阅读说明：本技术 调节胃肠道病症中的wnt信号传导 (Modulating WNT signaling in gastrointestinal disorders ) 是由 李阳 鲁成钢 海伦·巴里鲍特 叶文琛 谢力勤 王绎杰 孟维旭 于 2020-03-11 设计创作，主要内容包括：本发明提供了用WNT信号传导路径的调节剂治疗胃肠道病症的方法。本发明还提供了给药方法和药物组合物。(The present invention provides methods of treating gastrointestinal disorders with modulators of the WNT signaling pathway. The invention also provides methods of administration and pharmaceutical compositions.)

1. A method of treating a subject having a gastrointestinal disorder comprising administering to the subject an engineered WNT signaling modulator.

2. The method of claim 1, wherein the engineered WNT signaling modulator is an engineered WNT agonist.

3. The method of claim 1, wherein the engineered WNT signaling modulator is selected from the group consisting of: engineered polypeptides, engineered antibodies containing at least one epitope binding domain, small molecules, siRNA, and antisense nucleic acid molecules.

4. The method of claim 2, wherein the engineered WNT agonist comprises one or more binding compositions that bind to one or more FZD receptors (FZD1-10) and one or more binding compositions that bind to one or more LRP receptors (LRP 5-6).

5. The method of claim 4, wherein the binding composition of the engineered WNT agonist comprises:

a) one or more binding compositions that bind to:

i)FZD5；

ii)FZD 8；

iii)FZD 1；

iv)FZD 2；

vi)FZD 7；

vi) FZD5 and FZD 8;

vii) FZD1, FZD2, and FZD 7;

viii) FZD1, FZD2, FZD7, FZD5, and FZD 8;

ix)FZD4；

x) FZD 9; or

xi) FZD 10; and

b) one or more binding compositions that bind to:

i)LRP5；

ii) LRP 6; or

iii)LRP5/6。

6. The method of claim 5, wherein the WNT agonist comprises one or more binding compositions that bind to FZD5 and FZD8 and one or more binding compositions that bind to LRP5 or LRP 6.

7. The method of claim 6, wherein the WNT agonist comprises a binding composition that binds to FZD5 and FZD8 and a binding composition that binds LRP 6.

8. The method of claim 5, wherein the WNT agonist comprises the variable heavy chain sequence of SEQ ID NO 1, 3, 5, 7, 9, 11 or 13; and the variable light chain sequence of SEQ ID NO 2,4, 6,8, 10, 12 or 14.

9. The method of any one of claims 1-8, wherein the engineered WNT signaling modulator repairs the intestinal epithelium and/or reduces expression of inflammatory cytokines.

10. The method of any one of claims 1-8, wherein the engineered WNT signaling modulator comprises a tissue targeting molecule.

11. The method of claim 10, wherein the tissue-targeting molecule is an antibody or fragment thereof that binds to a tissue-specific cell surface antigen.

12. The method of claim 11, wherein the tissue-targeting molecule is selected from the group consisting of: GPA33, CDH17 and MUC-13 polypeptides and functional fragments or variants thereof.

13. The method according to any one of claims 1 to 8, wherein the modulator of WNT signaling is administered with a binding composition that specifically binds an inflammatory molecule.

14. The method of claim 13, wherein the binding composition that specifically binds the inflammatory molecule is an antagonist of the inflammatory molecule.

15. The method of claim 14, wherein the antagonist of the inflammatory molecule is an antagonist of TNF α, IL-12 and IL-23, or IL-23.

16. The method of any one of claims 1 to 8, wherein the gastrointestinal disease is inflammatory bowel disease.

17. The method of claim 16, wherein the inflammatory bowel disease is selected from the group consisting of: crohn's Disease (CD), CD with fistula formation and Ulcerative Colitis (UC).

18. A method of treating a subject having a gastrointestinal disorder comprising administering to the subject a tissue-specific WNT signaling-enhancing molecule.

19. The method of claim 18, wherein the WNT signal-enhancing molecule is an engineered molecule comprising:

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.

20. The method of claim 19, wherein the one or more E3 ubiquitin ligases is selected from the group consisting of: zinc and ring finger protein 3(ZNRF3) and ring finger protein 43(RNF 43).

21. The method of claim 19, wherein said first domain comprises an R-spondin (rspo) polypeptide.

22. The method of claim 21, wherein the RSPO polypeptide is selected from the group consisting of: RSPO-1, RSPO-2, RSPO-3, and RSPO-4.

23. The method of claim 21, wherein the RSPO polypeptide comprises a first furin domain and a second furin domain.

24. The method of claim 23, wherein said second furin domain is wild-type or mutated to have lower binding to G protein-coupled receptor 4-6(LGR4-6) containing a leucine-rich repeat.

25. The method of claim 18, wherein the WNT signal-enhancing molecule comprises a tissue-targeting molecule.

26. The method of claim 25, wherein the tissue-targeting molecule is an antibody or fragment thereof that binds to a tissue-specific cell surface antigen.

27. The method of claim 26, wherein the tissue-targeting molecule is selected from the group consisting of: GPA33, CDH17 and MUC-13 polypeptides and functional fragments and variants thereof.

28. The method of claim 27, wherein the WNT signaling enhancing molecule comprises the heavy chain sequence of SEQ ID NOs 17, 20, or 23; and the light chain sequence of SEQ ID NO 16, 19 or 22.

29. The method of any one of claims 18 to 28, wherein the WNT signaling-enhancing molecule is administered with a binding composition that specifically binds an inflammatory molecule.

30. The method of claim 29, wherein the binding composition that specifically binds the inflammatory molecule is an antagonist of the inflammatory molecule.

31. The method of claim 30, wherein the antagonist of the inflammatory molecule is an antagonist of TNF α, IL-12 and IL-23, or IL-23.

32. The method of any one of claims 18 to 28, wherein the gastrointestinal disease is inflammatory bowel disease.

33. The method of claim 32, wherein the inflammatory bowel disease is selected from the group consisting of: crohn's Disease (CD), CD with fistula formation and Ulcerative Colitis (UC).

34. A method of treating an individual having a gastrointestinal disorder, the method comprising administering to the individual an engineered WNT agonist and an engineered tissue-specific WNT signal-enhancing combination molecule.

35. The method of claim 34, wherein the combinatorial molecule comprises:

a) an engineered WNT agonist selected from the group consisting of: a FZD5 binding composition, a FZD8 binding composition, a FZD1 binding composition, a FZD2 binding composition, a FZD7 binding composition, an LRP5 binding composition, an LRP6 binding composition, and an LRP5/6 binding composition; and

b) the 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.

36. The method of claim 35, wherein the E3 ubiquitin ligase is selected from the group consisting of: zinc and ring finger protein 3(ZNRF3) and ring finger protein 43(RNF 43).

37. The method of claim 35, wherein said first domain comprises an R-spondin (rspo) polypeptide.

38. The method of claim 37, wherein the RSPO polypeptide is selected from the group consisting of: RSPO-1, RSPO-2, RSPO-3, and RSPO-4.

39. The method of claim 37, wherein the RSPO polypeptide comprises a first furin domain and a second furin domain.

40. The method of claim 39, wherein said second furin domain is wild-type or mutated to have lower binding to G protein-coupled receptor 4-6(LGR4-6) containing a leucine-rich repeat.

41. The method of claim 34, wherein the combinatorial molecule incorporates a tissue targeting molecule.

42. The method of claim 41, wherein the tissue-targeting molecule is an antibody or fragment thereof that binds to a tissue-specific cell surface antigen.

43. The method of claim 42, wherein the tissue-targeting molecule is selected from the group consisting of: GPA33, CDH17 and MUC-13 polypeptides and functional fragments and variants thereof.

44. The method according to any one of claims 34 to 42, wherein the combination molecule is administered with a binding composition that specifically binds to an inflammatory molecule.

45. The method of claim 44, wherein the binding composition specific for the inflammatory molecule is an antagonist of the inflammatory molecule.

46. The method of claim 45, wherein the antagonist of the inflammatory molecule is an antagonist of TNF α, IL-12 and IL-23, or IL-23.

47. The method of any one of claims 34 to 42, wherein the gastrointestinal disease is inflammatory bowel disease.

48. The method of claim 47, wherein the inflammatory bowel disease is selected from the group consisting of: crohn's Disease (CD), CD with fistula formation and Ulcerative Colitis (UC).

49. A polypeptide that specifically binds to frizzled receptor 5(FZD5) and frizzled receptor 8(FZD8), wherein the polypeptide comprises one or more sequences having at least 80%, at least 90%, or at least 95% homology to the sequence set forth in or encoded by any of SEQ ID nos. 33-40.

