in epithelial regeneration (Beumer and Clevers (2016), Development 143: 3639-3649).

[0112] ISCs located at the base of the intestinal crypts are also known as columnar base cells (CBCs) and are interposed by WNT-secreting Paneth cells (Cheng and Leblond (1974) Am. J. Anat. 141: 537-561). Periemacial mesenchymal cells also secrete some WNT proteins, fulfilling a redundant stem cell niche function in vivo (Farin, el al. (2012) Gastroenterol. 143: 1518-1529). In the presence of WNT signaling, ISCs divide to produce self-regenerating stem cells and differentiated daughter cells, which first undergo some rapid transit-amplifying (TA) division before differentiating into functional cell types. A quiescent stem cell population called +4 cells also exists in the intestinal crypts, which can contribute to epithelial regeneration when the CBC is damaged (Tian, ​​el al. (2011) Nature 478: 255-259). Constraint to individual lineages and terminal differentiation occur when TA cells migrate along the crypt-villous axis and detach from WNT-producing cells.

[0113] III. Manipulated WNT Agonists This disclosure provides engineered WNT agonists and intends to use engineered WNT agonists to stimulate, agonize, or promote WNT signaling, for example, through the standard WNT / β-catenin signaling pathway. Such engineered WNT agonists may also be referred to as WNT / β-catenin signaling agonists or Wnt mimics.

[0114] Several challenges exist regarding the manipulation of Wnt proteins for clinical application. Firstly, Wnt proteins are difficult to produce and do not possess typical drug-like properties. Secondly, while in vivo overexpression of RSPO, which amplifies Wnt signaling, or application of exogenous RSPO has been reported to be beneficial for intestinal epithelial regeneration in various injury models (Zhao et al., 2007), it has also been reported that increased proliferation of normal intestinal epithelial cells is induced (Yan Kelley S. et al., 2017).

[0115] This disclosure addresses the first challenge by providing synthetic Wnt mimics with drug-like properties, particularly in the form of recombinant bispecific antibodies that mimic endogenous Wnt ligands, combining Fzd and Lrp to stimulate signaling. The Wnt mimics of this disclosure can diffuse freely, approach damaged tissue, and guide tissue repair at sites where Wnt signaling is needed.

[0116] This disclosure addresses the second challenge by providing a Wnt mimetic that can repair damaged intestinal epithelium without being combined with RSPO. Unlike RSPO, the Wnt mimetic of this disclosure does not induce excessive proliferation of normal intestinal epithelium.

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

[0118] In some embodiments, the Wnt agonists disclosed herein support the proliferation and differentiation of stem cells in the damaged intestine or colonic crypts of patients with moderate to severe IBD. In some embodiments, the Wnt agonists disclosed herein have the potential to accelerate the repair of the intestinal barrier, thereby resulting in a reduction of bacterial invasion through the intestinal epithelium, as well as immune cell activation and inflammation reduction, thus enabling the treatment of inflammatory bowel disease.

[0119] In some embodiments, the Wnt agonists disclosed herein have several simultaneously present beneficial effects: activating the Wnt signaling pathway in intestinal stem cells 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 agonists disclosed herein are bispecific antibodies targeting Fzd5 / 8 and Lrp6. Fzd5 has been previously reported to be highly expressed in intestinal mucosal cells derived from IBD patients. Fzd5 is also highly expressed in a mouse model of dextran sulfate sodium (DSS)-induced colitis. In some embodiments, the Wnt agonists disclosed herein bind to DSS-injured intestinal cells and stimulate Wnt signaling, as measured by the expression of Axin2, a downstream target gene in the Wnt pathway. In some embodiments, the Wnt agonists disclosed herein bind to Fzd5 / 8 and Lrp6 on intestinal stem cells to activate Wnt signaling.

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

[0122] In some embodiments, the Wnt mimetic of this disclosure expands the progenitor cell population in epithelium. In some embodiments, the Wnt mimetic of this disclosure expands the progenitor cell population by increasing the expression of cell cycle genes in the cell population. The progenitor cell population may include, for example, normal progenitor cells responding to injury, and progenitor cells whose cellular state has been altered, such as dedifferentiated progenitor cells. In some embodiments, the Wnt mimetic of this disclosure substantially expands the progenitor cell population in epithelium within about 24 hours.

[0123] In some embodiments, the Wnt mimetic of this disclosure accelerates the differentiation of progenitor cells into mature cell types. In some embodiments, the Wnt mimetic of this disclosure accelerates the differentiation of progenitor cells, such as gastrointestinal progenitor cells, into intestinal cells, goblet cells, enteroendocrine cells, or tuft cells. In some embodiments, the Wnt mimetic of this disclosure accelerates the differentiation of progenitor cells into intestinal cells. In some embodiments, substantial differentiation of progenitor cells into mature cell types occurs within approximately 24 hours, 36 hours, 48 ​​hours, or 60 hours after treatment with the Wnt mimetic of this disclosure. In some embodiments, substantial differentiation of progenitor cells into mature cell types occurs within approximately 48 hours after treatment with the Wnt mimetic of this disclosure. In some embodiments, the Wnt mimetic of this disclosure accelerates the differentiation of progenitor cells into mature cell types while simultaneously reducing the expression of high levels of inflammatory genes.

[0124] In some embodiments, the breakdown of the intestinal barrier triggers the influx of luminal pathogens and an inflammatory response, thereby leading to further tissue damage. Disease modification in IBD can be measured by the levels of inflammatory cytokines present in the injured tissue and serum. In some embodiments, treatment of epithelial tissue injury with the Wnt mimetic of the Disclosure reduces the production of inflammatory cytokines. In some embodiments, treatment of damaged epithelial tissue with the Wnt mimetic of the 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 mimetic of the Disclosure or to no treatment.

[0125] In some embodiments, the Disclosure provides a Wnt mimetic capable of effectively repairing damaged epithelium without inducing excessive proliferation of normal epithelium. In some embodiments, the Wnt mimetic of the Disclosure alone does not affect the proliferation of normal epithelium. In some embodiments, the epithelium is colonic or small intestinal epithelium. In some embodiments, the Disclosure provides a Wnt mimetic capable of repairing damaged epithelium with higher efficacy than treatments including RSPO. In some embodiments, the Disclosure provides a Wnt mimetic capable of repairing damaged epithelium with higher efficacy than treatments including RSPO and the Wnt mimetic of the Disclosure. In some embodiments, the Wnt mimetic of this 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 treatment comprising RSPO, or treatment comprising RSPO and the Wnt mimetic of this disclosure. The effectiveness of repairing damaged epithelium may be determined by a histological severity score, where a higher score indicates more severe damage, or by a disease activity index (DIA), which may be calculated based on an average score of weight loss, stool consistency, and degree of intestinal bleeding.

[0126] In some embodiments, the Disclosure provides Fzd5, 8, and Lrp6-specific Wnt mimics (e.g., R2M13-26 or R2M13-h26). In some embodiments, the Fzd5, 8, and Lrp6-specific Wnt mimics of the Disclosure are capable of activating Wnt signaling in epithelial cells. Activation of Wnt signaling is known in the Art and can be measured by gene expression using the scRNA-seq (single-cell RNA sequencing) method described in the Disclosure. In some embodiments, epithelial cells are colonic or intestinal epithelial cells. In some embodiments, epithelial cells comprise a plurality of stem cells or progenitor cells.

[0127] The engineered WNT agonist comprises one or more binding domains that bind to one or more FZDs or their epitopes, and one or more binding domains that bind to 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 (or receptor) to which it binds.

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

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

[0130] In one embodiment, the disclosure provides VHH domains that bind to LRP5 and / or LRP6. Exemplary sequences of these VHH domains are provided in Table 1. The VHH-binding domains may be derived from any of the disclosed sequences. In certain embodiments, the VHH-binding domains are humanized. The disclosure intends to provide an engineered WNT agonist comprising one or more disclosed VHH domains, including functional fragments and variants of such VHH domains having at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to any of the humanized VHH domains disclosed herein, as well as any of the VHH sequences disclosed herein. In certain embodiments, the VHH domain comprises three CDR sequences: GRIFAIYDIA, IRVVTEIDYADSVKG, and RPWGSRDEY. In certain embodiments, the manipulated Wnt agonist contains 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 manipulated Wnt agonist is a bispecific antibody-like molecule containing 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-free and, for example, contain the LALAPG mutation.

[0131] In another aspect, the Disclosure provides FZD-binding domains that bind to one or more FZDs. Exemplary sequences of these FZD-binding domains are provided in Table 3 with respect to the VH and VL domains derived from the anti-FZD antibody R2M13. The Disclosure intends to provide engineered WNT agonists comprising one or more functional fragments and variants of the VH or VL domains disclosed herein, and having at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to either of the VH or VL sequences disclosed herein. Furthermore, the Disclosure intends to provide engineered WNT agonists comprising one or more functional fragments and variants of the heavy or light chain sequences provided in Table 3, and having at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to either 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 light chain CDR sequences: GGTFTYRYLH(CDRH1), GIIPIFGTGNYAQKFQG(CDRH2), and SMVRVVPYYYGMDV(CDRH3), any CDR provided herein.

[0132] In relevant embodiments, this disclosure envisions an engineered WNT agonist comprising 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; or one or more (e.g., two or three) of the CDRs present in the heavy or light chain disclosed herein. In certain embodiments, the engineered WNT agonist comprises all four, five, or 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 comprises all six, seven, eight, or nine of the CDRs shown for the engineered WNT agonist disclosed herein, for example, in Figure 6 or Table 3.

[0133] This disclosure includes polypeptides comprising or consisting of sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity with the binding domains provided herein, as well as polypeptides comprising two or more, for example, three, of the CDR sequences disclosed herein, for example, the following CDRs: GRIFAIYDIA, IRVVTEIDYADSVKG, and RPWGSRDEY (VHH, respectively). The present invention provides polypeptides comprising CDR1-3) and binding to LRP5 or LRP6, or polypeptides comprising the following CDRs:RASQSISSYLN(CDRL1), AASSLQS(CDRL2), and QQSYSTPLT(CDRL3) and binding to one or more FZDs in combination with a heavy chain, or polypeptides comprising the following CDRs:GGTFTYRYLH(CDRH1), GIIPIFGTGNYAQKFQG(CDRH2), and SMVRVVPYYYGMDV(CDRH3) and binding to one or more FZDs in combination with a light chain. This disclosure also includes an FZD-binding domain that binds to one or more FZDs, comprising two heavy chains and two light chains, each heavy chain comprising two or more of the following CDRs:GGTFTYRYLH(CDRH1),GIIPIFGTGNYAQKFQG(CDRH2), andSMVRVPYYYGMDV(CDRH3), and each light chain comprising two or more of the following CDRs:RASQSISSYLN(CDRL1),AASSLQS(CDRL2), and QQSYSTPLT(CDRL3). In certain embodiments, the FZD-binding domain is an antibody, and the heavy chain further comprises an Fc domain, for example, an IgG1 Fc domain, which may be modified. This disclosure further provides polypeptides comprising or comprising sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity with the variable weight or variable light domains disclosed, for example, in SEQ ID NOs: 1-25, Figure 6, or Table 3.This disclosure further provides polypeptides comprising or comprising sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the VHH domains disclosed, for example, in SEQ ID NOs: 1-25, Figure 6, or Table 3. This disclosure further provides polypeptides comprising or comprising sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the heavy chain, light chain, or fusion polypeptides disclosed, for example, in SEQ ID NOs: 1-25, Figure 6, or Table 3. In any embodiment of the polypeptide variants disclosed herein, the CDR is not modified compared to the original or parent sequence.

[0134] Furthermore, this disclosure provides polypeptides described herein, as well as functional fragments and variants thereof, for example, polynucleotide sequences encoding one or more functional fragments and variants bound to FZD or LRP5 / 6, a VH domain, and a VL domain.

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

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

[0137] The manipulated WNT agonist may take on a variety of different structural conformations, each containing one or more, e.g., two FZD-binding domains and one or more, e.g., two LRP5 / 6-binding domains. The FZD-binding domains and LRP5 / 6-binding domains may be fused directly to each other, or they may be fused via a linker, e.g., a peptide linker. Alternatively, the FZD-binding domains and LRP5 / 6-binding domains may form complexes with each other.

[0138] In a particular embodiment, the manipulated WNT agonist comprises two heavy chains and two light chains, where the light chains contain fused VHH and adopt an antibody-like conformation, with the two heavy chains linked to each other by disulfide bonds and the two light chains linked to the heavy chains by disulfide bonds.

[0139] The manipulated WNT agonists may adopt other antibody-like structures or conformations, including those found in a variety of functional fragments, which include, but are not limited to, any of those disclosed herein.

[0140] As is well known in this field, antibodies are immunoglobulin molecules capable of specifically binding to targets such as carbohydrates, polynucleotides, lipids, and polypeptides, at least through an epitope-binding domain located in the variable region of the immunoglobulin molecule. As used herein, this term encompasses not only intact polyclonal or monoclonal antibodies, but also their fragments containing an epitope-binding domain (e.g., dAb, Fab, Fab', (F(ab')2, Fv, single-chain (scFv), camelid antibodies, Nanobodies® (Nab; also known as sdAb or VHH domain), DVD-Ig, its synthetic variants, naturally occurring variants, fusion proteins containing an epitope-binding domain, humanized antibodies, chimeric antibodies, and any other modified configurations of immunoglobulin molecules containing an antigen-binding site or fragment (epitope-recognition site) of the required specificity; “diabodies” constructed by gene fusion; polyvalent or polyspecific fragments (WO94 / 13804; P. Holliger et al., Proc. Natl. Acad. Sci. USA 90 6444-6448, (1993) is also a specific form of antibody intended herein. Minibodies containing scFv conjugated to a CH3 domain are also included herein (S. Hu et al., Cancer Res., 56, 3055-3061, 1996). 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, See also: 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 protease papain preferentially cleaves IgG molecules to produce several fragments, two of which (F(ab) fragments) each contain a covalent heterodimer with an intact antigen-binding site. The enzyme pepsin can cleave IgG molecules to yield several fragments, including the F(ab')2 fragment, which contains both antigen-binding sites. Fv fragments for use in certain embodiments of this disclosure may be produced by preferential proteolytic cleavage of IgM, and less frequently by proteolytic cleavage of IgG or IgA immunoglobulin molecules. However, Fv fragments are more generally derived by recombinant techniques known in the art. Fv fragments contain a non-covalent VH::VL heterodimer with an antigen-binding site that retains much of the antigen-recognition and binding ability 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 their antigen-binding fragments described herein comprise heavy-chain and light-chain CDR sets, each positioned between heavy-chain and light-chain framework region (FR) sets that provide support to the CDRs and define the spatial relationships of the CDRs relative to each other. As used herein, the term “CDR set” refers to three hypervariable regions of the heavy-chain or light-chain V region. Proceeding from the N-terminus of the heavy-chain or light-chain, these regions are referred to as “CDR1,” “CDR2,” and “CDR3,” respectively. Thus, the antigen-binding site comprises six CDRs, each comprising a CDR set derived from the heavy-chain and light-chain V region. A polypeptide containing a single CDR (e.g., CDR1, CDR2, or CDR3) is referred herein to as a “molecular recognition unit.” Crystallographic analysis of numerous antigen-antibody complexes has demonstrated that 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 the four adjacent amino acid sequences that frame the CDR of a CDR set in a heavy or light chain V region. Some FR residues can come into contact with the bound antigen; however, FRs, particularly those directly adjacent to the CDR, are primarily responsible for folding the V region into the antigen-binding site. Within the FR, 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 CDR is displayed as a protruding loop motif that forms the antigen-binding surface. It is generally understood that there are conserved structural regions in the FR that influence the folding shape of the CDR loop to be a certain “standard” structure, regardless of the exact CDR amino acid sequence. Furthermore, certain FR residues are known to be involved in non-covalent interdomain contacts that stabilize the interaction between the heavy and light chains of the antibody.