50. The polypeptide of claim 49, wherein the polypeptide comprises an antibody or antibody binding fragment.

51. The polypeptide of claim 50, wherein the antibody or antibody-binding fragment comprises at least 5 or all 6 CDRs present in any one of the following sequence combinations: 33 and 34; 35 and 36; 37 and 38; or SEQ ID NOs 39 and 40.

52. The polypeptide of claim 50, wherein the polypeptide comprises six of the CDRs present in any one of the following sequence combinations: 33 and 34; 35 and 36; 37 and 38; or SEQ ID NOs 39 and 40, wherein one or more of the CDRs comprise one, two or three amino acid modifications, optionally site mutations, amino acid deletions or amino acid insertions.

53. An engineered WNT agonist comprising:

(a) one or more binding domains that bind to FZD5 and FZD8, wherein at least one of the one or more binding domains comprises the polypeptide of any one of claims 49-52; and

(b) one or more binding domains that bind to LRP5, LRP6, or both LRP5 and LRP 6.

54. An engineered WNT agonist comprising a polypeptide sequence having at least 80%, at least 90%, or at least 95% homology to any one of SEQ ID NOs 7-14.

55. The engineered WNT agonist of claim 54, comprising:

(a) a polypeptide sequence having at least 80%, at least 90% or at least 95% homology to SEQ ID No. 7 and a polypeptide sequence having at least 80%, at least 90% or at least 95% homology to SEQ ID No. 8;

(b) a polypeptide sequence having at least 80%, at least 90% or at least 95% homology to SEQ ID No. 9 and a polypeptide sequence having at least 80%, at least 90% or at least 95% homology to SEQ ID No. 10;

(c) a polypeptide sequence having at least 80%, at least 90% or at least 95% homology to SEQ ID NO. 11 and a polypeptide sequence having at least 80%, at least 90% or at least 95% homology to SEQ ID NO. 12; or

(d) A polypeptide sequence having at least 80%, at least 90% or at least 95% homology with SEQ ID NO. 13 and a polypeptide sequence having at least 80%, at least 90% or at least 95% homology with SEQ ID NO. 14.

56. A combination molecule comprising:

a) the engineered WNT agonist of any one of claims 53-55; 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.

57. A pharmaceutical composition comprising a polypeptide according to any one of claims 49-52, an engineered WNT agonist according to any one of claims 53-55, or a combination molecule according to claim 56.

58. A method of treating a subject having a gastrointestinal disorder comprising administering to the subject an engineered WNT agonist according to any one of claims 53 to 55, a combination molecule according to claim 56, or a pharmaceutical composition according to claim 57.

59. The method of claim 58, wherein the gastrointestinal disorder is an inflammatory bowel disease optionally selected from the group consisting of: crohn's Disease (CD), CD with fistula formation and Ulcerative Colitis (UC).

Technical Field

The present invention provides modulators of WNT signaling as a treatment for gastrointestinal disorders, particularly inflammatory bowel disease.

Cross reference to related applications

This application claims priority from united states provisional application No. 62/816,729, filed on 3/11/2019, and united states provisional application No. 62/888,749, filed on 8/19/2019, each of which is incorporated herein by reference in its entirety.

Statement regarding sequence listing

The sequence listing associated with the present application is provided in text format in lieu of a paper copy and is hereby incorporated by reference into this specification. The names of the text files containing the sequence listing are: SRZN _014_02WO _ ST25. txt. The text file is about 102KB, created 3 months and 11 days 2020, being submitted electronically via the EFS-Web.

Background

Adult intestinal epithelium is characterized by a continuous replacement of epithelial cells by a stereoscopic cycle of cell division, differentiation, migration and shedding that occurs during crypt-villus transit times of 5-7 days. Putative growth factors that regulate proliferation within the adult intestinal stem cell niche have not been fully identified, although studies have been directed to the intracellular role of catenin/Lef/Tcf signaling within the proliferating crypt compartment.

Various pathological conditions affect intestinal cells. Inflammatory Bowel Disease (IBD) may involve either or both the small and large intestines. Crohn's disease and ulcerative colitis are the most famous forms of IBD, and both belong to the class of "idiopathic" inflammatory bowel diseases, as their pathology is unknown. "active" IBD is characterized by acute inflammation. "chronic" IBD is characterized by crypt distortion and architectural changes in scarring. Crypt abscesses can occur in various forms of IBD.

Ulcerative Colitis (UC) involves the colon, a diffuse mucosal disease with a predominance of distal sites. It is virtually always the rectum and may involve other portions of the colon extending proximally from the rectum in a continuous pattern. The cause of UC is unknown. Patients with long-term UC have an increased risk of acquiring colon cancer. Patients with UC are also at risk for liver disease, including sclerosing cholangitis and cholangiocarcinoma.

Crohn's disease may involve any part of the gastrointestinal tract, but most often involves the distal small intestine and the colon. Inflammation is usually transmural and can result in anything from small ulcers (aphthous ulcers) on lymphoid follicles to deep fissile ulcers through transmural scarring and chronic inflammation. One third of the cases have granulomas, and sites outside the colon such as lymph nodes, liver and joints may also have granulomas. Transmural inflammation results in fistulas between the intestinal loop and other structures. Inflammation is usually segmental and the unrelated gut separates the involved regions of the gut. The etiology is unknown, although infection and immune mechanisms have been proposed.

WNT proteins form a highly conserved family of secretory signaling molecules that regulate cell-cell interactions during embryogenesis. WNT genes and WNT signaling are also implicated in cancer. Insight into the mechanism of WNT action has emerged from several systems: genetics in drosophila and caenorhabditis elegans; biochemistry in cell culture and ectopic gene expression in xenopus embryos. Many of the WNT genes in mice have been mutated, resulting in highly specific developmental defects. As is currently understood, WNT proteins bind to receptors of the Frizzled (Frizzled) family on cell surfaces. Through several cytoplasmic relay components, signals are transduced to β -catenin, which then enters the nucleus and forms a complex with TCF to activate transcription of WNT target genes. Expression of WNT proteins varies, but is often associated with developmental processes, such as in embryonic and fetal tissues.

The functional redundancy and the necessity of conditional inactivation strategies have hampered the exploration of the physiological functions of WNT proteins in adult organisms. Dickkopf-1(Dkk1) was recently identified as an initial member of the secreted protein family that effectively antagonizes 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) Nature Cell Biol 3: 683-6). Dkk1 associates with both the WNT co-receptor LRP5/6 and the transmembrane protein Kremen, and the resulting ternary complex results in impairment of rapid LRP6 internalization and WNT signaling due to the absence of the functional frizzled receptor/LRP 6 WNT receptor complex Mao et al (2001) nature 411:321-5(Semenov) et al (2001) contemporary biology (Curr Biol) 11: 951-61; and Mao et al (2002) Nature 417: 664-7).

Transgenic mice with Tcf locus knock-out show loss of the proliferating stem cell compartment in the small intestine during late embryogenesis. However, knockouts are fatal and therefore have not been studied in adults. In chimeric transgenic mice that allowed for analysis of adults, expression of constitutively active NH 2-truncated β -catenin stimulated proliferation in the crypts of the small intestine, although NH 2-truncated β -catenin or Lef-1/β -catenin fusions induced increased crypt apoptosis. The cause of the intestinal stem cell deficiency is unknown as a number of factors regulate beta-catenin/Lef/Tcf-dependent transcription, including the non-frizzled receptors GPCR and PTEN/PI-3-kinase. The development of agents for regulating intestinal epithelial growth is of great interest for clinical purposes.

The search for WNT agonists has been hindered by the fact that they are not naturally soluble diffusible molecules. The present invention provides methods of specifically modulating WNT signaling through specific FZD receptors with engineered soluble WNT agonists to achieve differential effects of epithelial regeneration.

Disclosure of Invention

The invention is based in part on the use of WNT agonists to modulate epithelial proliferation of the gastrointestinal tract, particularly in inflammatory bowel disease.

The invention provides a method of treating a subject suffering from a gastrointestinal disorder comprising administering to the subject an engineered WNT signaling modulator. In certain embodiments, the modulator of WNT signaling is an engineered WNT agonist. In further embodiments, the engineered WNT agonist is selected from the group consisting of: engineered polypeptides, engineered antibodies containing at least one epitope binding domain, small molecules, siRNA, and antisense nucleic acid molecules. In further embodiments, the engineered WNT agonists comprise binding compositions that bind to one or more FZD receptors (FZD1-10) and binding compositions that bind to one or more LRP (LRP5-6) receptors. In yet another embodiment, a binding composition of an engineered WNT agonist comprises: to FZD5, FZD8, FZD1, FZD2, FZD7, FZD5,8, FZD1,2,7, or FZD1,2,7, 5, 8; FZD 4; FZD 9; or one or more binding compositions of FZD 10; or one or more binding compositions that bind to LRP5, LRP6, or LRP5 and 6. In another embodiment, an engineered WNT agonist comprises one or more binding compositions that bind to FZD5 and/or FZD 8; and one or more binding compositions that bind to LRP5 and/or LRP 6. In another embodiment, the engineered WNT agonist comprises a binding composition that binds to FZD5 and FZD8, and a binding composition that binds LRP 6. In further embodiments, the WNT agonist has the variable heavy chain sequence of SEQ ID NOs 1, 3, 5, 7, 9, 11, or 13; and the variable light chain sequence of SEQ ID NO 2,4, 6,8, 10, 12 or 14. In another embodiment, the engineered WNT agonist reduces inflammatory cytokine expression in the intestine or colon and/or repairs the intestinal epithelium. In some embodiments, the engineered WNT agonist comprises a tissue targeting molecule. In another embodiment, 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 (GPA 33; representative sequence is NCBI polypeptide reference sequence NP-005805.1), cadherin-17 (CDH 17; representative sequence is NCBI polypeptide reference sequence NP-004054.3) and mucin 13 (cell surface associated (Muc-13; representative sequence is NCBI polypeptide reference sequence NP-149038.3) or functional fragments or variants thereof CD with fistula formation and Ulcerative Colitis (UC).

The invention also provides a method of treating a subject having a gastrointestinal disorder comprising administering to the subject a tissue-specific WNT signaling-enhancing molecule. In certain embodiments, the WNT signal 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 another embodiment, the E3 ubiquitin ligase is selected from the group consisting of: zinc and ring finger protein 3(ZNRF3) and ring finger protein 43(RNF 43). In another embodiment, the first domain comprises an R-spondin (RSPO) polypeptide. In another embodiment, 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 have lower binding to G protein-coupled receptor 4-6(LGR4-6) containing a leucine-rich repeat. In certain embodiments, the engineered agonist or Wnt signaling enhancing molecule is incorporated with 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 certain embodiments, the tissue-targeting molecule is selected from the group consisting of: GPA33, CDH17 and MUC-13, or a functional fragment or variant thereof. In some embodiments, a WNT agonist is administered with a binding composition that specifically binds an inflammatory molecule. In certain embodiments, the binding composition specific for the inflammatory molecule is an antagonist of the inflammatory molecule. In additional embodiments, the antagonist of an inflammatory molecule is an antagonist of TNF, IL-12 and IL-23, or IL-23. In some embodiments, the gastrointestinal disorder is 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).

In another embodiment, the invention provides a method of treating an individual having a gastrointestinal disorder comprising administering to the individual an engineered WNT agonist and an engineered tissue-specific WNT signaling enhancing molecule. The engineered WNT agonist and the engineered tissue-specific WNT signaling enhancing molecule may be administered simultaneously or at different times. In some embodiments, the individual comprises an effective amount of both during the overlapping time period. In certain embodiments, the engineered WNT agonist comprises one or more binding compositions that bind to FZD5, FZD8, FZD1, FZD2, FZD7, FZD5 and 8, or FZD1,2, and 7, and one or more binding compositions 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 fragment thereof that binds to a tissue-specific cell surface antigen. In further embodiments, the tissue-targeting molecule is selected from the group consisting of: GPA33, CDH17 and MUC-13, or a functional fragment or variant thereof. In certain embodiments, the engineered WNT signal-enhancing molecule comprises a first domain that binds to one or more E3 ubiquitin ligases; and a second domain that binds to a tissue-specific receptor. In further embodiments, the E3 ubiquitin ligase is selected from the group consisting of: zinc and ring finger protein 3(ZNRF3) and ring finger protein 43(RNF 43). In some embodiments, the first domain comprises an R-spondin (rspo) polypeptide. In other embodiments, the RSPO polypeptide is selected from the group consisting of: RSPO-1, RSPO-2, RSPO-3, and RSPO-4. In another embodiment, the RSPO polypeptide comprises a first furin domain and a second furin domain. In yet another embodiment, the second furin domain is wild-type or mutated to have lower binding to a G protein-coupled receptor 4-6 containing a leucine-rich repeat (LGR 4-6). In further embodiments, the WNT signal enhancing molecule has the heavy chain sequence of SEQ ID NO 17, 20 or 23; and the light chain sequence of SEQ ID NO 16, 19 or 22. In some embodiments, the engineered WNT agonist and the engineered tissue-specific WNT signaling enhancing molecule are administered with a binding composition that specifically binds an inflammatory molecule. In further embodiments, the binding composition specific for an inflammatory molecule is an antagonist of an inflammatory molecule. In still other embodiments, the antagonist of an inflammatory molecule is an antagonist of TNF, IL-12 and IL-23, or IL-23. In certain embodiments, the gastrointestinal disorder is 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).

In another embodiment, the invention provides a method of treating an individual having a gastrointestinal disorder comprising administering to the individual an engineered WNT agonist and an engineered tissue-specific WNT signaling-enhancing combination molecule. In certain embodiments, the combinatorial molecule comprises: a) an engineered WNT agonist comprising one or more binding compositions that bind to FZD5, FZD8, FZD1, FZD2, FZD7, FZD5 and 8, or FZD1,2 and 7 and one or more binding compositions that bind to LRP5, LRP6, or LRP5, and b) an engineered WNT signaling enhancing molecule comprising a first domain that binds to one or more E3 ubiquitin ligase 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(RNF 43). In some embodiments, the first domain comprises an R-spondin (rspo) polypeptide. In other embodiments, the RSPO polypeptide is selected from the group consisting of: RSPO-1, RSPO-2, RSPO-3, and RSPO-4. In another embodiment, the RSPO polypeptide comprises a first furin domain and a second furin domain. In yet another embodiment, the second furin domain is wild-type or mutated to have lower binding to a G protein-coupled receptor 4-6 containing a leucine-rich repeat (LGR 4-6). In some embodiments, the combination molecule incorporates a tissue targeting molecule. In certain embodiments, the tissue-targeting molecule is an antibody or fragment thereof that binds to a tissue-specific cell surface antigen. In further embodiments, the tissue-targeting molecule is selected from the group consisting of: GPA33, CDH17 and MUC-13, or a functional fragment or variant thereof. In further embodiments, the WNT signal enhancing molecule has the heavy chain sequence of SEQ ID NO 17, 20 or 23; and the light chain sequence of SEQ ID NO 16, 19 or 22. In some embodiments, the combination molecule is administered with a binding composition that specifically binds to an inflammatory molecule. In further embodiments, the binding composition specific for an inflammatory molecule is an antagonist of an inflammatory molecule. In still other embodiments, the antagonist of an inflammatory molecule is an antagonist of TNF, IL-12 and IL-23, or IL-23. In certain embodiments, the gastrointestinal disorder is 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).

In particular embodiments of any of the methods disclosed herein, the WNT agonist is selected from those disclosed in any one of: PCT application publication nos. WO 2016/040895; US application publication nos. US 2017-0306029; US application publication nos. US 2017 and 0349659; PCT application publication nos. WO 2019/126398; or PCT application publication No. WO 2020/01030. In particular embodiments of any of the methods disclosed herein, the tissue-specific WNT signaling enhancing molecule is selected from those disclosed in any one of: PCT application publication nos. WO 2018/140821; U.S. application publication No. US 2020-0048324; or PCT application publication No. WO 2020/14271, all of which are incorporated herein by reference in their entirety.

In another embodiment, the disclosure provides a polypeptide that specifically binds to frizzled receptor 5(FZD5) and frizzled receptor 8(FZD8), wherein the polypeptide comprises a sequence having at least 80%, at least 90%, or at least 95% homology to the sequence set forth in any one of SEQ ID nos. 33-40. In some embodiments, the polypeptide comprises an antibody or antibody binding fragment. In some embodiments, the polypeptide comprises at least 5 or all 6 of the CDRs present in any of the sequences set forth in any of SEQ ID NOs 33-40. In some embodiments, the polypeptide comprises six CDRs present in any of the sequences set forth in any one of SEQ ID NOs 33-40, wherein one or more of the CDRs optionally comprises one, two, or three amino acid modifications, optionally a site mutation, an amino acid deletion, or an amino acid insertion.