[0144] "Monoclonal antibodies" refer to a homogeneous population of antibodies composed of amino acids (naturally occurring and non-naturally occurring) involved in the selective binding of epitopes. Monoclonal antibodies are highly specific and target a single epitope. The term "monoclonal antibody" encompasses not only intact and full-length monoclonal antibodies, but also any other modified configurations of immunoglobulin molecules containing antigen-binding fragments (epitope recognition sites) with the required specificity and binding ability to an epitope, including fragments thereof (e.g., Fab, Fab', F(ab')2, Fv, etc.), single chains (scFv), Nanobodies®, their variants, fusion proteins containing antigen-binding fragments of monoclonal antibodies, humanized monoclonal antibodies, chimeric monoclonal antibodies, and engineered WNT agonists disclosed herein. This term is not intended to be limited in terms of the source of antibodies or the manner in which they are produced (e.g., by hybridoma, phage selection, recombinant expression, transgenic animals, etc.). The term encompasses the entire immunoglobulin and its fragments, etc., as described above under the definition of "antibody."

[0145] In certain embodiments, single-chain Fv or scFV antibodies are intended for use with manipulated Wnt agonists. For example, kappa bodies (Ill et al., Prot. Eng. 10: 949-57 (1997)); mini bodies (Martin et al., EMBO J 13: 5305-9 (1994)); dia bodies (Holliger et al., PNAS 90: 6444-8 (1993)); or Janusins ​​(Traunecker et al., EMBO J 13: 5305-9 (1994)); The antibodies described in 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 in accordance with the teachings of this application regarding the selection of antibodies having desired specificity. In further embodiments, bispecific or chimeric antibodies containing the ligands of the Disclosure can be produced. For example, a chimeric antibody may contain CDRs and framework regions derived from different antibodies, but a bispecific antibody can be produced that binds to one or more FZD receptors through one binding domain and specifically to a second molecule through a second binding domain. These antibodies can be produced by recombinant molecular biology techniques or physically conjugated together.

[0146] Single-chain Fv(scFv) polypeptides are covalently linked VH::VL heterodimers expressed from gene fusions containing genes encoding VH and VL, linked by peptide-encoding linkers. Huston et al. (1988) Proc. Nat. Acad. Sci. USA 85 (16): 5879-5883. Several methods have been described for determining the chemical structure to convert naturally assembled but chemically separate light and heavy polypeptide chains derived from the antibody V region into scFv molecules that fold into a three-dimensional structure substantially similar to that of the antigen-binding site. See, for example, U.S. Patents 5,091,513 and 5,132,405 to Huston et al., and U.S. Patent 4,946,778 to Ladner et al.

[0147] In certain embodiments, the antibodies described herein are in the form of diabodies. A diabody is a polypeptide polymer, each polypeptide comprising a first domain containing a binding region for an immunoglobulin light chain and a second domain containing a binding region for 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 polymer with the second domain of another polypeptide in the polymer (WO94 / 13804).

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

[0149] When bispecific antibodies are used, they can be manufactured in various ways (Holliger, P. and Winter G., Current Opinion Biotechnol. 4, 446-449 (1993)), for example, they may be conventional bispecific antibodies prepared chemically or from hybrid hybridomas, or any of the bispecific antibody fragments described above. The diabody and scFv may be constructed using only the variable domain and without the Fc region, thereby potentially reducing the influence of anti-idiotype reactions.

[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) with appropriate binding specificity can be easily selected from a library using phage display (WO94 / 13804). If one arm of a diabody is kept constant with specificity to, for example, antigen X, the other arm can vary, allowing for the creation of a library in which antibodies with appropriate specificity are selected. Whole bispecific antibodies can be constructed by the knob-into-hole operation (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 UniBody®, which is an IgG4 antibody from which the hinge region has been 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 area than current smaller antibody formats. IgG4 antibodies are inactive and therefore considered not to interact with the immune system. Fully human IgG4 antibodies can be modified by removing the hinge region of the antibody to obtain a halving fragment with stability properties distinct from the corresponding intact IgG4 (GenMab, Utrecht). By halving the IgG4 molecule, only one region on UniBody® remains that can bind to a congener antigen (e.g., a disease target), and therefore UniBody® binds monovalently to only one site on the target cell.

[0152] In certain embodiments, the antibodies of this disclosure may take the form of a single-domain (sdAb) or a VHH antibody fragment (also known as Nanobody®). The sdAb or VHH technology was originally developed following the discovery and identification that camelids (e.g., camels and llamas) possess fully functional antibodies consisting solely of heavy chains and therefore lacking light chains. These heavy-chain-only antibodies contain a single variable domain (VHH) and two constant domains (CH2, CH3). Cloned and isolated single variable domains possess full antigen-binding ability and are highly stable. These single variable domains, along with their unique structural and functional properties, form the basis of "Nanobodies®". sdAb or VHH are encoded by a single gene and are efficiently produced in almost all prokaryotic and eukaryotic hosts, e.g., E. coli (see, e.g., U.S. Patent No. 6,765,087), fungi (e.g., Aspergillus or Trichoderma), and yeasts (e.g., Saccharomyces, Kluyvermyces, Hansenula, or Pichia (see, e.g., U.S. Patent No. 6,838,254)). The production process is scalable, and several kilograms of Nanobodies® are produced. sdAb or VHH can be formulated as a ready-to-use solution 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. sdAb or VHH are single-domain antigen-binding fragments of heavy-chain-only antibodies specific to camelids.

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

[0154] In certain embodiments, the antibodies or antigen-binding fragments disclosed herein are humanized. This refers to chimeric molecules generally prepared using recombinant techniques, having an antigen-binding site derived from a non-human species immunoglobulin and the rest of the immunoglobulin structure of a molecule based on the structure and / or sequence of a human immunoglobulin. The antigen-binding site may consist of either a complete variable domain fused to a constant domain, or only a CDR grafted onto a suitable framework region within the variable domain. The epitope-binding site may be wild-type or modified by one or more amino acid substitutions. This eliminates the constant region as an immunogen in a human organism, but leaves open the possibility of an immune response to the exogenous variable region (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 not only on providing a human-derived constant region but also on modifying the variable region and reconstructing it to be as close to human form as possible. It is known that both the heavy and light chain variable regions contain three complementarity-determining regions (CDRs) that vary depending on the epitope in question and determine binding ability, flanked by four framework regions (FRs) which are relatively conserved in a given species and are presumed to provide scaffolding for CDRs. When a non-human antibody is prepared for a specific epitope, the variable region can be "reconstructed" or "humanized" by grafting CDRs derived from the non-human antibody onto the FRs present in the human antibody being modified. The application of this method to various antibodies is described in Sato, K., et al., (1993) Cancer Res 53: 851-856;Riechmann, L., et al., (1988) Nature 332: 323-327;Verhoeyen, M., et al. 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., This is reported in (1992) J Immunol 148: 1149-1154. In some embodiments, the humanized antibody preserves all CDR sequences (e.g., a humanized mouse antibody containing all six CDRs derived from the mouse antibody). In other embodiments, the humanized antibody has one or more CDRs (one, two, three, four, five, or six) that are modified with respect to the original antibody, and these are also called one or more CDRs "derived" from one or more CDRs from the original antibody.

[0156] In certain embodiments, the antibodies of this disclosure may be chimeric antibodies. In this regard, a chimeric antibody consists of an antigen-binding fragment of an antibody that is operably linked to or otherwise fused with heterologous Fc portions of different antibodies. In certain embodiments, the heterologous Fc domain is of human origin. In other embodiments, the heterologous Fc domain may be derived from a different Ig class than 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 consist of CH2 and CH3 domains derived from one or more different Ig classes. As described above with respect to humanized antibodies, the antigen-binding fragment of a chimeric antibody may contain 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 it may contain the entire variable domain (VL, VH, or both).

[0157] The structure and location of immunoglobulin CDRs and variable domains can be determined by referring to Kabat, EA et al., Sequences of Proteins of Immunological Interest. 4th Edition. US Department of Health and Human Services. 1987, and its revised edition, which is now available online (immuno.bme.nwu.edu).

[0158] In some embodiments, the manipulated WNT agonist comprises one or more Fabs or their antigen-binding fragments and one or more VHHs or sdAbs or their antigen-binding fragments (or one or more scFvs or their antigen-binding fragments). 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 exist in complete 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 exists in complete IgG format, with 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 IgG in any combination of these positions.

[0159] Fab can be converted to a complete IgG format containing both Fab and Fc fragments by, for example, using genetic engineering to generate a fusion polypeptide that includes Fab fused to an Fc region, i.e., Fab exists in a complete IgG format. The Fc region of the complete IgG format may originate from any of various different Fc fragments, 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 (including mutations in the core hinge region that interfere with the formation of half an IgG4 molecule), 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, as well as Fc-γ-dependent antibody-dependent cell-mediated cytotoxicity (ADCC), for both mouse IgG2a and human IgG1. These LALA-PG substitutions enable more accurate conversion between mice and primates of the results obtained with the “effector-free” antibody framework scaffold. In any particular embodiment of the IgG 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 manipulated WNT agonists that are bivalent to one or more Fzd receptors and both LRP5 and / or LRP6 are provided, including but not limited to those provided in Table 3. VHH or sdAb (or scFv) may 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 intended that VHH or sdAb (or scFv) may be fused to both the N-terminus and C-terminus of the heavy chain and / or light chain, the N-terminus of the light chain and the N-terminus of the light chain, the N-terminus of the heavy chain and the C-terminus of the light chain, or the C-terminus of the heavy chain and the N-terminus of the light chain. In other relevant embodiments, two or more VHH or sdAb (or scFv) may be fused together via a linker moiety as needed, or fused to Fab or IgG at one or more of these positions. In the relevant embodiments, the manipulated WNT agonist has a hetero-IgG format, where Fab is present as a semiantibody, and one or more VHH or sdAb (or scFv) are fused to one or more of the following: the N-terminus of Fc, the N-terminus of Fab, the C-terminus of Fc, or the C-terminus of Fab. In certain embodiments, Fab or its antigen-binding fragment (or IgG) is directly fused to VHH or sdAb (or scFv) or its antigen-binding fragment, while in other embodiments, the binding region is fused via a linker moiety.

[0161] In various embodiments, the engineered WNT agonist comprises one or more Fabs or their antigen-binding fragments that bind to one or more FZD receptors, and one or more Fabs or their antigen-binding fragments that bind to LRP5 and / or LRP6. In certain embodiments, the engineered WNT agonist comprises two Fabs or their antigen-binding fragments that bind to one or more FZD receptors, and two Fabs or their antigen-binding fragments that bind to / or LRP5 and / or LRP6. In certain embodiments, one or more Fabs exist in full IgG format, and in certain embodiments, both Fabs exist in full IgG format. In certain embodiments, the Fabs in full IgG format specifically bind to one or more FZD receptors, and the other Fabs specifically bind to LRP5 and / or LRP6. In certain embodiments, the Fabs specifically bind to one or more FZD receptors, and the Fabs in full IgG format specifically bind to LRP5 and / or LRP6. In certain embodiments, the Fabs specifically bind to LRP5 and / or LRP6, and the Fabs in full IgG format specifically bind to one or more FZD receptors. In certain embodiments, the Fab is fused to the N-terminus of IgG, for example, the heavy chain or the light chain N-terminus, via a linker as needed. In certain embodiments, the Fab is fused to the N-terminus of the heavy chain of IgG, but not to the light chain. In certain embodiments, two heavy chains may be fused together directly or via a linker. In other relevant embodiments, two or more VHH or sdAb may be fused together via a linker moiety as needed, and may be fused to Fab or IgG at one or more of these positions. In relevant embodiments, the manipulated WNT agonist has a hetero-IgG format, in which one of the Fabs exists as a semi-antibody, and the other Fabs are fused to one or more of the N-terminus of Fc, the N-terminus of Fab, or the C-terminus of Fc.In certain embodiments, a Fab or its antigen-binding fragment is directly fused to another Fab or IgG or its antigen-binding fragment, while in other embodiments, the binding region is fused via a linker portion.

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

[0163] In certain embodiments, the FZD-binding domain, LRP5 / 6-binding domain, and / or the manipulated WNT agonist bind 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 manipulated WNT agonists of the present invention, their binding domains, antibodies, or other agents may be assayed for specific binding by any method known in the art. Immunoassays that may 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 blotting, radioimmunoassay, ELISA, "sandwich" immunoassay, immunoprecipitation assay, precipitation reaction, gel diffusion precipitate reaction, immunodiffusion assay, agglutination assay, complement fixation assay, immunoradioquantification, fluorescence 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. An ELISA assay involves preparing an antigen, coating the wells of a 96-well microtiter plate with the antigen, adding an antibody or other conjugate 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 drug is not conjugated with a detectable compound, and instead, a second conjugated antibody that recognizes a first antibody or drug is added to the wells. In some embodiments, instead of coating the wells with the antigen, the wells can be coated with the antibody or drug, and after adding the antigen to the coated wells, a second antibody conjugated with a detectable compound can be added. Those skilled in the art will be familiar with parameters that can be modified to increase the detected signal, as well as with other known variations of ELISA in the art (see, for example, 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 drug to a target antigen and the off-rate of the antibody-antigen interaction can be determined by competitive binding assays. One example of a competitive binding assay is a radioimmunoassay, which involves incubating a labeled antigen (e.g., FZD, LRP) or a fragment or variant thereof with the antibody of interest in the presence of an increasing amount of unlabeled antigen, and then detecting the antibody bound to the labeled antigen. The antibody affinity and binding / off-rate can be determined from data obtained by scatchard plot analysis. In some embodiments, BIAcore kinetic analysis is used to determine the binding / association rate (on-rate) and dissociation rate of an antibody or drug. BIAcore kinetic analysis involves analyzing the binding and dissociation of an antibody to a chip on which an antigen is immobilized.

[0167] The engineered WNT agonists of the present invention are biologically active with respect to binding to one or more FZD receptors and one or more LRP5 and LRP6, and with respect to activation of WNT signaling. The term “WNT agonist activity” refers to the ability of an agonist to mimic the effect or activity of the WNT protein that binds to the frizzled protein and / or LRP5 or LRP6. The ability of the engineered WNT agonists disclosed herein to mimic WNT activity can be confirmed by several assays. WNT agonists typically initiate a response or activity similar to or identical to that initiated by the receptor’s native ligand. 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, e.g., the engineered WNT agonists 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 times, at least 1.2 times, at least 1.3 times, at least 1.4 times, at least 1.5 times, at least 2 times, at least 5 times, at least 10 times, at least 20 times, at least 50 times, or at least 100 times compared to the level of WNT / β-catenin signaling in the same cell type in the absence of the manipulated WNT agonist. 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., they specifically bind to two or more different epitopes, such as one or more FZD receptors and LRP5 and / or LRP6. In certain embodiments, the engineered WNT agonist binds to FZD5 and / or FZD8, as well as LRP5 and / or LRP6.

[0169] In certain embodiments, the manipulated WNT agonists disclosed herein are polyvalent and include, for example, two or more regions, each specifically binding to the same epitope, e.g., two or more regions binding to epitopes in one or more FZD receptors, and / or two or more regions binding to epitopes in LRP5 and / or LRP6. In certain embodiments, the engineered WNT agonist includes a ratio of the number of regions that bind to one or more FZD receptors to the number of regions that bind to LRP5 and / or LRP6, approximately 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 polyvalent.

[0170] In certain embodiments, this disclosure provides novel tissue-specific WNT signal-enhancing molecules capable of enhancing WNT activity in a tissue- or cell-specific manner. These can be used alone or in combination with one or more manipulated WNT agonists disclosed herein. In certain embodiments, the tissue-specific WNT signal-enhancing molecule is a bifunctional molecule comprising 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 and second domains may be any portion that can bind to a ligase complex or a targeted tissue or cell, respectively. For example, each of the first and second domains may be, but are 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 native ligand or its variant, fragment, or derivative. In certain embodiments, the native ligand may be a polypeptide, a small molecule, an ion, an amino acid, a lipid, or a sugar molecule. The first and second domains may be of the same type, or they may be of different types. In certain embodiments, the tissue-specific WNT signaling-enhancing molecule binds to a tissue or cell-specific cell surface receptor. In certain embodiments, the tissue-specific WNT signaling-enhancing molecule increases or enhances WNT signaling by, for example, at least 50%, at least 2x, at least 3x, at least 5x, at least 10x, at least 20x, at least 30x, at least 40x, or at least 50x compared to a negative control.

[0171] Tissue-specific WNT signal-enhancing molecules may have different formats. In certain embodiments, the 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 may be directly fused or fused via a linker. In certain embodiments, the tissue-specific WNT signal-enhancing molecule comprises a dimer or polymer comprising two or more polypeptides, e.g., two or more fusion proteins each containing a first domain and a second domain, where the two or more polypeptides are linked, e.g., via a linker moiety or by linkages between amino acid residues in each of the two or more polypeptides, e.g., by intermolecular disulfide bonds between cysteine ​​residues.

[0172] In certain embodiments, the tissue-specific WNT signal-enhancing molecule is an antibody (or its antigen-binding fragment) comprising an antibody heavy chain and a light chain, constituting either a first or second domain, wherein the other domain (i.e., the second or first domain) is fused 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 fused 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 a structure may be referred to herein as an additional IgG scaffold or format. 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 to or attached to either the heavy chain or light chain of the antibody that binds to ZNRF3 / RNF43. In another example, a tissue-specific WNT signal-enhancing molecule could be an antibody that binds to a tissue or cell-specific receptor, where a binding domain that binds to ZNRF3 / RNF43 is fused to 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 gut-specific WNT signal-enhancing molecule is an antibody or antigen-binding fragment that binds to GPA33, CDH17, or MUC-13, and the binding domain that binds to ZNRF3 / RNF43 is fused to or added to either the heavy or light chain of the antibody or antigen-binding fragment. In certain embodiments, the binding domain that binds to ZNRF3 / RNF43 comprises Fu1 and Fu2 domains, and the Fu1 and Fu2 domains optionally include one or more amino acid modifications, including any of those disclosed herein, e.g., F105R and / or F109A.

[0174] In certain embodiments, the tissue-specific WNT signaling-enhancing molecule comprises a first domain ("action module") that binds to ZNRF3 / RNF43, and a second domain ("targeting module") that binds to a tissue or cell-specific receptor, for example, with high affinity. In certain embodiments, each of these two domains has substantially reduced activity or is inactive in itself to enhance WNT signaling. However, when the tissue-specific WNT signaling-enhancing molecule engages with a target tissue expressing the tissue-specific receptor, the 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 final result is enhancement of WNT signaling in a tissue-specific manner.

[0175] In certain embodiments, the action module is a binding factor to the ZNRF3 / RNF43 E3 ligase, and the action module can be designed based on R-spongins, including but not limited to human R-spongins-1 to 4. In certain embodiments, the action module is an R-spongin, for example, wild-type R-spongins-1 to 4, optionally human R-spongins-1 to 4, or a variant or fragment thereof. In certain embodiments, it is a variant of any of R-spongins-1 to 4 having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with respect to the corresponding wild-type R-spongins-1 to 4 sequences. In certain embodiments, the action module includes or comprises a furin domain 1 of an R-spongin, for example, any of R-spongins-1 to 4, that binds to ZNRF3 / RNF43. Extended versions of furin domain 1 (including, but not limited to, those having mutant furin domain 2 that no longer bind to LGR4-6 or have reduced binding to LGR4-6), or engineered antibodies, or any other derivatives, or any engineered polypeptide other than an antibody 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-sponge.

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

[0177] In further embodiments, the action module includes another inhibitory moiety, such as a nucleic acid molecule that reduces or inhibits ZNRF3 / RNF43 activity or expression, e.g., antisense oligonucleotides; small interfering RNA (siRNA); small hairpin RNA (shRNA); microRNA (miRNA); or ribozymes. As used herein, “antisense” refers to a nucleic acid sequence complementary to a nucleic acid sequence, regardless of length. In certain embodiments, antisense RNA refers to a single-stranded RNA molecule that can be introduced into individual cells, tissues, or subjects and results in a reduction of target gene expression by a mechanism that does not necessarily rely on endogenous gene silencing pathways. Antisense nucleic acids may contain a modified backbone, e.g., phosphorothioates, phosphorodithioates, or others known in the art, or may contain non-natural internucleoside junctions. Antisense nucleic acids may include, for example, locked nucleic acids (LNAs). In certain embodiments, the other inhibitory moiety inhibits the activity of one or both of ZNRF3 / RNF43, or 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, such as a cell-specific surface receptor, which may be, for example, a native 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, for example, an organ, tissue, or cell type, where it is desired to enhance WNT signaling to treat or prevent a disease or disorder. In certain embodiments, the expression of the cell-specific surface molecule in a target organ, tissue, or cell type, for example, an organ, tissue, or cell type, where it is desired to enhance WNT signaling to treat or prevent a disease or disorder, is increased or enhanced compared to, for example, one or more other untargeted 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, each compared to one or more other organs, tissues, or cell types. For example, in certain embodiments, a cell surface receptor is considered a tissue-specific or cell-specific cell surface molecule if it is expressed in a target organ, tissue, or cell at a level at least 2 times, at least 5 times, at least 10 times, at least 20 times, at least 30 times, at least 40 times, at least 50 times, at least 100 times, at least 500 times, or at least 1000 times higher than the level expressed in one or more, five or more, all other organs, tissues, or cells, or at the average level of all other organs, tissues, or cells. In certain embodiments, the tissue-specific or cell-specific cell surface molecule is a cell surface receptor, such as a polypeptide receptor, which includes a region located within the cell surface membrane and extracellular region to which a targeting module can bind.In various embodiments, the methods described herein may be carried out by specifically targeting cell surface molecules expressed only in a target tissue or a subset of tissues containing a target tissue, or by specifically targeting cell surface molecules that are expressed at a high level in the target tissue compared to all, the majority, or a substantial number of other tissues, for example, cell surface molecules that are expressed at a higher level in the target tissue than at least two, at least five, at least ten, or at least twenty other tissues.

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

[0180] In certain embodiments, the components of the manipulated WNT agonist can be combined with a WNT signal-enhancing molecule to confer higher tissue specificity.

[0181] This invention is partly based on the use of engineered WNT agonists, particularly for regulating gastrointestinal epithelial proliferation in inflammatory bowel disease.

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

[0183] In certain embodiments, the engineered WNT agonist includes one or more binding domains that bind to one or more FZD receptors (FZD1-10) and one or more binding domains that bind to one or more LRP receptors (LRP5-6). In further embodiments, the binding domains of the engineered WNT agonist include one or more binding domains that bind to FZD5, FZD8, FZD1, FZD2, FZD7, FZD5, 8, FZD1, 2, 7 or FZD1, 2, 7, 5, 8; FZD4; FZD9; or FZD10, as well as one or more binding domains that bind to LRP5, LRP6, or LRP5 and 6. In further embodiments, the engineered WNT agonist includes one or more binding domains that bind to FZD5 and / or FZD8, as well as one or more binding domains that bind to LRP5 and / or LRP6. In further embodiments, the manipulated WNT agonist includes binding domains that bind to FZD5 and FZD8, and binding domains that bind to LRP6. In further embodiments, the WNT agonist includes the heavy chain sequence of SEQ ID NOs. 1, 3, 5, 7, 9, 11, 13, 15, or 17 or a variable heavy chain region derived therefrom; and the light chain sequence of SEQ ID NOs. 2, 4, 6, 8, 10, 12, 14, 16, or 18 or a variable light chain region derived therefrom.

[0184] In some embodiments, the manipulated WNT agonist includes a tissue-targeting molecule. In 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; representative sequence is NCBI polypeptide reference sequence NP_005805.1), cadherin-17 (CDH17; representative sequence is NCBI polypeptide reference sequence NP_004054.3), and mucin 13 (cell surface-related (Muc-13; representative sequence is NCBI polypeptide reference sequence NP_149038.3)) or its functional fragments or variants. In certain embodiments, the WNT agonist is administered with a binding domain that specifically binds to the inflammatory molecule. In further embodiments, the binding domain that specifically binds to 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.

[0185] In another related embodiment, the Disclosure provides a combination molecule comprising: a) an engineered WNT agonist as disclosed herein; and b) an engineered WNT signaling-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 polypeptides that specifically bind to Frizzled 5 (FZD5) and Frizzled 8 (FZD8), 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 described in SEQ ID NOs: 1 to 18. In some embodiments, the polypeptide comprises an antibody or antibody-conjugated fragment, e.g., one or more variable heavy chains or variable light chains. In some embodiments, the antibody or antibody-conjugated fragment comprises at least five or 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 CDRs present in any of these sequence combinations, one or more of the CDRs comprising one, two, or three amino acid modifications, optionally point mutations, amino acid deletions, or amino acid insertions.

[0187] This disclosure also provides engineered WNT agonists disclosed herein; as well as combination molecules comprising engineered WNT signaling-enhancing molecules comprising a first domain that binds to one or more E3 ubiquitin ligases and a second domain that binds to tissue-specific receptors.

[0188] In some embodiments, the manipulated WNT agonists of the Disclosure promote cell differentiation toward intestinal cells (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 intestinal cells. In some embodiments, a lineage trajectory inference tool called Slingshot is used to complement timestamp-based observations of cell differentiation. In some embodiments, Slingshot predicts the direction of cell differentiation from the initial starting group. As an example, cells predicted by Slingshot progress in one direction toward TA1, cup, brush, and enteroendocrine cells, and in the other direction toward intestinal cells. In some embodiments, predicted lineage trajectory pseudo-time series values ​​show that the percentage of samples treated with the manipulated WNT agonist that progress further along the intestinal cell lineage trajectory is higher compared to control-treated cells; Figure 28E provides an example of predicted pseudo-time series values. In some embodiments, the predictions for this intestinal cell lineage coincide with actual timestamp data.

[0189] In some embodiments, a reliable standard for verifying improved differentiation is that, given a number of days after inducing injury, the expression of mature, differentiated cell type markers in the manipulated WNT agonist-treated group more closely resembles that of the naive, uninjured colon compared to the control group. In some embodiments, improved differentiation is observed 6 days after treatment with the manipulated WNT agonist. In some embodiments, the samples treated with the manipulated WNT agonist include intestinal cells, goblet cells, enteroendocrine cells, tuft cells, or a combination thereof.

[0190] The evidence disclosed herein demonstrates that the Wnt mimetic molecules of this disclosure possess desired properties, including the ability to restore affected intestinal tissue to normal physiological function. In some embodiments, short-term treatment with the Wnt mimetic molecules of this disclosure (e.g., R2M13-26 or R2M13-h26) induces rapid restoration of epithelial tissue. For example, in a severe DSS model, a single injection of R2M13-26 at various doses restored normal histology of damaged colon tissue. With R2M13-h26, severely damaged epithelial barriers in an acute DSS model were completely restored within 6 days of treatment.

[0191] In addition to barrier and colon tissue restoration, in some embodiments, treatment with the Wnt mimetic of the Disclosure reduces inflammatory cytokines and disease activity indices, thereby eliminating the vicious cycle of barrier breach, microbial pathogen infiltration, tissue inflammation, and injury. In some embodiments, the Wnt mimetic of the Disclosure directly affects epithelial cells, expanding the progenitor cell pool and accelerating differentiation into all mature differentiated cell types. In some embodiments, the Wnt mimetic of the Disclosure restores Wnt signaling and stem cell niches in damaged colon tissue without further impact on the crypts after repair.

[0192] In some embodiments, treatment with the Wnt mimetic of this disclosure alone does not affect normal intestinal epithelium. In such embodiments, hyperplasia may be induced by RSPO. Together, this disclosure provides a Wnt activator with optimal tissue repair and bioactivity.

[0193] As an example, treatment with R2M13-26, a Fzd5,8-specific Wnt mimetic, resulted in rapid mucosal healing within a few days, improving tissue histology and disease activity, while simultaneously reducing symptoms of inflammation and colitis. 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 leveraging the specificity of the FZD receptor is a viable option for inducing tissue-layer-specific pathway activation. Simultaneously with the induction of Wnt target genes, R2M13-26 also induced robust increases in cell cycle gene expression across a wide range of progenitor cells, whether normal stem / progenitor cells responding to injury or altered cellular states consistent with dedifferentiation. These transcriptome changes manifested as a transient expansion of the progenitor cell pool, accelerated differentiation of appropriate secretory and absorptive lineages into colonic epithelium, and reconstruction of the epithelial barrier. The direct impact on epithelial regeneration and barrier restoration secondarily led to a decrease in inflammatory signals and infiltrating immune cells.

[0194] The circumstances of injury / damage can set stages in epithelial progenitor cell expansion. As disclosed in the accompanying examples, injury triggers inflammatory responses in all tissue layers, in addition to affecting developmental signaling pathways such as EGF and Notch. In epithelium, the interferon-gamma and NF-κB pathways become activated post-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 the NF-κB and Wnt pathways together can further promote the process of dedifferentiation toward progenitor cells in the gut (Schwitalla et al., 2013). As an example, in the DSS model of this disclosure, Wnt signaling was dramatically reduced in colonic epithelium, likely due to a reduction in the expression of certain Wnts and an increase in several Wnt antagonists. In this example, R2M13-h26 was able to overcome this defect in Wnt signaling. Therefore, R2M13-h26-induced Wnt pathway activation can, in cooperation with these inflammatory signals, enhance progenitor cell proliferation, even if transient.

[0195] Unlike the effects of RSPO, which affect both non-injured and damaged epithelium (Kim et al., 2005; Yan Kelley S. et al., 2017; Zhao et al., 2007), targeted receptor-level Wnt signaling agonism using the Wnt mimetic of this disclosure can promote specific crypt proliferation in the context of damaged tissue. For example, widespread proliferation was observed in the small and colon when the Wnt mimetic R2M13-26 was introduced together with RSPO2 in a DSS model. Improvement of DSS-induced colitis was observed with RSPO in mice, but excessive proliferation also occurred with RSPO treatment. Surprisingly, we found that the Wnt mimetic itself can specifically induce the expression of β-catenin target genes and the proliferation of epithelial cells in the injured colon.