In related embodiments, the present disclosure provides an engineered WNT agonist comprising: (a) (ii) one or more binding domains that bind to FZD5 and FZD8, wherein at least one of the one or more binding domains comprises a polypeptide having a sequence at least 80%, at least 90%, or at least 95% homologous to the sequence set forth in any one of SEQ ID NOs 33-40; and (b) one or more binding domains that bind to LRP5, LRP6, or both LRP5 and LRP 6. In some embodiments, the engineered WNT agonist comprises a polypeptide sequence that is at least 80%, at least 90%, at least 95%, or at least 98% homologous to any one of SEQ ID NOs 7-14. In some embodiments, the engineered WNT agonist comprises: a polypeptide sequence having at least 80%, at least 90% or at least 95% homology to SEQ ID No. 7 and a polypeptide sequence having at least 80%, at least 90% or at least 95% homology to SEQ ID No. 8; a polypeptide sequence having at least 80%, at least 90% or at least 95% homology to SEQ ID No. 9 and a polypeptide sequence having at least 80%, at least 90% or at least 95% homology to SEQ ID No. 10; a polypeptide sequence having at least 80%, at least 90% or at least 95% homology to SEQ ID NO. 11 and a polypeptide sequence having at least 80%, at least 90% or at least 95% homology to SEQ ID NO. 12; or a polypeptide sequence having at least 80%, at least 90% or at least 95% homology to SEQ ID NO. 13 and a polypeptide sequence having at least 80%, at least 90% or at least 95% homology to SEQ ID NO. 14.

In another related embodiment, the present disclosure provides a combinatorial molecule comprising: a) an engineered WNT agonist 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. In another embodiment, the disclosure provides a pharmaceutical composition comprising a polypeptide, an engineered WNT agonist, or a combinatorial molecule disclosed herein.

In related embodiments, the disclosure provides polypeptides that specifically bind to frizzled receptor 5(FZD5) and frizzled receptor 8(FZD8), wherein the polypeptides comprise one or more sequences having at least 80%, at least 90%, at least 95%, or at least 98% homology to the sequence shown in or encoded by any of SEQ ID nos. 33-40. In some embodiments, the polypeptide of claim 49, wherein the polypeptide comprises an antibody or antibody binding fragment. In some embodiments, the antibody or antibody binding fragment comprises at least 5 or all 6 CDRs present in any one of the following sequence combinations: 33 and 34; 35 and 36; 37 and 38; or SEQ ID NOs 39 and 40. In some embodiments, the polypeptide comprises six CDRs present in any one of the following combinations of sequences: 33 and 34; 35 and 36; 37 and 38; or SEQ ID NOs 39 and 40, wherein one or more of the CDRs comprise one, two or three amino acid modifications, optionally site mutations, amino acid deletions or amino acid insertions. In another embodiment, the present disclosure provides an engineered WNT agonist comprising: one or more binding domains that bind to FZD5 and FZD8, wherein at least one of the one or more binding domains comprises a polypeptide that specifically binds frizzled receptor 5(FZD5) and frizzled receptor 8(FZD8), e.g., any of the disclosures herein; and one or more binding domains that bind to LRP5, LRP6, or both LRP5 and LRP 6. The present disclosure also provides a combinatorial molecule comprising: a) an engineered WNT agonist 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.

In related embodiments, the present disclosure provides a method of treating an individual having a gastrointestinal disorder, the method comprising administering to the individual an engineered WNT agonist, an engineered WNT signal-enhancing molecule, and/or a combination molecule disclosed herein, or a pharmaceutical composition comprising an engineered WNT agonist or a combination molecule disclosed herein. In some embodiments, the gastrointestinal disorder is an inflammatory bowel disease optionally selected from the group consisting of: crohn's Disease (CD), CD with fistula formation and Ulcerative Colitis (UC). Any of the methods disclosed herein can be practiced using any of the engineered WNT agonists, engineered WNT signaling enhancing molecules, and/or combinatorial molecules disclosed herein.

Drawings

FIGS. 1A-1L show a cross-sectional view as taken through2.5 expression of the mouse intestinal curl receptors (FZD)1, 2, 3, 4, 5, 6,7, 8, 9 and 10 (fig. 1A-1J) and FZD5 and FZD7 (fig. 1K and 1L) in the human colon detected by HD Assay-Red. The number of red dots in the image indicates the FZD receptor expression level. Enlarged views of the selected regions are shown in the inset of fig. 1K (FZD5) and 1L (FZD 7).

Figure 2 shows the activity of recombinant soluble WNT agonist in tissue culture cells. WNT agonists were tested for signaling activity by a Super TOPFlash luciferase reporter (STF) assay. Dose response curves for R2M3-26, 1RC07-03, and R2M13-03 luciferase reporter gene activities were measured as shown for the figures.

Figures 3A-3E show the activity of different FZD receptor specific recombinant WNT agonists on mouse gut organoids. Mouse intestinal organoids were treated with R2M3-26 (FIG. 3A), 18R5-DKK1C scFv (FIG. 3B), C) R2M13-03 (FIGS. 3C and D)1RC07-03 (FIG. 3D) in the presence of 1. mu.M IWP2 (porcine inhibitor) in basal medium. Figure 3E shows control organoids treated with only 1 μ M IWP 2. F: normal organoids grown in basal medium. The scale bar in fig. 3A, 3B and 3E is at 200 microns, fig. 3C and 3D: at 400 microns.

Figure 4 shows immunohistochemical staining of mouse small intestine organoids after treatment with 100nM of R2M3-26, showing staining with anti-Ki 67 (red) and anti-E-cadherin (green) to demonstrate cell proliferation after WNT agonist treatment.

Figure 5A shows a schematic of the experimental protocol used for in vivo studies in a mouse model of Dextran Sodium Sulfate (DSS) -induced acute colitis. Red arrows indicate daily Body Weight (BW), fecal score, and fecal occult blood test. Top arrows (day 4 and 7) and bottom arrows (day 4, day 5, day 6, day 7, day 8 and day 9) above the bars indicate twice weekly and daily treatment times, respectively. Figures 5B and 5C show graphs of body weight and stool scores over time treated with WNT agonists and/or R-Spondin 2(RSPO 2-Fc). For fig. 5B, the top-to-bottom line at day 9 corresponds to: no DSS, RSPO2-hFc/R2M 326 daily, RSPO2-hFc/R2M 3262/week, R2M3-26(10mpk) 2/week, RSPO2-hFc 3mpk daily, anti-GFP, and RSPO2-hFc 3mpk 2/week. For FIG. 5C, day 9 top-to-bottom lines correspond to RSPO2-hFc 3mpk daily, RSPO2-hFc 3mpk 2/week, anti-GFP, R2M-26(10mpk) 2/week, RSPO2-hFc/R2M 3262/week, and RSPO2-hFc/R2M 326/week. RSPO2-Fc/R2M3-26 combined treatment, twice weekly or daily, significantly improved DAI at day 9 compared to negative controls. R2M3-26 alone and in combination significantly improved body weight on day 10 (. P values, 0.05;. P values <0.01,. P values <0.001,. P values <0.0001)

FIGS. 6A-6E show pathological image analysis of R2M3-26 and RSPO2-Fc treated colitis models, alone and in combination.

FIGS. 7A-7E show semi-quantitative analysis of the extent of colitis after treatment with R2M3-26 and RSPO2-Fc, alone and in combination. R2M3-26 treatment significantly reduced histological scores on mucosal erosion, inflammatory severity, crypt proliferation and goblet cell loss on day 10(, P value, 0.05;. P value < 0.01;. P value). Such as, for example, Geboes et al (2000) gastrointestinal disorders (Gut) 47: 404 and 409.

Figure 8 shows a histological image of a cross section of the small intestine. R2M3-26 alone did not cause proliferation of the small intestine, while RSPO2-Fc alone and R2M3-26 in combination induced proliferation.

Figures 9A-9J show that R2M3-26, RSPO2-Fc, and combined treatment of R2M3-26 and RSPO2-Fc all reduced serum inflammatory cytokine levels of IFN- γ, IL-1 β, IL-12P70, and TNF- α (, P values, 0.05;, <0.001, > P values, < 0.0001). For each figure, the bars from left to right are as follows: blue-no DSS treatment; green-GFP control; purple-R2M 3-26(10mpk) 2X/week; orange-RSPO 2-hFc (3mpk) 2X/week; black-RSPO 2-hFc (3mpk) daily; brown-RSPO 2-hFc (3mpk) + R2M3-26(10mpk) 2X/week; and dark blue-RSPO 2-hFc (3mpk) + R2M3-26(10mpk) daily.

Figures 10A-10B show weight loss and stool scores in DSS-induced acute colitis models (4% DSS for 7 days followed by 1% DSS until termination). For fig. 10A, the top-to-bottom line at day 10 corresponds to: no DDS, R2M3-26(10mpk) 2/week, R2M13-26(10mpk) 2/week, CO 7-263 mpk 2/week, RSPO2/R2M 3-262/week, DSS PBS and anti-GFP. For fig. 10B, the top-to-bottom line at day 10 corresponds to: RSPO2/R2M 3-262/week, anti-GFP, DSS PBS, R2M3-26(10mpk) 2/week, R2M3-26(10mpk) daily, CO 7-263 mpk 2/week, and R2M 13-2610 mpk 2/week. In the DSS-treated group, R2M3-26, R2M13-26, and 1RC07-26 treatments significantly improved body weight (fig. 10A) and stool score (fig. 10B) at day 10 compared to negative controls (PBS or GFP). (value of P, 0.05; < 0.01; < 0.001; < 0.0001).