[0196] As an example, activation of the Wnt pathway by R2M13-26 did not lead to crypt hyperproliferation or enlargement. This is a clear contrast to the effects of RSPO treatment as well as hereditary gene mutants. As previously reported, genetically removing the negative regulator Apc or expressing a constitutively active mutant of beta-catenin prevented crypt proliferation and differentiation in an unregulated manner (Barker Nick et al., 2009; Krausova & Korinek, 2014; Mah, Yan, & Kuo, 2016). However, the Wnt mimetic of this disclosure avoids these consequences by mimicking endogenous Wnt signaling and initiating pathway activation at the receptor level, in contrast to permanent genetic alterations that circumvent negative feedback. R2M13-26 enables the activation of the negative feedback mechanism by influencing the pathway at the receptor level. In this example, Axin2, which contributes to the destruction complex, was induced. Similarly, the expression of the Wnt-targeting gene E3 ubiquitin ligase Rnf43 was increased, thereby promoting the removal of the FZD receptor from the cell surface. Furthermore, in this example, R2M13-26 increased the expression of several cyclin-dependent kinase inhibitors, which potentially limited proliferation.

[0197] IV. Pharmaceutical Compositions Pharmaceutical compositions comprising the engineered WNT agonist molecules described herein and one or more pharmaceutically acceptable diluents, carriers, or excipients are also disclosed. In another embodiment, the disclosure provides pharmaceutical compositions comprising the polypeptides, engineered WNT agonists, or combination molecules disclosed herein and one or more pharmaceutically acceptable diluents, carriers, or excipients.

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

[0199] In some embodiments, WNT agonists are engineered recombinant polypeptides incorporating various epitope-binding fragments that bind to various molecules within the WNT signaling pathway. For example, FZD and LRP antibody fragments (e.g., Fab, scFv, sdAb, VHH, etc.) can be conjugated together onto a single molecule, either directly or using linkers of various sizes.

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

[0201] Conversely, WNT agonists may also be recombinant polypeptides incorporating epitope-binding fragments that bind to various molecules within the WNT signaling pathway and enhance WNT signaling. For example, a WNT agonist may be an antibody or fragment thereof that binds to the FZD receptor and / or LRP receptor and enhances WNT signaling. FZD and LRP antibody fragments (e.g., Fab, scFv, sdAb, or VHH) can be conjugated together directly or onto a single molecule using linkers of various sizes.

[0202] In further embodiments, pharmaceutical compositions are also disclosed that include an expression vector comprising a polynucleotide containing a nucleic acid sequence encoding a WNT agonist molecule described herein, such as a viral vector, and one or more pharmaceutically acceptable diluents, carriers, or excipients.

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

[0204] The target molecules, either alone or in combination, can be combined with pharmaceutically acceptable carriers, diluents, excipients, and reagents that are generally safe, non-toxic, and desirable, and are acceptable for use in mammals, e.g., humans or primates. Such excipients may be solids, liquids, semi-solids, or, in the case of aerosol compositions, gases. Examples of such carriers, diluents, and excipients include, but are not limited to, water, saline solution, Ringer's solution, dextrose solution, and 5% human serum albumin. Complementary active compounds may also be incorporated into the formulations. The solutions or suspensions used in the formulation may include sterile diluents, such as water for injection, saline solution, non-volatile oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antibacterial compounds, such as benzyl alcohol or methylparaben; antioxidants, such as ascorbic acid or sodium bisulfite; chelate compounds, such as ethylenediaminetetraacetic acid (EDTA); buffers, such as acetic acid, citric acid, or phosphoric acid; detergents, such as Tween® 20 for preventing aggregation; and compounds for adjusting osmotic pressure, such as sodium chloride or dextrose. The 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 composition may further comprise a sterile aqueous solution or dispersion, and a sterile powder for the immediate preparation of a sterile injectable solution or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water or phosphate buffered saline (PBS). In some cases, the composition can be fluid so that it can be drawn into a syringe or delivered from a syringe to a subject. In certain embodiments, this is stable under the conditions of manufacture and storage and protected against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be, for example, a solvent or dispersion medium including water, ethanol, polyols (such as glycerol, propylene glycol and liquid polyethylene glycol, etc.), and suitable mixtures thereof. Suitable fluidity can be maintained, for example, by coating, such as the use of lecithin, by maintaining the required particle size in the case of a dispersion, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it is preferable to include in the composition isotonic agents such as sugars, polyalcohols such as mannitol, sorbitol, sodium chloride. Extended absorption of the internal composition can be brought about by including in the composition agents that delay absorption, such as aluminum monostearate and gelatin.

[0206] The sterile solution is prepared by incorporating the WNT agonist, such as an antibody or its antigen-binding fragment (or the encoding polynucleotide or cells containing it), in the required amount in a suitable solvent together with one or a combination of the ingredients listed above, and then, if necessary, filter sterilizing. Generally, the dispersion is prepared by incorporating the active compound in a sterile vehicle containing the basic dispersion medium and the other required ingredients from those listed above. In the case of a sterile powder for the preparation of a sterile injectable solution, the method of preparation is vacuum drying and lyophilization, which yields a powder of the active ingredient plus any additional desired ingredients from its pre-sterile filtered solution.

[0207] In one embodiment, pharmaceutical compositions, such as controlled release formulations comprising an implant and a microencapsulated delivery system, are prepared using a carrier that protects an antibody or its antigen-binding fragment from 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 preparing such formulations will be apparent to those skilled in the art. These materials can also be obtained commercially. Liposome 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 the pharmaceutical composition in dosage unit form. As used herein, a dosage unit form refers to physically discrete units suitable as unit dosages for the subject to be treated; each unit contains a predetermined amount of the active antibody or its antigen-binding fragment calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specifications for the dosage unit forms are determined by and directly depend on the unique characteristics of the antibody or its antigen-binding fragment, the particular therapeutic effect to be achieved, and the inherent limitations in the art of making such active antibody or its antigen-binding fragment for the treatment of individuals.

[0209] The pharmaceutical composition can be included in a container, pack or dispenser, such as a syringe, for example a pre-filled syringe, together with instructions for use for administration.

[0210] The pharmaceutical compositions of the present disclosure include any pharmaceutically acceptable salt, ester, or salt of such ester, or any other compound that can (directly or indirectly) provide a biologically active antibody or its antigen-binding fragment upon administration to an animal, including a human.

[0211] This disclosure includes pharmaceutically acceptable salts of the WNT agonist molecules described herein. The term “pharmaceutically acceptable salt” means a physiologically and pharmaceutically acceptable salt of the compounds of this disclosure: i.e., a salt that retains the desired biological activity of the parent compound and does not impart any undesirable toxicological effects thereto. 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 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). For a general overview of appropriate 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, and calcium. Amines include N-N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine (see, e.g., Berge et al., "Pharmaceutical Salts", J. Pharma Sci., 1977, 66, 119). 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 a salt in the conventional manner. The free acid form can be regenerated by contacting the salt form with the acid and isolating the free acid in the conventional manner. The free acid form differs somewhat from its respective salt form in certain physical properties, e.g., solubility in polar solvents, but in other respects, the salt is equivalent to its respective free acid for the purposes of this disclosure.

[0213] In some embodiments, the pharmaceutical compositions provided herein include a therapeutically effective amount of the WNT agonist molecule described herein or a pharmaceutically acceptable salt thereof, in a mixture with pharmaceutically acceptable carriers, diluents and / or excipients, such as saline, phosphate-buffered saline, phosphates and amino acids, polymers, polyols, sugars, buffers, preservatives and other proteins. Exemplary amino acids, polymers and sugars include octylphenoxypolyethoxyethanol compounds, polyethylene glycol monostearate 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' solutions, cysteine, arginine, carnitine, alanine, glycine, lysine, valine, leucine, polyvinylpyrrolidone, polyethylene and glycols. Preferably, this formulation is stable at 4°C for at least 6 months.

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

[0215] V. How to use This disclosure provides methods for using engineered WNT agonists and / or tissue-specific WNT signaling-enhancing molecules in various therapeutic situations, for example, to modulate the WNT signaling pathway, for example, to enhance WNT signaling, as well as the administration of engineered WNT agonists and / or tissue-specific WNT signaling-enhancing molecules.

[0216] Treatment methods using manipulated WNT agonist molecules and / or tissue-specific WNT signal-enhancing molecules are also provided herein.

[0217] In certain embodiments, engineered WNT agonists may be used to increase Wnt signaling in tissues or cells. Accordingly, in some embodiments, the present invention provides a method for increasing or enhancing Wnt signaling in tissues or cells, comprising the step of contacting the tissue or cells with an effective amount of an engineered WNT agonist disclosed herein or a pharmaceutically acceptable salt thereof, wherein the engineered WNT agonist is a Wnt signaling pathway agonist. In some embodiments, the contacting step is performed in vitro, ex vivo, or in vivo. In certain embodiments, the cells are cultured cells, and the contacting step is performed in vitro. In certain embodiments, the method further includes the step of contacting the tissue or cells with one or more Wnt polypeptides or Norrin polypeptides.

[0218] The engineered WNT agonists disclosed herein may be used to treat diseases, disorders, or conditions by, for example, increasing Wnt signaling in targeted cells, tissues, or organs. Accordingly, in some embodiments, 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 a therapeutic benefit, in a subject requiring such treatment, the method comprising the step of 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: an engineered WNT agonist; one or more polynucleotides comprising a nucleic acid sequence encoding the engineered WNT agonist, e.g., DNA or mRNA, optionally modified mRNA; a vector comprising a nucleic acid sequence encoding the engineered WNT agonist, e.g., an expression vector or viral vector; or cells comprising a nucleic acid sequence encoding the engineered WNT agonist. In certain embodiments, the disease or condition is a pathological disease or disorder, or an injury, e.g., an injury resulting from a wound. In certain embodiments, the wound may be the result of another therapeutic treatment. In certain embodiments, the disease or condition involves or benefits from damaged tissue repair, healing, or regeneration. In some embodiments, the contact step is performed in vivo, i.e., the composition in question is administered to the subject.

[0219] Wnt signaling plays a crucial 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. Manipulated 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 signaling and Wnt signaling enhancers 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 affected or damaged tissue, for use in tissue regeneration, for use in cell growth and proliferation, and / or for use in tissue manipulation.

[0220] Human diseases associated with mutations in the Wnt pathway provide strong evidence for the enhancement of Wnt signaling in the treatment and prevention of these diseases. Preclinical in vivo and in vitro studies provide further evidence of the involvement of Wnt signaling in many disease states, further supporting the use of engineered WNT agonists in various human diseases. For example, the compositions of the present invention may be used to promote or enhance bone growth or regeneration, bone grafting, fracture healing, osteoporosis and osteoporotic fractures, spinal fusion, spinal cord injuries including vertebral compression fractures, preoperative spinal surgery optimization, osseointegration of orthopedic devices, tendon-osteogenesis, 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 for the treatment of alopecia; the treatment of hearing loss, including the enhancement of regeneration of sensory organs, such as the regeneration of inner and outer hair cells; the treatment of vestibular dysfunction; the treatment of macular degeneration; the treatment of retinopathy, including vitreoretinopathy and diabetic retinopathy; other diseases of retinal degeneration; Fuchs dystrophy; and other corneal diseases; as a result of stroke, traumatic brain injury, Alzheimer's disease, multiple sclerosis, multiple dystrophy, sarcopenia or cachexia, and other conditions affecting the degeneration or integrity of the blood-brain barrier.

[0221] In certain embodiments, the present invention provides a method for treating a subject having a disease or disorder related to reduction of WNT signaling or in which increased WNT signaling may be beneficial, the method comprising the step of administering to the subject an effective amount of an engineered WNT agonist or a pharmaceutical composition comprising an engineered WNT agonist. In certain embodiments, the disease or disorder includes, but is not limited to, oral mucositis, short bowel syndrome, inflammatory bowel disease (IBD), graft-versus-host disease (GVHD), alcoholic hepatitis, celiac disease, radiation-induced gastrointestinal mucositis and chemotherapy-induced gastrointestinal mucositis; treatment of metabolic syndrome, dyslipidemia, diabetes, pancreatitis, conditions in which the pancreatic exocrine or endocrine tissue is damaged; treatment of conditions in which enhanced epidermal regeneration is desired, e.g., epidermal wound healing, diabetic foot ulcers, syndromes involving tooth, nail, or skin dysplasia, and conditions in which angiogenesis is beneficial; myocardial infarction, coronary artery disease, heart failure; immunodeficiency, graft-versus-host disease, acute kidney injury, The following conditions are 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 or after hepatitis C or B virus infection or antiviral therapy, alcoholic liver disease, alcoholic hepatitis, non-alcoholic liver disease with steatohepatitis or steatohepatitis, treatment for hearing loss including reduction of inner and outer hair cells, vestibular dysfunction, macular degeneration, vitreoretinopathy, diabetic retinopathy, other retinal degenerative diseases, Fuchs dystrophy, treatment for 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 manipulated WNT agonists and compositions of the present invention may also be used for the treatment of oral mucositis, short bowel syndrome, Crohn's disease (CD), and ulcerative colitis (UC), in particular inflammatory bowel disease (IBD), including CD with fistula formation, and other gastrointestinal disorders; for the treatment of metabolic syndrome, dyslipidemia, diabetes, pancreatitis, and conditions in which the exocrine or endocrine tissue of the pancreas is damaged; for conditions in which enhanced epidermal regeneration is desired, such as epidermal wound healing, diabetic foot ulcers, and syndromes involving tooth, nail, or skin malformations, and in which angiogenesis is beneficial; for the treatment of myocardial infarction, coronary artery disease, and heart failure; for the treatment of hematopoietic cell growth, such as enhanced hematopoietic stem cell transplantation from bone marrow, peripheral blood mobilization, immunodeficiency, and graft-versus-host disease; for the treatment of acute kidney injury and chronic kidney disease; and for the treatment of lung diseases, chronic obstructive pulmonary disease (COPD), pulmonary fibrosis including idiopathic pulmonary fibrosis, and enhanced regeneration of lung tissue. The compositions of the present invention may also be used for the regeneration of hepatocytes, for example, to enhance liver regeneration, treat cirrhosis, enhance liver transplantation, treat acute liver failure, chronic liver disease caused by or after hepatitis C or B virus infection or antiviral therapy, alcoholic liver disease including alcoholic hepatitis, and non-alcoholic liver disease with steatohepatitis or fatty liver disease. The compositions of the present invention can treat diseases and disorders in which regenerative cell growth is desired, without limitation.

[0223] Human genetics involving loss-of-function or gain-of-function mutations in Wnt signaling components provides strong evidence supporting the enhancement of Wnt signaling for bone growth. Conditions in which enhanced bone growth is desired may include, but are not limited to, fractures, grafts, periprosthetic devices, osteoporosis, osteoporotic fractures, spinal fusion, vertebral compression fractures, preoperative optimization for spinal surgery, osteonecrosis of the jaw, dental implants, periodontal disease, and maxillofacial reconstruction. Manipulated WNT agonists enhance and promote Wnt signaling, which is critical 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, resulting in the activation of stem cells in the bone marrow.

[0224] In some embodiments, bone regeneration is enhanced by contacting a responsive cell population, such as bone marrow, bone progenitor cells, or bone stem cells, with an effective dose of an engineered WNT agonist disclosed herein. Methods for regenerating bone tissue benefit from the administration of an engineered WNT agonist, which may be systemic or localized. In some such embodiments, the contact is carried out in vivo. In other such embodiments, the contact is carried out ex vivo. The molecule may be localized to the site of action by loading it onto a matrix, for example, which may be biodegradable as needed and may provide sustained release of the active agent as needed. Matrix carriers include, but are not limited to, absorbable collagen sponges, ceramics, hydrogels, polymeric microspheres, nanoparticles, and bone cement.

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

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

[0227] The methods disclosed herein, including methods for treating or preventing a bone disease or disorder, include a method comprising providing to a subject in need thereof both an engineered WNT agonist and an antiresorptive agent. In certain embodiments, the method is 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, the method comprising providing to the subject an effective amount of an engineered WNT agonist, wherein the engineered WNT agonist is an agonist of the Wnt signaling pathway. In certain embodiments, the method further comprises providing to the subject an antiresorptive agent. In certain embodiments, the subject is diagnosed with or at risk of osteoporosis, optionally postmenopausal osteoporosis. Various antiresorptive agents are known in the art and include, but are not limited to, those disclosed herein.

[0229] When a modified WNT agonist is provided to a subject in combination with another therapeutic agent, such as a bone resorption inhibitor, the two agents may be provided in the same pharmaceutical composition or in different pharmaceutical compositions. The two agents may be provided to the 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 the subject during overlapping periods.

[0230] Compositions comprising one or more engineered WNT agonists disclosed herein (or polynucleotides encoding engineered WNT agonists, or vectors or cells containing polynucleotides encoding engineered WNT agonists) may be used for in vivo treatment of skeletal tissue defects. "Skeletal tissue defect" means a defect in bone or other skeletal connective tissue at any site where restoration of bone or connective tissue is desired, regardless of the origin of the defect, for example, whether it is the result of surgical intervention, tumor removal, ulceration, implantation, fracture, or other traumatic or degenerative condition. The compositions of the present invention may be used as part of a regimen for restoring cartilage function of connective tissue to repair defects or lesions of cartilage tissue such as degenerative wear and arthritis, trauma to tissue, meniscus replacement, meniscectomy, dislocation of joint due to torn ligament, joint misalignment, fracture, or genetic disorders.

[0231] Manipulated WNT agonists may also be used to treat periodontal disease, a leading cause of tooth loss and associated with several systemic conditions. In some embodiments, tooth or underlying bone regeneration is enhanced by contact with a responsive cell population. In some such embodiments, the contact is performed in vivo. In other such embodiments, the contact is performed ex vivo, followed by implantation of activated stem cells or progenitor cells. Molecules may be localized to the site of action by loading them onto a matrix, for example, which is biodegradable as needed and provides sustained release of the active agent as needed. Matrix carriers include, but are not limited to, absorbable collagen sponges, ceramics, hydrogels, bone cements, polymeric microspheres, and nanoparticles.

[0232] Multiple studies have shown that Wnt signaling and the biology of R-spongin can promote sensory hair cell regeneration in the inner ear after injury, aging, or degeneration. The compositions of the present invention may also be beneficial for the loss of sensory hair cells in the inner ear that is associated with hearing loss or vestibular dysfunction. In the inner ear, the auditory organs house mechanosensitive hair cells required to convert sound vibrations into electrical impulses. The vestibular organs, consisting of the semicircular canals (SSC), utricle, and saccule, also contain sensory hair cells to detect head position and movement. The compositions of the present invention may be used to enhance auditory regeneration, for example, in injection; in a matrix or other depot system; or in other topical applications to the ear.

[0233] Manipulated WNT agonists can also be used for the regeneration of retinal tissue. In the adult mammalian retina, Müller glial cells can regenerate retinal cells, including photoreceptor cells, for example, after neurotoxic injury in vivo. Wnt signaling and Wnt signaling enhancers can promote the proliferation of Müller glial-derived retinal progenitor cells after injury or during degeneration. The compositions of the present invention can also be used for the regeneration of tissues and other cell types in the eye. For example, the compositions of the present invention may be beneficial for age-related macular degeneration (AMD), other retinal degenerative diseases, corneal diseases, Fuchs dystrophy, vitreoretinopathy, and genetic diseases. AMD is characterized by a progressive decline in central visual field and visual acuity. Fuchs dystrophy is characterized by a progressive loss of corneal endothelial cells. Wnt signaling and Wnt signaling enhancement can promote the regeneration of corneal endothelium, retinal epithelium, and other ocular tissues. In other embodiments, the compositions of the present invention may be used for retinal regeneration and the treatment of macular degeneration, for example, in injection; in a matrix or other depot system; or in other topical applications to the eye.

[0234] Certain populations of proliferative cells for homeostatic regeneration of hepatocytes, such as Axyn 2-positive cells in the pericentral region, have been identified through lineage follow-up studies. Lineage follow-up studies have also identified further potential hepatic progenitor cells, including but not limited to Lgr-positive cells. Self-regenerating hepatocytes, as well as other populations of potential progenitor cells, including Lgr5-positive and Axyn 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 hepatic failure, as well as chronic liver disease, have shown hepatocyte recovery and regeneration that benefit from enhancing Wnt signaling.

[0235] In certain embodiments, compositions comprising the engineered WNT agonists disclosed herein (or polynucleotides encoding engineered WNT agonists, or vectors or cells containing polynucleotides encoding engineered WNT agonists) may be used to promote liver regeneration, reduce fibrosis, and / or improve liver function. In certain embodiments, compositions and methods disclosed herein may be used to increase liver weight, increase the ratio of liver to body weight, increase the number of PCNA and pH3-positive nuclei in the liver, increase the expression of Ki67 and / or cyclin D1 in the liver, increase hepatocyte proliferation and / or mitosis, reduce fibrosis after chronic liver injury, or increase hepatocyte function.

[0236] In certain embodiments, compositions of the present invention may be used for the treatment of acute liver failure, acute alcoholic liver injury, chronic liver disease caused by or after hepatitis C or B virus infection or antiviral therapy, chronic alcoholic liver disease, alcoholic hepatitis, non-alcoholic fatty liver disease and non-alcoholic steatohepatitis (NASH), cirrhosis and severe chronic liver disease of all causes, and for enhancing hepatocyte regeneration. Methods for regenerating liver tissue benefit from the administration of compounds of the present invention, which may be systemic or localized. These include, but are not limited to, methods of systemic administration and methods of localized administration, such as by injection into liver tissue, by injection into veins or blood vessels leading to the liver, or by implantation of sustained-release formulations.

[0237] In certain embodiments, compositions comprising the 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 any liver disease or disorder, including but not limited to, or to treat or prevent liver injury or disorder resulting from any of the following: acute liver failure (all causes), chronic liver failure (all causes), cirrhosis, and hepatic fibrosis. Hepatitis (all causes), portal hypertension, alcoholic liver disease including alcoholic hepatitis, non-alcoholic steatohepatitis (NASH), non-alcoholic 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 graft" in liver surgery and transplantation. (for size) Syndrome, congenital liver disease and disorder, genetic disorder, degeneration, aging, drugs, or any other liver disorder or defect resulting from injury.

[0238] Wnt signaling plays a crucial role in the regeneration of various epithelial tissues. Treatment with the compounds of the present invention is beneficial for various epidermal conditions. Mucositis occurs when there is a breakdown of rapidly dividing epithelial cells lining the gastrointestinal tract, making the mucosal tissue susceptible to ulceration and infection. The inner layer of epithelium covering the mouth, known as 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. Furthermore, 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, and syndromes involving tooth, nail, or skin malformations. The molecules of the present invention can be used for all of these conditions, in which case regenerative cells are brought into contact 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. Contact may include, for example, topical application of gels, lotions, creams, etc., to a targeted site, including intradermal and subcutaneous applications.

[0239] In addition to the skin and gastrointestinal tract, Wnt signaling and its enhancement and promotion play important roles in the repair and regeneration of tissues, including the pancreas, kidneys, and lungs, in preclinical models. Manipulated WNT agonists may be beneficial for various disease conditions involving the exocrine and endocrine parts of the pancreas, kidneys, or lungs. Manipulated WNT agonists may be used in the treatment of metabolic syndrome; diabetes mellitus; acute or chronic pancreatitis; exocrine pancreatic insufficiency; acute kidney injury; chronic kidney disease; chronic obstructive pulmonary disease (COPD); pulmonary fibrosis, including but not limited to idiopathic pulmonary fibrosis (IPF); and other conditions causing loss of pulmonary epithelial tissue. Methods for regenerating these tissues benefit from the administration of the compounds of the present invention, which may be systemic or localized.

[0240] Epidermal Wnt signaling, in coordination with signaling mediated by other developmental factors, is crucial for adult hair follicle regeneration. Alopecia is a common problem, androgenic alopecia, often called male pattern baldness, is the most common form of hair loss in men. In some embodiments, hair follicle regeneration is enhanced by contacting a responsive cell population with the molecules of the present invention. In some such embodiments, the contact is carried out in vivo. In other such embodiments, the contact is carried out ex vivo. The molecules may be localized to the site of action, for example, in the form of a topical lotion, gel, or cream.

[0241] Stroke, traumatic brain injury, Alzheimer's disease, multiple sclerosis, and other conditions affecting the blood-brain barrier (BBB) ​​can be treated with engineered WNT agonists. Angiogenesis is crucial for ensuring oxygen and nutrients reach many tissues throughout the body, and is particularly important for the central nervous system (CNS) because nerve tissue is highly susceptible to hypoxia and ischemia. CNS endothelial cells that form the BBB differ from endothelial cells in non-nerve tissues in that they are highly polarized cells held together by tight junctions and express specific transporters. Wnt signaling modulates CNS angiogenesis and / or function. For conditions where the BBB is impaired, administration of the compounds of the present invention, which may be systemic or localized, may be beneficial, for example, by direct injection, intrathecal administration, or implantation of a sustained-release formulation. Furthermore, Wnt signaling is actively involved in neurogenesis and plays a role in post-injury neuroprotection. The compositions of the present invention can also be used to treat spinal cord injury, other spinal cord diseases, stroke, traumatic brain injury, and other conditions.

[0242] Wnt signaling also plays a role in angiogenesis. Manipulated WNT agonists may be beneficial in treating conditions where angiogenesis is advantageous, such as myocardial infarction, coronary artery disease, heart failure, diabetic retinopathy, and conditions resulting from genetic disorders. Methods for regenerating these tissues benefit from the administration of the compounds of the present invention, which may be systemic or localized.

[0243] In certain embodiments, the method of the present invention promotes tissue regeneration in tissue that has suffered damage or a reduction or loss of tissue or cells. The loss or damage may be anything that causes a reduction in cell number, including disease or injury. For example, trauma may be constituted by an accident, autoimmune disorder, side effects of treatment, or pathological condition. Tissue regeneration increases the number of cells in the tissue, preferably allows for the reconstruction of connections between cells in the tissue, and more preferably restores the functionality of the tissue.

[0244] The terms “administer,” “introduce,” or “deliver,” as used herein, refer to the delivery of a composition to cells, to cells, tissues and / or organs of interest, or to the subject. Such administering or introducing steps may be carried out 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 topically, for example, externally or intramuscularly. In some embodiments, the composition is administered to target tissue, for example, bone, joint, ear tissue, eye tissue, gastrointestinal tract, skin, wound site, or spinal cord. The methods of the present invention can be carried out in vivo or ex vivo. In some embodiments, the step of contacting target cells or tissue with a WNT agonist is carried out ex vivo, accompanied by subsequent implantation of cells or tissue, for example, activated stem cells 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] Dosage and dosage regimens may depend on various factors readily determined by a physician, such as the nature of the disease or disorder, the characteristics of the subject, and the subject's medical history. In certain embodiments, the amount of manipulated WNT agonist administered or provided to a subject is in the range of 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 body weight of the subject. In certain embodiments of any of the methods disclosed herein, the WNT agonist is administered intravenously or subcutaneously 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, subjects are administered approximately 0.5 to 100 mg of WNT agonist per kg of body weight, or approximately 2 to 50 mg of WNT agonist per kg of body weight, for example, approximately 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 35 mg / kg, 40 mg / kg, 45 mg / kg, or 50 mg / kg of WNT agonist. In certain embodiments, subjects are administered approximately 25 mg, 75 mg, 250 mg, 750 mg, 1500 mg, or 2250 mg of WNT agonist. In certain embodiments, subjects are administered R2M13-h26 intravenously or subcutaneously at a dose of approximately 3 to 30 mg per kg of body weight 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.

[0247] The terms “treatment,” “to treat,” etc., are used herein to generally mean obtaining a desired pharmacological and / or physiological effect. This effect may be prophylactic in terms of completely or partially preventing a disease or its symptoms, e.g., reducing the likelihood of the disease or its symptoms occurring in a subject, and / or therapeutic in terms of partial or complete cure of the disease and / or adverse effects resulting from the disease. “Treatment,” as used herein, covers any treatment of a disease in a mammal and includes: (a) preventing the onset of the disease in a subject that is susceptible to the disease but has not yet been diagnosed with it; (b) inhibiting the disease, i.e., halting its development; or (c) mitigating 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 a disease or injury. Treatment of an ongoing disease is particularly targeted, where the treatment stabilizes or reduces undesirable clinical symptoms in the patient. Such treatment is preferably performed before complete loss of function in the affected tissue. The treatment is preferably administered during the symptomatic stage of the disease, and in some cases, after the symptomatic stage. In some embodiments, the method of the subject results in therapeutic benefits, such as prevention of the onset of disability, cessation of the progression of disability, or reversal of the progression of disability. In some embodiments, the method of the subject includes a step of detecting that the therapeutic benefit has been achieved. Those skilled in the art will understand that such measures of therapeutic effectiveness are applicable to the specific disease being modified and will recognize appropriate detection methods to be used to measure therapeutic effectiveness.

[0248] In certain embodiments, the method disclosed herein results in one or more of the following PK / PD parameters after administration of the manipulated 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 days; 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 the manipulated 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 manipulated WNT agonists, optionally in combination with Norrin or R-spongin polypeptides. In certain embodiments, cells or tissues are contacted ex vivo, in vitro, or in vivo. Such methods may be used, for example, to generate cells, tissues, or organoids for therapeutic use, which are to be transplanted or grafted into a subject. These may also be used to generate cells, tissues, or organoids for research use. Manipulated WNT agonists are widely applied in non-therapeutic methods, such as in vitro research methods.

[0250] In certain embodiments, engineered WNT agonists, including those disclosed herein, may be used to preserve cells, tissues, organs, or organoids, for example, tissues or organs for transplantation. For example, cells, tissues, organs, or organoids can be brought into contact with the engineered WNT agonist in vivo or ex vivo. In situations where cells, tissues, or organs are being preserved for transplantation, cells, tissues, organs, or organoids can be brought into contact with the engineered WNT agonist while they are still in the donor (i.e., before removal from the donor) and / or after removal from the donor. By means of this, the viability of cells, tissues, or organs can be maintained or enhanced, for example, during storage or before transplantation to a recipient. In certain embodiments, cells, tissues, or organs are perfused with a composition or solution containing the engineered WNT agonist. In certain embodiments, a particular organ tissue is brought into contact with a WNT superagonist molecule to maintain the viability of that tissue. In certain embodiments, the organ tissue is a donor organ tissue to be transplanted to a recipient in need of transplantation. In certain embodiments, donor organ tissue is perfused in vivo with a solution containing the manipulated WNT agonist disclosed herein, for example, before the organ tissue is removed from the donor. In certain embodiments, donor organ tissue is perfused ex vivo with a solution containing the manipulated WNT agonist disclosed herein, for example, during storage or transport from donor to recipient. In certain embodiments, organ tissue that has been in contact with the manipulated WNT agonist remains viable for transplantation at least 10%, at least 20%, at least 50%, or at least 100% longer than organ tissue that has not been in contact with the manipulated WNT agonist. In certain embodiments, the organ tissue is liver tissue.