Figures 11A-11H show that WNT agonist treatment repaired colonic epithelial lesions in the DSS model. Histological evaluation of the transverse colon of DSS model mice showed colonic epithelial lesions including inflammation extending from the mucosa to the serosa, crypt hyperplasia, goblet cell loss and ulceration. The R2M3-26, R2M13-26, and 1RC07-26 treatments effectively repaired the colonic epithelium, reducing epithelial erosion, goblet cell loss, and neutrophil migration.

Figures 12A-12H show cross sections of the small intestine in DSS colitis model mice that were not treated, treated with WNT agonists or a combination of WNT agonists and RSPO 2-hFc. R2M3-26, R2M13-26 or 1RC07-3 did not cause small intestine hyperplasia, whereas treatment with R2M3-26 in combination with RSPO2-Fc induced small intestine hyperplasia.

Figures 13A-13F show that WNT agonist treatment reduced inflammatory cytokine levels of TNF- α, IL-6 and IL-8 in serum and colon tissues (; P value <0.01,; P value <0.001,; P value < 0.0001). For each figure, the bars from left to right are shown below.

Figures 14A-14B show that R2M13-26 treatment shows dose-dependent efficacy in DSS models with DAI. R2M13-26 treatment, 0.3, 1, 3, 10mpk, twice weekly, and 1, 3, 10, 30mpk, once weekly, both reduced DAI in a dose response pattern (figure 14B) (. P value, 0.05;. P value <0.01,. P value < 0.001. P value < 0.0001). For fig. 14A, the line at day 10 time point is associated with a top-down legend. For FIG. 14B, the line at day 10 time point is related from top to bottom to: DSS anti-GFP 10mpk 2/week, R2M 13-261 mpk 1/week, R2M 13-2630 mpk 1/week, R2M 13-2610 mpk 1/week and R2M 13-263 mpk 1/week.

Figures 15A-15J show histological evaluation of cross-sections of the transverse colon of DSS model mice. Colonic epithelial lesions included neutrophil infiltration, edema, crypt hyperplasia, goblet cell loss, and ulceration (fig. 15B). R2M13-26 treatments with different doses and frequency all showed improved colonic histology, repair of epithelial erosion, and reduction of goblet cell loss and neutrophil migration in DSS colitis mice.

Figures 16A-16C show that R2M13-26 treatment at different doses and frequencies all reduced inflammatory cytokine levels in serum of TNF- α, IL-6 and IL-8 (, P values, 0.05;, < 0.01;, < 0.001;, < 0.0001).

Figures 17A-17C show that R2M13-26 treatment at different doses and frequencies all reduced inflammatory cytokine levels of TNF- α, IL-6 and IL-8 in colon tissue (, P values, 0.05;, < 0.01;, < 0.001;, < 0.0001).

FIG. 18: activity of four FZD5, 8-specific WNT agonists. FZD5, 8-specific agonist signaling activity was tested by Super TOPFlash luciferase reporter (STF) assay. Dose response curves for 57SE8-26, 57SB8-26, 174R-E01-26 and 57SA10-26 luciferase reporter activity were measured as indicated and compared to the activity of R2M13-26 in the same assay.

Figures 19A-19D show the efficacy of four FZD5, 8-specific WNT agonists in an acute DSS model. FIG. 19A shows that treatment with the four FZD5, 8-specific WNT agonists all showed efficacy by reducing the disease activity index in a DSS model (DAI; see Geboes et al (2000) gastrointestinal disorders 47: 404-409). WNT agonist treatment at 10mpk twice weekly significantly reduced DAI compared to anti-GFP control (. P values <0.01,. P values <0.001,. P values < 0.0001). The top to bottom line on day 8 corresponds to: anti-GFP, 57SE8-26, 57SB8-26, 174RE01-26, R2M13-26, 57SA10-26 and no DSS.

Figures 19B-19D show that four different FZD5, 8-specific WNT agonist treatments all reduced inflammatory cytokine levels of TNF- α, IL-6 and IL-8 in serum compared to the same dose of R2M13-26 (.; P values <0.01,; P values <0.001,; P values < 0.0001).

Figure 20 shows the antibody clone C14 in IgG form (see e.g. WO2016168607a1) bound to a MUC-13 expressing human intestinal HT29 cell line. The other two MUC-13 binding agents, C4 and C7 (see e.g. WO2016168607a1), which were also expressed as full length antibodies, did not show specific binding to human intestinal cells HT29 (fig. 20A-20C). Non-specific binding was assessed using a HEK293 cell line that does not express MUC-13 (FIGS. 20D-20F). Cell surface binding of MUC-13 antibody was examined by FACS at 10 nM. C14 showed significant FACS translocation on HT29 cells, but not on HEK293 cells, indicating specific binding.

FIG. 21 shows the testing of the signaling activity of MUC-13 targeted mutant RSPO2(mutRSPO2) fusions by a SuperTOPFlash luciferase reporter (STF) assay in HT29 cells or HEK293 cells. MutRSPO2 has an amino acid mutation in the Furin2 binding domain, thus reducing binding to LGR1-4 (see e.g. WO 2020014271). Dose response curves for C4-mutRSPO2, C7-mutRSPO2, and C14-mutRSPO2 luciferase reporter activity were measured as shown for the graphs. C14-mutRSPO exhibited a specific left shift of the dose response curve only in HT29 cells, where EC50 was comparable to wild-type Fc-RSPO 2.

FIG. 22 shows the maintenance of human small intestine organoid growth when wild-type RSPO in culture was replaced by C14-mutRSPO. Human small intestine organoids were grown in basal medium with RSPO-1 replaced by non-epithelial cell (e.g. hepatocyte) targeted mutRSPO1(ASGR1-mutRSPO 1; see e.g. WO 2020014271; and WO 20140821) (fig. 22A-22C) in the concentration dilution series shown or by C14-mutRSPO2 (fig. 22D-22F) at the same concentration. While organoids grown in ASGR1-mutRSPO1 stopped growing and began to denature, similar to the results observed when grown in basal medium without any RSPO (fig. 22G), C14-mutRSPO was able to maintain organoid growth similar to intesticult (stem cell Technologies) medium with wild-type RSPO (fig. 22H).

Detailed Description

As used herein, including the appended claims, the singular forms of words such as "a" and "the" include corresponding plural referents unless the context clearly dictates otherwise.

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

I. And (4) defining.

The "activity" of a molecule may describe or refer to the binding of the molecule to a ligand or receptor, catalytic activity, the ability to stimulate gene expression, antigenic activity, modulation of the activity of other molecules, and the like. "activity" of a molecule may also refer to activity that modulates or maintains interactions between cells (e.g., adhesion) or maintains cellular structure (e.g., cell membrane or cytoskeleton). "Activity" may also refer to specific activity, e.g., [ catalytic activity ]/[ mg protein ] or [ immunological activity ]/[ mg protein ], and the like.

As used herein, the terms "administering" or "introducing" or "providing" as used herein refer to the delivery of a composition to a cell, cells, tissue organoid and/or organ of an individual or to an individual. Such administration or introduction may occur in vivo, in vitro, or ex vivo.

As used herein, the term "antibody" means an isolated or recombinant binding agent comprising an essential variable region sequence that specifically binds to an epitope of an antigen. Thus, an antibody is any form of antibody or fragment thereof that exhibits a desired biological activity, e.g., binds to a specific target antigen. It is therefore used in the broadest sense and specifically encompasses monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, nanobodies, bifunctional antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, including but not limited to scFv, Fab, and Fab2, so long as they exhibit the desired biological activity.

An "antibody fragment" comprises a portion of an intact antibody, for example, the antigen binding or variable region of an intact antibody. Examples of antibody fragments include Fab, Fab ', F (ab')2, and Fv fragments; a bifunctional antibody; linear antibodies (e.g., Zapata et al, Protein engineering 8(10):1057-1062 (1995)); single chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments. Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each having a single antigen-binding site; and residual "Fc" fragments, the designation reflecting their ability to crystallize readily. Pepsin treatment produces F (ab')2 fragments that have two antigen combining sites and are still capable of cross-linking antigens.

The term "antigen" refers to a molecule or a portion of a molecule that is capable of being bound by a selective binding agent, such as an antibody, and additionally a molecule or a portion of a molecule that is capable of being used in an animal to produce an antibody that is capable of binding to an epitope of the antigen. In certain embodiments, a binding agent (e.g., a WNT-replacement molecule or binding region thereof or a WNT antagonist) is considered to specifically bind an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and/or macromolecules.