[0251] In certain embodiments, engineered WNT agonists, including those disclosed herein, are used for the enlargement and / or maintenance of ex vivo tissue, e.g., skin tissue. In certain embodiments, the tissue is isolated from a donor or patient. The tissue can be brought into contact with an engineered WNT agonist in vivo or ex vivo (e.g., maintained or cultured in the presence of the engineered WNT agonist). In certain embodiments, the tissue is brought into contact ex vivo, e.g., by perfusing with a composition containing the engineered WNT agonist.

[0252] In another embodiment, engineered WNT agonists, including those disclosed herein, may be used to generate or maintain organoids or organoid cultures. For example, an organoid culture can be brought into contact with an engineered WNT agonist by culturing the organoids in a medium containing the engineered WNT agonist, for example. In a particular embodiment, an organoid culture is generated, grown, or maintained by contacting one or more engineered WNT agonists disclosed herein. In a particular embodiment, the engineered WNT agonist is present in a culture medium used to grow or maintain organoid tissue.

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

[0254] Wnt signaling is a crucial component of stem cell culture. Examples include the stem cell culture media 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 manipulated WNT agonists disclosed herein are suitable alternatives to or can be combined with R-sponge for use in these stem cell culture media.

[0255] Accordingly, in one embodiment, the Disclosure provides a method for enhancing the proliferation of stem cells, comprising the step of contacting stem cells with one or more manipulated WNT agonists disclosed herein. In one embodiment, the Disclosure provides a cell culture medium comprising one or more manipulated WNT agonists disclosed herein. In some embodiments, the cell culture medium may be any cell culture medium already known in the Art, which typically comprises Wnt or R-spongin, but in which Wnt or R-spongin is replaced (completely or partially) with or supplemented with one or more manipulated WNT agonists 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), which are incorporated herein by reference in their entirety.

[0256] Stem cell culture media often contain additional growth factors. Therefore, this method may further include a step of supplying growth factors to stem cells. 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 mitogenic factor for various ectoderm and mesoderm cultured cells, both in vivo and in It has a remarkable effect on the differentiation of certain cells in vitro as well as several fibroblasts under cell culture. EGF precursor cells exist as membrane-bound molecules that are cleaved by proteolysis to produce a 53-amino acid peptide hormone that stimulates cells. Therefore, EGF or other mitotic growth factors can be supplied to stem cells. During stem cell culture, mitotic growth factors can be added to the culture medium every two days, and at the same time, the culture medium is preferably replaced every four days. Generally, mitotic growth 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 α FGF; vii) TGF-alpha and KGF; viii) TGF-alpha and FGF7; ix) or TGF-alpha and α FGF. In certain embodiments, the Disclosure comprises a stem cell culture medium comprising, for example, one or more of the manipulated WNT agonists disclosed herein in combination with, as may be, one or more of the growth factors or combinations thereof described herein.

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

[0258] In some embodiments, manipulated WNT agonists are 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 (U.S. Patent 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] This invention is partly based on the use of engineered WNT agonists, particularly for regulating gastrointestinal epithelial proliferation in inflammatory bowel disease.

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

[0261] In certain embodiments, the present invention also provides a method for treating a subject suffering from a gastrointestinal disorder, comprising the step of administering to the subject a tissue-specific WNT signaling-enhancing molecule. In certain embodiments, the WNT signaling-enhancing 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 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 another embodiment, the first domain comprises an R-spongin (RSPO) polypeptide. In further embodiments, the RSPO polypeptide is selected from the group consisting of RSPO-1, RSPO-2, RSPO-3, and RSPO-4. In certain embodiments, 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, a tissue-targeting molecule is incorporated into the engineered agonist or Wnt signal-enhancing molecule. In further embodiments, the tissue-targeting molecule is an antibody or 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 functional fragments or variants 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 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. In some embodiments, the gastrointestinal disease is an inflammatory bowel disease. In further embodiments, the inflammatory bowel disease is selected from the group consisting of Crohn's disease (CD), CD with fistula formation, and ulcerative colitis (UC).

[0262] In another embodiment, the present invention provides a method for treating a subject suffering from a gastrointestinal disorder, comprising the step of 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 may be administered at the same time or at different times. In some embodiments, the subject receives effective amounts of both the engineered WNT agonist and the engineered tissue-specific WNT signal-enhancing molecule for 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, as well as 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 manipulated WNT signal-enhancing molecule includes 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 includes an R-spongin (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 includes a first furin domain and a second furin domain. In 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 agonists are disclosed in Table 3.In some embodiments, the engineered WNT agonist and the engineered tissue-specific WNT signaling-enhancing molecule are administered together 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 even 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, inflammatory bowel disease is selected from the group consisting of Crohn's disease (CD), CD with fistula formation, and ulcerative colitis (UC).

[0263] In another embodiment, the present invention provides a method for treating a subject suffering from a gastrointestinal disorder, comprising the step of administering to the subject an engineered WNT agonist and an engineered tissue-specific WNT signal-enhancing 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-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. 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-spongin (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 even 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, a tissue-targeting molecule is incorporated into the combination 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 functional fragments or variants 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 to the inflammatory molecule is an antagonist of the inflammatory molecule.In further embodiments, the antagonist of the inflammatory molecule is a TNFα antagonist, an IL-12 antagonist, an IL-12 and IL-23 antagonist, or an IL-23 antagonist. In certain embodiments, the gastrointestinal disease is inflammatory bowel disease. In further embodiments, inflammatory bowel disease is selected from the group consisting of Crohn's disease (CD), CD with fistula formation, and ulcerative colitis (UC).

[0264] In any particular embodiment of the methods disclosed herein, the manipulated WNT agonist is selected from those disclosed in: PCT Publication WO2016 / 040895; U.S. Publication US2017-0306029; U.S. Publication US2017-0349659; PCT Publication WO2019 / 126398; or PCT Publication WO2020 / 01030. In any particular embodiment of the methods disclosed herein, the tissue-specific WNT signal-enhancing molecule is selected from those disclosed in: PCT Publication WO2018 / 140821; U.S. Publication US2020-0048324; or PCT Publication WO2020 / 14271. All of these are incorporated herein by reference in their entirety.

[0265] In relevant embodiments, the Disclosure provides a method for treating a subject suffering from a gastrointestinal disorder, comprising the step of administering to the subject an engineered WNT agonist, an engineered WNT signaling enhancer 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 selected as appropriate from the group consisting of Crohn's disease (CD), CD with fistula formation, and ulcerative colitis (UC). Any method disclosed herein may be carried out using any of the engineered WNT agonists, engineered WNT signaling enhancers, 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, subjects are administered intravenously at a dose of approximately 3 to 30 mg of R2M13-h26 per kg of body weight 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 a disulfide bond. In certain embodiments, the method is used to treat IBD, e.g., moderate to severe IBD, using a WNT agonist disclosed herein, e.g., R2M13-h26. In certain embodiments, IBD is selected from the group consisting of Crohn's disease, Crohn's disease with fistula formation, or ulcerative colitis.

[0267] Any method disclosed herein may also be carried out using a combination of a WNT agonist molecule and a tissue-specific WNT signal-enhancing molecule, or a combination molecule containing, for example, both the WNT agonist molecule and the tissue-specific WNT signal-enhancing molecule (combination molecule) described herein. In one embodiment, the WNT agonist molecule and / or the tissue-specific WNT signal-enhancing molecule, or the combination molecule, is provided to a subject having a disease involving inappropriate or deregulated WNT signaling. In certain embodiments, the method disclosed herein includes the step of providing the WNT agonist molecule and / or the tissue-specific WNT signal-enhancing molecule alone, in combination, or as a combination molecule to a subject in need. In certain embodiments, the WNT agonist molecule and the tissue-specific WNT signal-enhancing molecule are provided to the subject in the same or different pharmaceutical compositions. In some embodiments, the WNT agonist molecule and the tissue-specific WNT signal-enhancing molecule are provided to the subject at the same time or at different times, for example, one before or after the other. In some embodiments, the method includes the step of providing a subject with an effective amount of a WNT agonist molecule and / or a tissue-specific WNT signal-enhancing molecule. In some embodiments, the effective amount of the WNT agonist molecule and the tissue-specific WNT signal-enhancing molecule are present in the subject for an overlapping period, for example, one day, two days, or one week. In other embodiments, the method disclosed herein includes the step of providing a subject requiring it with a combination molecule comprising a WNT agonist molecule and a tissue-specific WNT signal-enhancing molecule (combination molecule).

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

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

[0270] WNT agonists and / or tissue-specific WNT signaling-enhancing molecules, or combination molecules, may be used to treat gastrointestinal disorders, including but not limited to inflammatory bowel diseases, including but not limited to Crohn's disease, Crohn's disease with fistula formation, and ulcerative colitis. In certain embodiments, WNT agonists may be used to treat gastrointestinal disorders, including but not limited to inflammatory bowel diseases, including but not limited to Crohn's disease with or without fistula formation, 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 disorders in which the epithelial barrier is impaired or the intestine is shortened. In particular, the present invention provides WNT / β-catenin signaling WNT / β-catenin agonists that enhance the regeneration of intestinal epithelium as a result of injury caused by these disorders. In certain embodiments, the WNT agonist is R2M13-h26.

[0271] Manipulated WNT agonists may also be used to modulate various tissue and / or cellular processes, as well as to modulate gene expression within tissues and / or cells. In certain embodiments, this disclosure provides a method for modulating gene expression, comprising the step of contacting a subject, organ, tissue, or cell with a manipulated WNT agonist, for example, as disclosed herein in Table 3. A manipulated WNT agonist can be administered to a subject, and an organ, tissue, or cell can be contacted with the manipulated WNT agonist in vivo, ex vivo, or in vitro. In certain embodiments, the method results in upmodulation or downmodulation of one or more genes in the WNT signaling pathway, including but not limited to any of the genes disclosed in Tables 4-8. 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 5-fold, at least 10-fold, or at least 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 interest 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 the corresponding cells or tissue not in contact with the manipulated WNT agonist.

[0272] In some embodiments, the Disclosure provides a method for modulating the expression of WNT pathway molecules in one or more tissues and / or cells of a subject having a gastrointestinal disorder, the method comprising the step of 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 manipulated Wnt agonist, the expression of WNT pathway molecules (genes or proteins) in one or more tissues and / or cells of interest increases 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 decreases by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%. In certain embodiments, the tissue is 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 entero cells (AltEntero), intestinal progenitor cells (EnteroPrecur), goblet cell 1, goblet cell 2, enteroendocrine cells, or tuft cells. In a particular embodiment, the WNT agonist is R2M13-h26.

[0273] In another embodiment, the Disclosure provides a method for stimulating tissue repair in a subject having a gastrointestinal disorder, comprising the step of administering to the subject an engineered WNT agonist or pharmaceutical composition disclosed herein. In a particular embodiment, tissue repair is stimulated (or modulated by the method) by modulation of at least one WNT pathway molecule selected from the group consisting of genes related to the cell cycle, genes related to the regeneration and differentiation of stem cells and progenitor cells, genes related to the repair and barrier restoration of epithelial cells, and / or genes listed in any of Tables 4-8. In certain embodiments, the genes related to the cell cycle are those provided in Table 4, or selected from 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 genes related to the regeneration and differentiation of stem cells and progenitor cells are those provided in Table 8, and selected from Axin2, Id1, Hmga2, Nhp2, Foxq1, and Adh1. In certain embodiments, the genes associated with epithelial cell repair and barrier restoration are those provided in Table 6, or selected from Apex1, Agr2, B3gnt7, Fcgbp, Muc2, Muc3, Tff3, Zg16, and Sprr2a3. In certain embodiments, after administration of the engineered Wnt agonist, the expression of the gene in one or more tissues and / or cells of interest is increased by at least 2-fold, at least 5-fold, at least 10-fold, or at least 20-fold, or decreased 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 Disclosure provides a method for reducing inflammation in a subject (or tissue or cells of such subject) having a gastrointestinal disorder, comprising the step of administering to the subject an engineered WNT agonist or pharmaceutical composition disclosed herein. In certain embodiments, inflammation is reduced by (or modulated by) a gene provided in Table 5, or by modulation of at least one WNT pathway molecule selected from the group consisting of Adamdec1, Atf3, Gpx2, Gsta3, Gstm1, Gdf15, Il18, Nox1, Reg4, Sycn, Selenbp1, Tgfbr2, and Timp3. In certain embodiments, inflammation is reduced in gastrointestinal tissue, and optionally in 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), intestinal progenitor cells (EnteroPrecur), goblet cell 1, goblet cell 2, or enteroendocrine cells. In certain embodiments, after administration of the engineered Wnt agonist, the expression of WNT pathway molecules in one or more tissues and / or cells of interest increases 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 decreases 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 a cell surface molecule.

[0276] The present invention also provides combination therapies with known and novel treatments for gastrointestinal disorders, particularly inflammatory bowel disease (IBD). For example, WNT agonists can be combined with several known therapies for IBD, including but not limited to 5-aminosalicylates (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 novel agents that may become available on the market for the aforementioned disorders. The therapeutic agents described above may be administered sequentially or simultaneously with the molecule of the present invention.

[0277] Therapeutic agents (e.g., modified WNT agonists and / or tissue-specific WNT signaling-enhancing molecules or combination molecules) may be administered before, during, or after the onset of a disease or injury. Treatment of a disease in progress, where the treatment stabilizes or reduces undesirable clinical symptoms in the patient, is of particular interest. Such treatment is preferably performed before complete loss of function in the affected tissue. The therapies of the subject are preferably performed during the symptomatic stage of the disease, and in some cases, after the symptomatic stage of the disease. In some embodiments, the methods of the subject result in therapeutic benefits, such as prevention of the onset of disability, cessation of the progression of disability, or reversal of the progression of disability. In some embodiments, the methods of the subject include a step of detecting that a therapeutic benefit has been achieved. Those skilled in the art will understand that such measures of therapeutic effectiveness are applicable to specific diseases to which the treatment is modified and will recognize appropriate detection methods used to measure therapeutic effectiveness.

[0278] All of the above-mentioned U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications mentioned herein and / or listed in the application data sheets are incorporated herein by reference in their entirety.

[0279] As stated above, specific embodiments of the Disclosure are described herein for illustrative purposes, but it should be understood that various modifications can be made without departing from the spirit and scope of the Disclosure. Therefore, the Disclosure is not limited to those provided for by the appended claims.

[0280] The scope of the present invention is best understood by referring to the following examples, which are not intended to limit the present invention to any particular embodiment. [Examples]

[0281] (Example 1) General method For example, the following standard methods in molecular biology were used: 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), complex carbohydrate 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, fusion protein production, and protein glycosylation are described; for example, 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, See NJ, pp. 384-391. For the production, purification, and fragmentation of polyclonal and monoclonal antibodies, see, for example, 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, as described above. Standard techniques for characterizing ligand / receptor interactions are available. See, for example, Coligan et al. (2001) Current Protocols in Immunology, Vol. 4, John Wiley, Inc., New York.