As used herein, the term "antigen-binding fragment" refers to a polypeptide comprising an immunoglobulin heavy and/or light chain, or(Nab) that binds to an antigen of interest, in particular to one or more FZD receptors, or to LRP5 and/or LRP 6. In this regard, an antigen-binding fragment of an antibody described herein can comprise 1,2, 3, 4, 5, or all 6 CDRs from the VH and VL of an antibody that binds one or more FZD receptors or LRP5 and/or LRP 6.

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

As used herein, the term "bifunctional antibody" refers to an antibody comprising a first arm with specificity for one antigenic site and a second arm with specificity for a different antigenic site, i.e., a bifunctional antibody is bispecific.

"bispecific antibodies" are used herein to refer to full-length antibodies produced by the four-source hybridoma technique (see Milstein et al, Nature, 305(5934):537 (1983)), by chemically binding two different monoclonal antibodies (see Staerz et al, Nature, 314(6012):628-631(1985)), or by knob-in-hole (knob-int-hole) or similar methods that introduce mutations in the Fc region (see Holliger et al, Proc. Natl. Acad. Sci. USA, 90 (14): 6444-6448(1993)), resulting in a variety of different immunoglobulin substances, only one of which is a functional bispecific antibody. Bispecific antibodies bind one antigen (or epitope) on one of their two binding arms (one HC/LC pair) and a different antigen (or epitope) on their second arm (the other HC/LC pair). According to this definition, a bispecific antibody has two different antigen-binding arms (in both specificity and CDR sequences) and is monovalent for each antigen to which it binds.

"comprising" means that the element is required in, for example, a composition, method, kit, etc., but that other elements may be included to form, for example, a composition, method, kit, etc., within the scope of the claims. For example, an expression cassette "comprising" 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 polyadenylation sequences, enhancer elements, other genes, linker domains, and the like.

"consisting essentially of means that the scope of, for example, the described compositions, methods, kits, etc., is limited to the specified materials or steps that do not materially affect the basic and novel characteristics of, for example, the compositions, methods, kits, etc. For example, an expression cassette "consisting essentially of" a gene encoding a therapeutic polypeptide operably linked to a promoter and polyadenylation sequence may include additional sequences, such as linker sequences, so long as they do not substantially affect the transcription or translation of the gene. As another example, a variant or mutant polypeptide fragment "consisting essentially of" the sequence has the amino acid sequence of the sequence plus or minus about 10 amino acid residues at the boundaries of the sequence of the full-length native polypeptide from which it is derived, e.g., 10, 9, 8,7, 6, 5, 4, 3, 2, or 1 residues less than the boundary amino acid residues, or 1,2, 3, 4, 5, 6,7, 8, 9, or 10 residues more than the boundary amino acid residues.

"consisting of" means a composition, method, or kit that excludes any element, step, or ingredient not specified in the claims. For example, a polypeptide or polypeptide domain "consisting of" the sequence contains only the sequence.

"control element" or "control sequence" refers to a nucleotide sequence involved in molecular interactions that contribute to the functional regulation of a polynucleotide, including the replication, duplication, transcription, splicing, translation or degradation of a polynucleotide. The modulation may affect the frequency, speed, or specificity of the process, and may be enhancing or suppressing in nature. Control elements known in the art include, for example, transcriptional regulatory sequences, such as promoters and enhancers. A promoter is a region of DNA that is capable of binding RNA polymerase under certain conditions and initiating transcription of a coding region that is typically located downstream (3' to) the promoter.

An "epitope" is a specific region on an antigen that an antibody recognizes and binds, and is also referred to as an "antigenic determinant". Epitopes are typically 5-8 amino acids long on the surface of the protein. Proteins are three-dimensional folded structures, and epitopes can be recognized only in the form in which they are present in solution or in their native form. When an epitope is composed of amino acids that are grouped together by a three-dimensional structure, the epitope is conformational or discontinuous. An epitope is a continuous or linear epitope if it is present on a single polypeptide chain. Depending on the epitope recognized by the antibody, it may bind only fragments or denatured segments of the protein, or it may also be capable of binding to the native protein.

The portion of an antibody or antibody fragment thereof that recognizes an epitope is referred to as an "epitope binding domain" or an "antigen binding domain". The epitope or antigen binding domain of an antibody or antibody fragment is in the Fab fragment and the effector functions in the Fc fragment. Six segments, called variable regions of heavy and light chains (V)_HAnd V_L) The Complementarity Determining Regions (CDRs) within, loop out of the framework (FR regions) globular structure of the rest of the antibody, and interact to form an exposed surface at one end of the molecule. This is the antigen binding domain. Generally, 4-6 CDRs will be directly involved in binding to antigen, but fewer CDRs may provide the primary binding motif.

An "expression vector" is a vector, such as a plasmid, minicircle, viral vector, liposome, etc., discussed herein or known in the art, which comprises a region encoding a gene product of interest and is used to effect expression of the gene product in a desired target cell. Expression vectors also include control elements, such as promoters, enhancers, UTRs, miRNA targeting sequences, and the like, operably linked to the coding region to facilitate expression of the gene product in a target. The combination of a control element and a gene or genes to which it is operably linked for expression is sometimes referred to as an "expression cassette," many of which are known and available in the art, or can be readily constructed from components available in the art.

As used herein, the term "FR set" refers to the four flanking amino acid sequences of the CDRs of the set of CDRs that make up the heavy or light chain V regions. Some FR residues may contact the bound antigen; however, the FR is primarily responsible for folding the V region into the antigen binding site, particularly FR residues immediately adjacent to the CDRs. Within the FR, certain amino residues and certain structural features are extremely highly conserved. In this regard, all V region sequences contain an internal disulfide loop of about 90 amino acid residues. When the V-region is folded into the binding site, the CDRs appear as prominent loop motifs, forming an antigen binding surface. It is generally accepted that there are conserved structural regions of the FR that affect the circular folding of the CDR into certain "canonical" structures, regardless of the precise CDR amino acid sequence. In addition, certain FR residues are known to be involved in non-covalent interdomain contacts that stabilize antibody heavy and light chain interactions.

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

"monoclonal antibody" refers to a homogeneous population of antibodies, wherein the monoclonal antibodies are composed of amino acids (both naturally occurring and non-naturally occurring) that are involved in selective binding of an epitope. Monoclonal antibodies are highly specific, being directed against a single epitope. The term "monoclonal antibody" encompasses not only intact monoclonal antibodies and full-length monoclonal antibodies, but also fragments thereof (e.g., Fab ', F (ab')2, Fv), single chains (scFv),variants thereof, fusion proteins comprising antigen-binding fragments of monoclonal antibodies, humanized monoclonal antibodies, chimeric monoclonal antibodies and antibodies comprising a peptide having the desired specificity and binding to the epitopeAny other modified configuration of immunoglobulin molecules that are antigen binding fragments of the epitope binding sites (epitope recognition sites) of the potential for translocation, including the WNT replacement molecules disclosed herein. . There is no intention to limit the source of the antibody or the manner in which the antibody is prepared (e.g., by hybridoma, phage selection, recombinant expression, transgenic animal, etc.). The term includes whole immunoglobulins and fragments and the like as described above under the definition of "antibody".

As used herein, the term "native" or "wild-type" refers to a nucleotide sequence, e.g., a gene or gene product, e.g., RNA or protein, that is present in a wild-type cell, tissue, organ, or organism. As used herein, the term "variant" refers to a reference polynucleotide or polypeptide sequence, e.g., a mutant of a native polynucleotide or polypeptide sequence, i.e., having less than 100% sequence identity to the reference polynucleotide or polypeptide sequence. In other words, a variant comprises at least one amino acid difference (e.g., amino acid substitution, amino acid insertion, amino acid deletion) relative to a 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 to the full-length native polynucleotide sequence, e.g., 75% or 80% or more, e.g., 85%, 90% or 95% or more, e.g., 98% or 99% identity to the full-length native polynucleotide sequence. As another example, a variant may be a polypeptide having 70% or more sequence identity to the full-length native polypeptide sequence, e.g., 75% or 80% or more identity, e.g., 85%, 90% or 95% or more, e.g., 98% or 99% identity to the full-length native polypeptide sequence. Variants may also include a reference sequence, e.g., a variant fragment of a native sequence, which shares 70% or greater sequence identity with the reference sequence, e.g., a fragment of the native sequence, e.g., shares 75% or 80% or greater identity, e.g., 85%, 90% or 95% or greater, e.g., 98% or 99% identity with the native sequence.

"operably linked" refers to the juxtaposition of genetic elements wherein the elements are in a relationship permitting them to operate in their intended manner. For example, a promoter is operably linked to a coding region if it helps to initiate transcription of the coding sequence. Intervening residues may be present between the promoter and the coding region so long as this functional relationship is maintained.