[0283] Flow cytometry methods, including fluorescence-activated cell sorting and detection systems (FACS®), are available; see, for example, 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 are available, including nucleic acid primers and probes, polypeptides, and antibodies, for example, for use as diagnostic reagents. See Molecular Probes (2003) Catalogue, Molecular Probes, Inc., Eugene, Oreg.; Sigma-Aldrich (2003) Catalogue, St. Louis, Mo.

[0284] Standard methods for histology of the immune system are described. For example, 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, See McGraw-Hill, New York, NY.

[0285] For example, software packages and databases are available for determining antigen fragments, leader sequences, protein folding, functional domains, glycosylation sites, and sequence alignments. Examples include GenBank, Vector NTI® Suite (Informax, Inc., Bethesda, Md.), GCG Wisconsin Package (Accelrys, Inc., San Diego, Calif.), DeCypher® (TimeLogic Corp., Crystal Bay, Nev.), and 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 See Heijne (1983) Eur. J. Biochem. 133: 17-21; von Heijne (1986) Nucleic Acids Res. 14: 4683-4690.

[0286] The exemplary methods and materials used in this disclosure are shown below.

[0287] RNA in situ hybridization: mRNA expression is converted to RNAscope in situ hybridization (ACD). Detection was performed using RNAscope Bio. The RNAscope probes used are listed below. For colorimetric quantitative visualization, images were acquired using a Leica DMi8 microscope equipped with a DFC7000T color camera, following the standard RNAscope® 2.5 HD Assay-Red protocol (www.acdbio.com). For fluorescence RNAscope in situ hybridization, the standard RNAscope Multiplex Fluorescent Reagent Kit v2 Assay protocol (ACD Bio Document#323100-USM) was followed, and it was used in combination with the TSA Plus Cyanine 3 System and TSA Plus Cyanine 5 System. Fluorescence 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 the Applied Biosystems TaqMan Fast Advanced Master Mix (Thermofisher, 4444557) was used for qPCR.

[0289] Affinity measurement: The binding kinetics of the Fzd binding moieties and Fab of R2M13 and R2M13-26 to the CRDs of Fzd5 and 8 were determined by biolayer interferometry (BLI) using an Octet Red 96 instrument (PALL ForteBio, Fremont, CA) at 30°C and 1000 rpm with a streptavidin (SA) biosensor. Biotinylated CRDs of Fzd, diluted to 25 nM in running buffer (PBS, 0.05% Tween®-20, 0.5% BSA, pH 7.2), were captured with the SA biosensor and then immersed in wells containing R2M13 Fab protein at various concentrations in running buffer, or in wells containing only running buffer as a reference channel. The KD for each binding factor was calculated using Octet System software based on fitting to a 1:1 binding model. The binding specificity of R2M13 IgG to 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 using an 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 mimetic cells was measured using Huh7 human hepatocytes containing a luciferase gene regulated by a WNT-responsive promoter, according to an established protocol (H. Chen, Lu, Lee, & Li, 2020) (Super TopFlash reporter assay, STF).

[0291] Organoid culture and proliferation assays: Mouse small intestinal organoids were maintained in mouse IntestiCult® Organoid Growth Medium (STEMCELL technologies) and passaged weekly until the day of the assay for Wnt mimicry activity (H. Chen, Lu, Lee, & Li, 2020). To assay organoid proliferation, organoids were dissociated by shaking for 10 minutes using Gentle Cell Dissociation Reagent (STEMCELL technologies), washed twice with cold PBS (Gibco), and resuspended 1:1 in Matrigel (Corning) on ​​ice. 25 μl of the cell resuspended in Matrigel was seeded in the center of each well of a pre-warmed 48-well tissue culture plate and allowed to coagulate at 37°C for 5 minutes. 300 μl of basic medium (Table 10), basic medium + IWP2 + anti-βGal, or basic medium + IWP2 + Wnt mimicry was applied to the wells. Each condition was repeated 5-6 times. The medium and treatment were changed once on day 4 after plating. On the 7th day, images of the 3D cultured organoids were obtained.

[0292] Animal management: Seven-week-old C57Bl / 6J female mice were obtained from Jackson Laboratories (Bar Harbor, ME, USA) and housed in cages of 4-5 mice each. All animal experiments followed the "Guidelines for the Care and Use of Laboratory Animals" prepared by the National Academy of Sciences. The experiment complied with the standards of the Care and Use of Laboratory Animals. The animal experiment protocol was approved by the Surrozen Institutional Animal Care and Use Committee. Mice were acclimatized for a minimum of two days before the start of the experiment. Mice were housed in a 12 / 12 light / dark cycle at 30%–70% humidity and room temperature 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) sodium dextran sulfate (DSS, MP Biomedicals, molecular weight 36-50 kDa, Ref#160110) in drinking water from day 1 to day 7 to induce colitis, and were switched to 1% DSS from day 8. Protein treatment was administered either once on day 7 or twice on days 4 and 7. The animals were sacrificed on day 10 to allow for the protein treatment process over 6 days. The colon was collected for histology and RT-qPCR. In one study, DSS-induced mouse weight loss in animals treated with anti-GFP was nearly 25% on day 9, and therefore, in compliance with IACUC regulations, the animals were switched to drinking water without DSS. The Disease Activity Index (DAI) was calculated based on the mean scores of weight loss, stool consistency, and the degree of intestinal bleeding (Wirtz Stefan et al., 2017). A scoring system based on malignancy classification was established on a scale of 0 to 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 retains shape; 2, soft; 3, very soft and moist; 4, watery diarrhea), and intestinal bleeding (0-1, negative hemocult test; 2, positive hemocult test; 3, visible traces of blood in stool; 4, macroscopic rectal bleeding).

[0294] Histology: The small and large intestines were resected, fecal contents removed, weighed, and measured in length. Desired small intestinal segments (duodenum, jejunum, ileum) and colonic segments (ascending colon, transverse colon, and descending colon) were excised and directly fixed overnight in 10% neutral buffered formalin (NBF). The tissues were then transferred to 70% ethanol and embedded in paraffin. The paraffin tissue blocks were then sectioned into 5 μM thick sections and stained with hematoxylin and eosin (H&E) for histological analysis. Pathological interpretation was performed by an independent pathologist.

[0295] Immunohistochemistry and indirect immunofluorescence: In short, formalin-fixed paraffin-embedded tissue sections, 5 microns thick, were deparaffinized on slides, followed by antigen recovery in citrate buffer (pH 6) in a steamer. The slides were then thoroughly washed with tap water, followed by a single wash with PBST. Next, the tissue sections were blocked at room temperature for 1 hour using serum-free protein blocks (Agilent, X090930-2), and then incubated in primary antibody. After incubation with primary antibody, the tissue sections were washed at least three times with 0.1% TX-100 in PBST (PBST), and then incubated in secondary antibody. Finally, the tissue sections were washed with PBST and mounted on coverslips using Vectashield Vibrance anti-fading mounting medium containing DAPI (Vector Laboratories, H-1800).

[0296] Fluorescence-activated cell sorting (FACS) Mouse colons were dissected as described below and resuspended in FACS buffer (HBSS, 2% FBS, 10 mM HEPES, 1 mM sodium pyruvate, and 1% Pen-strep or antibiotic / antifungal solution). Prior to FACS, cells were passed through a 40 micron filter and DAPI was added to distinguish between live and dead cells. Prior to targeted 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, sequencing. For an acute DSS model, mice were treated with 4% DSS in drinking water throughout the duration of the experiment. Animals treated with DSS were administered 10 mpk of R2M13-26 or anti-GFP antibody on day 4 of DSS treatment. Cells were collected in three replicates each from two unharmed naive mice (without DSS) on days 5 and 6, as well as from DSS animals treated with anti-GFP and DSS animals treated with R2M13-26 at each time point. Each animal was considered one replicate.

[0298] The transverse colon was isolated from each animal, and fecal matter was removed. After a brief wash with cold PBS, the colon was cut longitudinally, the tube was opened, and the tissue was flattened into a 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 in a shaker at 37°C and 150 rpm for 15 minutes. After 15 minutes, the tube containing the sample was shaken vigorously for 10 seconds to release more epithelial cells. The epithelial cells suspended in the suspension were removed and placed in a new tube, and centrifuged at 200 rcf for 2 minutes.

[0299] Next, the residual tissue containing the remaining epithelium and stroma / lamina propria was incubated in 8-12.5 mL of lamina propria dissociation buffer (10 mM HEPES, 0.2% FBS, DNAse1 (80 U / mL), Liberase® (0.2 mg / mL), and AdvDMEM / F12 with 1% antibiotic / antifungal agent) at 37°C for 30 minutes with horizontal shaking at 150 rpm. After pelleting, the epithelial cells were resuspended in 1 mL of TrypLE with DNase1, incubated at 37°C for 5 minutes, and ground with a P1000 pipette for 30 seconds. After grounding, 10 mL of PBS + 50 U / mL of DNAse1 was added to the epithelial cells, and the mixture was centrifuged at 4°C and 500 rcf, and the supernatant was removed. Next, the epithelial cells were washed once with FACS buffer (HBSS, 2% FBS, 10 mM HEPES, 1 mM sodium pyruvate, and 1% Pen-strep or antibiotic / antifungal solution), followed by 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 the volume was reduced to 1 mL, and the sample was ground with a P1000 until the solution was homogenized and all tissue fragments had dissociated. After grinding, the sample was centrifuged at 4°C and 500 rcf for 5 minutes, washed with FACS buffer, and then resuspended in 1 mL of FACS buffer in preparation for FACS.

[0300] All cells were filtered through a 40-micron filter and then subjected 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 (in a 1:5 ratio), counted using a hemocytometer, and then captured. A standard 10× Genomics Chromium 3'v3 scRNA-seq reagent (PN1000075) was used. Approximately 4000–4500 cells were loaded per channel. Cells from one individual animal repeat were captured per channel. Standard 10× Genomics Chromium 3'v3 scRNA-seq RT, cDNA amplification, and sequencing library preparation protocols were followed. Sequencing of the multiplexed sequencing library was performed using 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 a 10×Genomics Cellranger (version 3.0.2) pipeline running STAR aligner against the mm10-3.0.0 version of the mouse transcriptome. The demultiplexed UMI count data were then evaluated, and after exploratory data analysis, low-quality cells and underexpressed genes were partially removed using the R package scone (version 1.14.0) and dataset-specific filtering cutoffs: only cells with a UMI greater than 1000, genes between 500 and 6500, and a UMI less than or equal to 60000 were retained, potentially removing empty droplets and limiting doublets. Cells with a mitochondrial gene percentage greater than the mean by more than one standard deviation were filtered. Only genes expressed in the top quartiles of at least three cells were obtained, resulting in 16039 genes. UMI count data was normalized using deconvolution scaling from the R package scran (version 1.18.5; Lun, Bach, & Marioni, 2016). After normalization, batch-specific cell populations were not observed when evaluated in the reduced dimensional space, and no strong correlation was found between QC metrics and observed gene expression principal components.

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

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

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

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

[0306] (Example 2) Manipulated Wnt agonist We synthesized IgG1-formatted Wnt agonists containing Wnt agonists in which a humanized Lrp5 / 6 binding domain was fused to the N-terminus of each light chain of an Fzd-conjugated antibody. Exemplary structures are shown in Figure 1. The Lrp5 / 6 binding domains were derived from various camelid single-chain antibody (VHH) binding domains selected from VHH03, VHH26, or VHH36. The VHH03 domain binds to Lrp5, the VHH26 domain binds to Lrp6, and the VHH36 domain binds to both Lrp5 and Lrp6. Camelid single-chain antibodies were humanized by retaining the CDR sequence but replacing other sequences with a human antibody backbone. The resulting LRP5 / 6 binding domains were modified to eliminate potential drawbacks.

[0307] Humanization of VHH26 was performed as follows. The VHH domain in camelids is thought to be difficult to humanize because it originates from single-chain homodimer antibodies lacking VL:CL or VH:CH interactions present in heterotetrameric human IgG1 antibodies. (Surface characteristics of VHH in camelids: Muyldermans (2013) Annu. Rev. Biochem. 82: 775-797; Vincke et al (2009)) The gene (J. Biol. Chem. 284: 3273-3284) has been evolutionarily redesigned to optimize the stability of the homodimeric properties of single-chain antibodies. Humanization of camelid VHH26 was initially performed by CDR grafting to the human germline sequence with the highest sequence identity (for a review, see: Safdari et al., (2013) Biotechnol. Genet. Eng. Rev. 27: 175-186). In the next step, several different humanized VHH26 constructs with revert mutations to camelid sequences were created to identify engineered VHH with optimal expression, homogeneity, and biophysical properties such as binding affinity to the Lrp6 receptor. The alignment of VHH26 (Table 1) and its closest human germline sequence IGHV3-23*01 is shown in Figure 2A. Table 1 lists the sequences of six different humanized VHH26 (H1-H6), and Figure 2B shows their alignments to their parent VHH26.

[0308] These six humanized VHH26 variants H1-H6 and parental VHH26 were transiently expressed in Expi293 cells with a C-terminal hexa-histidine tag (80 mL scale). The proteins were purified using His-Complete resin (Roche, USA) according to standard procedures. The expression levels and homogeneity of VHH26 and its humanized variants were analyzed by SDS-PAGE and SEC (size exclusion chromatography). To determine the affinity of Lrp6 to VHH26, the 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) were determined by biolayer interferometry (BLI) using an Octet Red 96 instrument (PALL ForteBio, Fremont, CA), 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 using an SA biosensor and then immersed in wells containing VHH26 protein at various concentrations in the running buffer, or in wells containing only the running buffer as a reference channel. The KD for each binding factor was calculated using Octet System software based on fitting to a 1:1 binding model. The 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 molecule being tested.

[0309] SDS-PAGE, SEC, and Octet-BLI profiles for VHH26 and its humanized variants are shown in Figures 3A-3B. SDS-PAGE analysis of Ni pulldown samples revealed that among the six VHH26 human variants, VHH26-H2, VHH26-H4, and VHH26-H5 showed 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 fraction of each of these peaks was investigated by Octet-BLI for their ability to interact with Lrp6. Table 2 lists the kinetic parameters such as kon, koff, and KD for the interaction between VHH26 constructs and the Lrp6E3E4 domain. Analysis of these parameters revealed that the VHH26-H5 humanized variant had only a slight effect on its binding affinity to Lrp6 compared to the parental VHH26 (Table 2; Figure 3B). For comparison, the alignment of parental VHH26 and VHH26-H5 is shown in Figure 2B.

[0310] Based on the above, VHH26-H5 was used in further experiments as a humanized LRP-binding domain fused with an Fzd-binding domain (e.g., a tetravalent, bispecific WNT agonist). The Fzd-binding domain was derived from the R2M13 antibody that binds to Fzd5 and Fzd8 and contained an effector-less Fc region that retains FcRn binding, e.g., LALAPG or N297G (Wang X et al., Protein Cell 2018, 9: 63-73). N297G is an unglycosylated form of IgG1 antibody in which Asn is substituted with Gly. In the case of R2M13-26 humanized N297, N297 corresponds to amino acid N302, and therefore the N297G mutation is sometimes called N302G instead. LALAPG represents three mutations in the Fc domain of IgG1. Using standard IgG1 sequence number assignment, 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 creating an IgG-like molecule containing the R2M13 antibody with VHH at both N-terminuses of the antibody light chain.