As used herein, the terms "polypeptide," "peptide," and "protein" refer to a polymer of amino acids of any length. The term also encompasses amino acid polymers that have been modified: including, for example, disulfide bond formation, glycosylation, lipidation, phosphorylation, or binding to a labeling component.

The term "polynucleotide" refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides or analogs thereof. Polynucleotides may include modified nucleotides, such as methylated nucleotides and nucleotide analogs, and may be interspersed with non-nucleotide components. If present, modification of the nucleotide structure may be performed before or after assembly of the polymer. The term polynucleotide as used herein refers interchangeably to double-stranded and single-stranded molecules. Unless otherwise stated or required, any embodiment of the invention described herein (which is a polynucleotide) encompasses a double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form.

A polynucleotide or polypeptide has a certain percentage of "sequence identity" to another polynucleotide or polypeptide, meaning that when aligned, the percentage of bases or amino acids is the same when compared to the two sequences. Sequence similarity can be determined in a number of different ways. To determine sequence identity, sequences can be aligned using methods and computer programs including BLAST available through the world wide web ncbi. Another alignment algorithm is FASTA, available in the Genetics Computing Group (GCG) package of Madison, Wis., USA, a capital division of Oxford Molecular Group, Inc. Other techniques for alignment are described in Enzymology, volume 266: computer Methods for Macromolecular Sequence Analysis (Computer Methods for Macromolecular Sequence Analysis) (1996), editor Dolitle, Academic Press, Inc., a subsidiary of Harbour Brace & Co., San Diego, Calif., USA, Calif., USA. Of particular interest are alignment programs that allow gaps in the sequence. Smith-Waterman is a type of algorithm that allows gaps in sequence alignments. See methods in molecular biology (meth. mol. biol.) 70:173-187 (1997). In addition, the GAP program using Needleman and Wunsch alignment methods can be used to align sequences. See journal of molecular biology (J.mol.biol.) 48: 443-.

Interestingly, the BestFit program that uses the native homology algorithm of Smith and Waterman to determine sequence identity ("Applied math in Applied Mathematics") 2: 482-.

Another program of interest is the FastDB algorithm. FastDB describes Current Methods in Sequence Comparison and Analysis (Current Methods in Sequence compatibility and Analysis), macromolecular Sequencing and Synthesis (macromolecular Sequencing and Synthesis), selection Methods and Applications (Selected Methods and Applications), pp.127 and 149, 1988, an R.Liss company. Percent sequence identity was calculated by FastDB based on the following parameters: mismatch penalty: 1.00; gap penalties: 1.00; gap size penalty: 0.33; and merge penalties: 30.0.

as used herein, "promoter" encompasses a DNA sequence that directs the binding of RNA polymerase and thereby promotes RNA synthesis, i.e., a minimal sequence sufficient to direct transcription. Promoters and corresponding protein or polypeptide expression may be ubiquitous, meaning strong activity or cell type-specific, tissue-specific or species-specific for a wide range of cells, tissues and species. A promoter may be "constitutive," meaning persistently active, or "inducible," meaning that the promoter can be activated or deactivated by the presence or absence of biological or non-biological agents. Also included in the nucleic acid constructs or vectors of the invention are enhancer sequences which may or may not be contiguous with the promoter sequence. Enhancer sequences affect promoter-dependent gene expression and may be located in the 5 'or 3' region of the native gene.

"recombinant" as applied to a polynucleotide refers to a polynucleotide that is the product of various combinations of cloning, restriction, or ligation steps, as well as other procedures that result in a construct that is different from a polynucleotide found in nature.

As used herein, "RNA interference" refers to the use of agents that reduce target gene expression by degrading target mrnas through endogenous gene silencing pathways (e.g., Dicer and RNA-induced silencing complex (RISC)). RNA interference can be achieved using a variety of agents, including shrnas and sirnas. "short hairpin RNA" or "shRNA" refers to a double-stranded artificial RNA molecule with hairpin turns that can be used to silence target gene expression via RNA interference (RNAi). Expression of shrnas in cells is typically achieved by delivery of plasmids or by viral or bacterial vectors. shRNA is a favorable mediator for RNAi because it has a relatively low degradation rate and turnover rate. Small interfering RNAs (sirnas) are a class of double-stranded RNA molecules, typically 20-25 base pairs in length, similar to mirnas, and operate within the RNA interference (RNAi) pathway. It prevents translation by interfering with the expression of a specific gene having a complementary nucleotide sequence by degrading the mRNA after transcription. In certain embodiments, the siRNA is 18, 19, 20, 21, 22, 23, or 24 nucleotides in length and has a2 base overhang at its 3' end. siRNA can be introduced into individual cells and/or culture systems and cause degradation of the target mRNA sequence. As used herein, "morpholino" refers to a modified nucleic acid oligomer in which standard nucleobases are bound to a morpholino ring and are linked by phosphorodiamidate linkages. Morpholinos bind to complementary mRNA sequences similar to siRNA and shRNA. However, morpholinos function by steric inhibition of mRNA translation and alteration of mRNA splicing rather than targeting complementary mRNA sequences for degradation.

The terms "treatment" and the like are generally used herein to mean obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of completely or partially preventing the disease or symptoms thereof, e.g. reducing the likelihood of developing the disease or symptoms thereof in the individual, and/or may be therapeutic in terms of a partial or complete cure for the disease and/or side effects caused by the disease. As used herein, "treatment" covers any treatment of a disease in a mammal and includes: (a) preventing the occurrence of a disease in an individual who may be predisposed to the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease, i.e., arresting its development; or (c) relieving the disease, i.e., causing regression of the disease. The therapeutic agent may be administered before, during or after the onset of the disease or injury. Treatment of developing diseases is of great interest, where treatment stabilizes or reduces undesirable clinical symptoms in the patient. It is desirable to perform such treatment before the affected tissue is completely lost of function. It is desirable to administer the subject therapy during and in some cases after the symptomatic phase of the disease.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology), microbiology, biochemistry and immunology, which are within the skill of the art. Such techniques are explained fully in, for example, the following documents: "" molecular cloning: a Laboratory Manual, second edition (Sambrook et al, 1989); "oligonucleotide synthesis" (compiled by m.j.gait, 1984); "Animal Cell Culture" (eds., R.Freshney, 1987); "Methods in Enzymology" (Academic Press, Inc.)); "Handbook of Experimental Immunology" (eds. D.M.Weir & C.C.Blackwell); "Gene Transfer Vectors for Mammalian Cells" (eds. J.M.Miller & M.P.Calos, 1987); "Molecular Biology Protocols in Molecular Biology" (ed. F.M. Ausubel et al, 1987); "" PCR: polymerase Chain Reaction (PCR), The Polymerase Chain Reaction, "(eds., Mullis et al, 1994); and "(Current Protocols in Immunology)" in (J.E.Coligan et al, 1991), each of which is expressly incorporated herein by reference.

Several aspects of the invention are described below with reference to example applications for illustrative purposes. It should be understood that numerous specific details, relationships, and methods are set forth to provide a full understanding of the invention. One skilled in the relevant art will readily recognize, however, that the invention can be practiced without one or more of the specific details, or with other methods. The present invention is not limited by the illustrated ordering of acts or events, as some acts may occur in different orders and/or concurrently with other acts or events. Moreover, not all illustrated acts or events are required to implement a methodology in accordance with the present invention.

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms "includes," having, "" with, "or variants thereof are used in either the detailed description and/or the claims, such terms are intended to be inclusive in a manner similar to the term" comprising.

The term "about" or "approximately" means within an acceptable range of deviation of the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 1 or greater than 1 standard deviation, according to practice in the art. Alternatively, "about" may mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value. Alternatively, particularly for biological systems or processes, the term may mean within an order of magnitude, preferably within 5 times the value, and more preferably within 2 times. Where a particular value is described in the application and claims, unless otherwise stated, it should be assumed that the term "about" means within an acceptable error range for the particular value.

All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. It should be understood that to the extent there is conflict, the present disclosure supersedes any disclosure of the incorporated publication.

It is further noted that the claims may be drafted to exclude any optional element. Thus, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely", "only", or use of a "negative" limitation in connection with the recitation of claim elements.

Unless otherwise indicated, all terms used herein have the same meaning as they would to one skilled in the art and the practice of the present invention will employ conventional techniques of microbiology and recombinant DNA technology within the knowledge of one skilled in the art.

Overview.

The present invention provides methods of modulating WNT signaling to ameliorate gastrointestinal disorders including, but not limited to, inflammatory bowel disease, including, but not limited to, crohn's disease with fistula formation, and ulcerative colitis. In particular, the invention provides agonists of WNT/β -catenin signalling for use in enhancing the regeneration of intestinal epithelium due to injury from these conditions.