[0311] Table 3 provides sequences of R2M13 heavy-chain IgG and R2M13 light-chain fused with various LRP5 / 6 VHH-binding domains and amino acid linkers present in various Wnt agonists. The heavy-chain and light-chain sequences present in the parent R2M13-03, R2M13-26, and R2M13-36 Wnt agonists without LALPG or N297G modifications are provided as SEQ ID NOs. 136-138 (light chains, respectively) and SEQ ID NO. 153 (heavy chain) in PCT application publication WO2019 / 126398, which is incorporated herein by reference in their entirety. The Wnt agonists shown contain two heavy chains and two light chains in antibody-type format, with these chains linked 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 which the Fzd binding factor R2M13 was paired with various humanized Lrp-binding domains in a fully engineered Wnt agonist format was determined using the Super TOPFlash luciferase reporter (STF) assay, which measures the activation of standard Wnt signaling in a Wnt-responsive Huh-7 reporter cell line (Huh-7STF). The results are shown in Figure 4. The R2M13-humanized_26-LALAPG construct ("R2M13-26 humanized LALAPG"; also referred to herein as R2M13-h26, R2M13-h26-LALAPG, or humanized LALPG) showed the highest activity of the humanized Lrp-binding domain. The R2M13 humanized_26-N297G construct (R2M13-26 humanized N297G; humanized N297G) was not stable. Humanized VHH03 and VHH36 showed significantly reduced efficacy in vitro when paired with R2M13, but their absolute EC50 values ​​were comparable to those of VHH26 paired with R2M13. Figure 6 shows the heavy and light chain sequences of the R2M13-humanized_26-LALAPG construct (R2M13-h26). This construct consisted of two heavy chains and two light chains linked by disulfide bonds. The effector function was removed by the LALAPG mutation in the Fc domain (see, e.g., Wang, et al. (2018) Protein Cell. 9:63-73). Various domains of the R2M13-h26 construct are shown, allowing for easy determination of the domains of other constructs based on these.

[0313] (Example 3) Dose-response of engineered Wnt agonists in animal models of acute colitis (DSS) 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 a mouse model of acute DSS colitis; to characterize the in vivo activity of R2M13-26 at various doses and frequencies in the mouse model of acute DSS colitis; and to evaluate the effects of R2M13-26 on 1) body weight, fecal score and occult blood, 2) epithelial / barrier repair by histology, and 3) inflammatory cytokines in serum and colon.

[0314] 86 female C57Bl / 6J mice, aged 6-8 weeks, were obtained from Jackson Laboratories (Bar Harbor, ME, USA) and housed in cages of 5 mice each. All animal experiments adhered to the standards of the "Guide for the Care and Use of Laboratory Animals" developed by the National Academy of Sciences. The animal experiment 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 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 3 days. The mouse groups were either left 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 agonist.

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

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

[0318] (Example 4) Manipulated Wnt agonists repair damaged colonic epithelium in an animal model of acute colitis with DSS. As outlined in Figure 7, various manipulated humanized Wnt agonists were tested in a DSS model of acute colitis. The 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 in cages of five. All animal experiments were conducted by the National Academy of Sciences. The animal experiment protocols adhered to the standards of the "Guide for the Care and Use of Laboratory Animals" created by the Surrozen Institutional Animal Care Committee. The experiment was approved by the (and Use Committee). Mice were acclimatized for at least two days before starting the experiment. Mice were housed in a 30%–70% humidity environment and at room temperature in the range of 20°C–26°C in a 12 / 12 hour 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). The mouse groups were either left untreated, treated with an isotype control antibody (anti-GFP) on days 4 and 7, or treated with the indicated engineered Wnt agonist at 1 mg / kg. All protein treatments resulted in comparable serum antibody exposure at the end of the study (Figure 7).

[0321] Control animals subjected to DSS developed severe colitis characterized by marked and persistent weight loss and bloody diarrhea, resulting in an increase in the disease activity index, as expressed by fecal scores. Treatment with humanized R2M13-26 and humanized R2M13-36 in either LALAPG or N297G form significantly improved body weight in DSS mice. A significant improvement in body weight was observed when using humanized R2M13-36-LALAPG compared to the parent construct. These constructs also significantly reduced the disease activity index (DAI) in DSS mice (Figure 8), decreased fecal scores, increased colon length and fecal size, 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. These levels were elevated in the DSS-treated group (Figure 10). Humanized R2M13-36-LALAPG showed a significant improvement in body weight compared to the parent construct. In addition, humanized R2M13-26-LALAPG (R2M13-h26-LALAPG) was demonstrated to restore ZO-1, an epithelial tight junction marker, in vivo (Figure 11), repair damaged colonic epithelium (Figure 12), and restore epithelial cell lineages including colonic cells, goblet cells, and tuft cells (Figure 13). Therefore, both humanized R2M13-26 and humanized R2M13-36 showed good efficacy in DSS mice.

[0322] (Example 5) Pharmacokinetics (PK) of manipulated Wnt agonists The pharmacokinetics (PK) of parental R2M13-26 (R2M13-26-LALAPG) and humanized R2M13-26 (R2M13-h26-LALAPG) after intravenous injection were determined by measuring the amount of antibody in the serum of rats at various time points after administration and comparing the results with data obtained from mice (Figure 14). The Cmax for humanized R2M13-26 (R2M13-h26) was higher than that for parental R2M13-26 (R2M13-26), and therefore the difference was maintained 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 parent R2M13-26 (40.0 mL / day / kg), and the half-life of humanized R2M13-26 (3.75 days) was longer than that of parent R2M13-26 (2.47 days).

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

[0324] Six-to-eight-week-old C57Bl / 6J female mice were obtained from Jackson Laboratories (Bar Harbor, ME, USA) and housed in cages of four to five mice each. All animal experiments adhered to the standards of the "Guide for the Care and Use of Laboratory Animals" developed by the National Academy of Sciences. The animal experiment 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 7 days of pure drinking water. This cycle was repeated three times. The mouse groups were treated with either an isotype control antibody (anti-GFP) or four doses of 10 mg / kg of R2M13-26-LALAPG (R2M13-26) on days 16, 19, 28, and 31. The animals were sacrificed on day 33.

[0326] In a 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 manipulated Wnt agonists on a DSS acute colitis model Examples 3 and 4 demonstrated the effectiveness of R2M13-26 and R2M13-h26 Wnt agonists, which are specific to Fzd5 and 8, in treating an acute mouse colitis (acute DSS) model. The objective of this study was to develop a more comprehensive understanding of the mechanism by which R2M13-26 affects colonic cells throughout the repair process, using a similar model system.

[0328] Six-to-seven-week-old C57Bl / 6J female mice were obtained from Jackson Laboratories (Bar Harbor, ME, USA) and housed in cages of four to five mice each. All animal experiments adhered to the standards of the "Guide for the Care and Use of Laboratory Animals" developed by the National Academy of Sciences. The animal experiment protocols were approved by the Surrozen Institutional Animal Care and Use Committee.

[0329] To induce acute colitis, female mice were given free access to drinking water containing 4.0% (w / v) dextran sulfate sodium (DSS, MP Biomedicals, MFCD00081551) for 7 days, and then to drinking water containing 1.0% (w / v) DSS for 3 days. On day 4, the mouse groups were either left untreated, treated with a control antibody (anti-GFP), or treated with a single intravenous injection of R2M13-26-LALAPG (R2M13-26). A total of 123 mice were divided into groups (13 on day 3, 13 on day 4, 26 on day 5, 24 on day 6, 26 on day 7, and 21 on day 10). 91 mice were used for histological endpoints, and 21 mice were used for scRNA-seq (a group excluded due to machine-related issues). Daily food intake, body weight (BW), fecal score, and occult blood were measured. At the end of the study, mice were treated as follows: Groups A-E: transverse colon was collected for qPCR and histology (Group A was completed on days 3 and 4, Group C on days 3, 4, 5, 6 and 7, and Groups B, D and E were completed on days 5, 6, 7 and 10). Assays / endpoints included RT-qPCR, histology, scRNA-seq, fecal score for stool consistency and occult blood, disease activity index (DAI) = (decreased BW + stool consistency + blood) / 3, serum inflammatory cytokines (TNF-α, IL-6, lipocalin 2), and anatomical pathology: ascending colon, transverse colon and descending colon, H&E. Histopathological scoring criteria included inflammation severity, extent of inflammation, mucosal erosion, crypt proliferation, and goblet cell depletion.

[0330] No difference was observed between PBS treatment and anti-GFP treatment on days 3–7 using DSS (data not shown). However, treatment with R2M13-26 showed healthier colon tissue at days 5–10, and animals treated with R2M13-26 showed notable histological improvement by day 7 (data not shown). R2M13-26 improved fecal score and BW reduction in DSS mice (data not shown), and therefore improved experimental colitis in mice.

[0331] RT-qPCR analysis was performed on bulk colon samples to evaluate changes in gene expression. Wnt induction studies showed a significant decrease in Axin2 induced by DSS. R2M13-26 induced Axin2 expression under conditions without DSS. Examination of proliferation markers showed a significant decrease in Ki67 induced by DSS on day 4, indicating 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 induced by DSS on day 4, which was rescued by R2M13-h26. Regarding clinical markers for IBD, significant upregulation of Gpx2 was observed on days 5 and 6.

[0332] For scRNA-seq experiments investigating gene expression in a DSS model, mice were treated with 4% DSS in drinking water throughout the duration of the experiment. Animals treated with DSS were administered 10 mpk of R2M13-26 or anti-GFP antibody on day 4 of DSS treatment. Three animals each were collected on days 5 and 6, 24 and 48 hours after administration, respectively, from the Wnt agonist-treated and anti-GFP-treated animals. Samples from two naive, uninjured animals were also collected on days 5 and 6. Colon, small intestine, spleen, and liver tissue were collected at the end of the experiment 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 samples to isolate epithelium only.

[0333] The transverse colon was isolated from each animal, and fecal matter was removed. After a brief wash with cold PBS, the colon was cut longitudinally, the tube was opened, and the tissue was flattened into a sheet. The tissue was then cut into 3-4 mm long fragments. The tissue fragments were incubated with 5 mM EDTA in pre-warmed (37°C) PBS in a shaker at 37°C and 150 rpm for 15 minutes. After 15 minutes, the tube containing the sample was shaken vigorously for 10 seconds to release more epithelial cells. The epithelial cells suspended in the suspension were removed and placed in a new tube, and the cells were pelleted by centrifugation at 200 rcf for 2 minutes to separate the epithelial cells 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 and 150 rpm with horizontal shaking for 30 minutes. After pelletizing, the epithelial cells were resuspended in 1 mL of TrypLE with DNase-1, incubated at 37°C for approximately 8 minutes, and ground approximately 25 times with a P1000 pipette. After grinding, the epithelial cells were centrifuged at 4°C and 500 rcf, and the supernatant was removed. The epithelial cells were then washed once with FACS buffer, followed by 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 the volume was reduced to 1 mL, and the sample was ground with a P1000 until the solution was homogenized and all tissue fragments had dissociated. After grinding, the sample was centrifuged at 4°C and 500 rcf for 5 minutes, washed with FACS buffer, and then resuspended in 1 mL of FACS buffer in preparation for FACS.

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

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

[0336] R2M13-26 exerts its effects primarily by directly affecting colonic epithelial cells, which is attributed to the high expression of FZD5 in intestinal epithelial cells and its enrichment in stem cell and progenitor cell populations. Comparison of expression across epithelial lineages and all cell types between R2M13-26 treatment and control treatment revealed increased expression of the following Wnt target genes (Table 7). Molecules showing at least a twofold increase between treatment and control across epithelial lineages and those documented in the literature as direct Wnt targets were selected. The majority of Wnt target genes were obtained from genetic engineering 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 showing significant changes across the entire epithelial lineage, we used scRNA-seq data to investigate specific cell types and compared gene expression between cells treated with R2M13-26 and control cells to identify Wnt target genes that were increased or decreased in each relevant cell type within the epithelial lineage. Differential expression analysis of this type 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 intestinal cells (AltEntero), intestinal progenitor cells (EnteroPrecur), goblet cells 1, goblet cells 2, enteroendocrine, and tuft cells. Table 7 summarizes the Wnt target genes modulated across the entire epithelial lineage and / or in specific epithelial subtypes, along with examples of log2 magnification changes. Figure 26B shows heatmaps of epithelial cells detected in scRNA-seq experiments.

[0338] Numerous molecules were identified that significantly increased or decreased in expression across aggregated epithelial lineages and / or cell types when comparing the R2M13-26 treatment with the control treatment. Molecules that showed at least a twofold 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] By cross-referencing the genes enlarged by treatment with R2M13-26 with a list of established cell cycle genes (Giotti et al., 2019), we identified genes involved in cell cycle progression and regulation that were enlarged by R2M13-26 treatment. 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 crucial aspect of its function (Davidson, 2010; Hirata 2013). R2M13-26 promoted the expansion of stem and progenitor cells, which are essential for the ability to regenerate the epithelium in injured colon epithelium. These data suggest that some of these genes are also direct Wnt targets (Table 8).

[0340] Wnt signaling is crucial for regulating the maintenance and regeneration of the stem cell and progenitor cell pool, as well as their differentiation, in addition to promoting the expansion of stem and progenitor cells to facilitate epithelial regeneration (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), Hmga2 (Nishino 2008; Parisi 2020), Foxq1 (Tu 2018; Zhang 2018), and Aldh1 (Tomita 2016) (Table 8). Furthermore, an effect on the expression of Areg (Fujii 2008; Mahtouk 2005; Suzuki 2010; Takahashi 2020), a ligand for EGFR signaling crucial for maintaining the intestinal stem cell niche, was also observed. 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 several 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 the proliferation of stem and progenitor cells as well as epithelial crypt regeneration (Drucker 1999; Markovic 2019; Zatorski 2019). These data suggest that Wnt signaling activation increases glucagon expression, which leads to elevated GLP-2 levels and contributes to the expansion of stem and progenitor cells.

[0341] In addition to regulating the self-renewal and differentiation of stem cells and progenitor cells, one of the important aspects of tissue repair and epithelial regeneration is the repair of intracellular and extracellular damage and the reconstruction of the epithelial barrier. For this purpose, some of the genes induced and / or increased by treatment with R2M13-26 are associated with these processes (Table 6). For example, Apex1 is critical for DNA repair (Park 2014). Dysfunction of mucus production and the mucus barrier is an important aspect of IBD (Antoni 2014; Dorofeyev 2013; Kim, Ho 2010). Some of the genes increased by treatment with R2M13-26 promote mucus secretion and mucus barrier establishment (B3gnt7, Agr2, Muc2, Muc3, Tff3, Fcgbp, and Zg16). These genes play crucial roles in mucus production, processing, and secretion (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). Furthermore, Sprr2a3 (Gibbs 1993), a member of a small proline-rich repeat protein group involved in epithelial barrier formation, was enriched.

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

[0343] In addition to influencing epithelial repair and regeneration by regulating stem cell and progenitor cell proliferation and differentiation, cell repair, and barrier formation, R2M13-26 promoted the expression of numerous 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 worsening of IBD.

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

[0345] (Example...

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