WNT ("wingless associated integration site" or "wingless and Int-1" or "wingless-Int") ligand and its signaling play a key role in controlling the development, homeostasis and regeneration of many important organs and tissues, including bone, liver, skin, stomach, intestine, kidney, central nervous system, breast, taste buds, ovary, cochlea, lung and many others (reviewed by Clevers, Loh and Nusse, 2014; 346: 1248012). Modulation of the WNT signaling pathway has potential for treatment of degenerative diseases and tissue injury.

One of the therapeutic challenges in modulating WNT signaling is the presence of multiple WNT ligands and WNT receptors, frizzled receptors 1-10(FZD 1-10), where many tissues express multiple and overlapping FZD. Typical WNT signaling involves Low Density Lipoprotein (LDL) receptor-related protein 5(LRP5) or Low Density Lipoprotein (LDL) receptor-related protein 6(LRP6) as co-receptors, which are widely expressed in various tissues, in addition to FZD.

R-spondyloproteins (R-spondin)1-4 are a family of ligands that amplify WNT signals. Each R-spinal protein acts through a receptor complex containing zinc and either ring finger 3(ZNRF3) or ring finger protein 43(RNF43) at one end and a G protein-coupled receptor 4-6(LGR4-6) containing leucine-rich repeats at the other end (reviewed, for example, by Knight and Hankenson 2014, Matrix Biology (Matrix Biology); 37: 157-161). R-vertebrates may also act through other mechanisms of action. ZNRF3 and RNF43 are two membrane-bound E3 ligases that specifically target WNT receptors (FZD1-10 and LRP5 or LRP6) for degradation. Binding of R-spondyloproteins to ZNRF3/RNF43 and LGR4-6 causes clearance or chelation of a ternary complex, which removes the E3 ligase from the WNT receptor and stabilizes the WNT receptor, resulting in enhanced WNT signaling. Each R-spondyloprotein contains two Furin (Furin) domains (1 and 2), with Furin domain 1 binding to ZNRF3/RNF43 and Furin domain 2 binding to LGR 4-6. Fragments of R-spondyloproteins containing furin domains 1 and 2 were sufficient to amplify WNT signaling. Although the R-spondyloprotein effect depends on the WNT signal, the effect of R-spondyloprotein is not tissue specific since both LGR4-6 and ZNRF3/RNF43 are widely expressed in various tissues.

Activation of WNT signaling by RSPO or by WNT agonists may be useful in the treatment of gastrointestinal disorders. Previous work in the literature suggests that RSPO can be used in the treatment of experimental colitis of the colon (j.zhao et al, 2007). WNT agonist molecules may also be used in the treatment of gastrointestinal disorders. In particular, active WNT signaling may provide a major stem cell maintenance signal and play a key role in regulating intestinal epithelial regeneration in homeostasis and injury. Two intestinal epithelial lineages, absorptive and secretory, define two major functions of the intestinal organs: secretory cells secrete hormones and provide resistance to food-borne microorganisms, toxins and antibiotics primarily through secretion of mucus and antimicrobial peptidesThe original important barrier. In contrast, absorptive cells carry out the uptake of dietary nutrients because they are predominantly located at the tip of the villus in the small intestine or at the top of the colon crypt, thus constituting the majority of the luminal surface area of the intestine (see, e.g., Trends in Cell Biol., in Santos et al (2018) printing),https://doi.org/10.1016/j.tcb.2018.08.001). Under steady state conditions, all cells in the intestinal epithelium regenerate within 3-10 days.

Different niche factors maintain ISC activity and unique non-epithelial and/or epithelial cells elaborate the various signals that make up the cellular niche. Such niche factors include canonical signals such as WNT, R-spondin, notch and Bone Morphogenic Protein (BMP), but also inflammatory and dietary effects. After injury, ISC niche adaptation goes beyond its homeostasis to explain pathogenic stimuli and convert them into epithelial regeneration. This regeneration is mediated by viable Lgr5+ ISC or other mature cell types such as intestinal, enteroendocrine, or Paneth (2016) (Development 143: 3639) -3649 that can switch back to Lgr5+ ISC to aid in epithelial regeneration.

Intestinal Stem Cells (ISCs), also known as Columnar Basal Cells (CBCs), at the bottom of the intestinal crypts are inserted with Pan's cells secreting WNT (Cheng and Leblond (1974), J.A. Neuroradiotherapeutic (am.J.Ant.) 141: 537-561). Mesenchymal cells surrounding the intestinal epithelium also secrete some WNT proteins, providing overlapping stem cell niche functions in vivo (Farin et al (2012): gastroenterology (143: 1518-1529)). In the presence of WNT signaling, ISCs divide to generate self-renewing stem cells and differentiate daughter cells, which first undergo several rapid Transit Amplification (TA) divisions and then differentiate into functional cell types. Quiescent stem cell populations also exist in intestinal crypt +4 cells, which can contribute to epithelial regeneration when CBC is impaired (Tian et al 2011 Nature 478: 255-. Individual lineage commitment and terminal differentiation occurs when TA cells migrate along the crypt-villus axis, away from WNT-producing cells.

In some embodiments, a WNT/β -catenin signaling agonist may comprise a binding agent or epitope binding domain that binds one or more FZD receptors and inhibits or enhances WNT signaling. In certain embodiments, the agent or antibody specifically binds to a cysteine-rich domain (CRD) within one or more human frizzled receptors to which it binds. In addition, binding agents containing epitope binding domains directed against LRP may also be used. In some embodiments, a WNT/β -catenin agonist has a binding agent or epitope binding domain that binds the E3 ligase ZNRF3/RNF 43. The E3 ligase agonist antibody or fragment thereof may be a single molecule or combined with other WNT antagonists such as FZD receptor antagonists, LRP receptor antagonists, and the like.

As is well known in the art, antibodies are immunoglobulin molecules capable of specifically binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., via at least one epitope binding domain located on the variable region of the immunoglobulin molecule. As used herein, the term encompasses not only intact polyclonal or monoclonal antibodies, but also fragments thereof (e.g., dAb, Fab', (F (ab))₂Fv, single chain (scFv),(Nabs; also known as sdAbs or VHH domains), DVD-Igs, synthetic variants thereof, naturally occurring variants, fusion proteins comprising epitope-binding domains, humanized antibodies, chimeric antibodies, and any other modified configuration of an immunoglobulin molecule comprising an antigen-binding site or fragment (epitope-recognition site) with the desired specificity. "bifunctional antibodies" are multivalent or multispecific fragments constructed by gene fusion (WO 94/13804; P.Holliger et al, Proc. Natl. Acad. Sci. USA 906444-6448, 1993), which are also specific forms of antibodies encompassed herein. Also included herein are minibodies comprising scFv conjugated to the CH3 domain (S.Hu et al, Cancer research (Cancer Res.), 56, 3055-3061, 1996). See, e.g., Ward, E.S. et al, Nature 341, 544-546 (1989); bird et al, Science, 242, 423, 426, 1988; huston et al, Proc. Natl. Acad. Sci. USA (PNAS USA)85, 5879-5883, 1988); PCT/US 92/09965; WO 94/13804; holliger et al, Proc. Natl. Acad. Sci. USA 906444-; reiter et al, Nature Biotech, 14, 1239-1245, 1996; hu et al, cancer research, 56, 3055, 3061, 1996.

The proteolytic enzyme papain preferentially cleaves IgG molecules to produce fragments, two of which (f (ab) fragments) each comprise a covalent heterodimer that includes an intact antigen binding site. The enzyme pepsin is capable of cleaving IgG molecules to provide several fragments, including F (ab')2 fragments, which comprise two antigen binding sites. Fv fragments according to certain embodiments of the present disclosure can be produced by IgM, and in rare cases by preferential proteolytic cleavage of IgG or IgA immunoglobulin molecules. However, Fv fragments are more commonly derived using recombinant techniques known in the art. Fv fragments include non-covalent VH:: VL heterodimers, which comprise an antigen binding site that retains most of the antigen recognition and binding ability of the native antibody molecule. Inbar et al (1972) Proc. Natl. Acad. Sci. USA 69: 2659-; hochman et al (1976) biochemistry (Biochem) 15: 2706-2710; and Ehrlich et al (1980) biochemistry 19: 4091-.

In certain embodiments, single chain Fv or scFV antibodies are contemplated. For example, Kappa antibodies (Ill et al, protein engineering (prot. eng.) 10: 949-57 (1997)); minibodies (Martin et al, J. Eur. Biotech (EMBO J) 13: 5305-9 (1994)); bifunctional antibodies (Holliger et al, Proc. Natl. Acad. Sci. USA 90: 6444-8 (1993)); or Janusins (Trautech et al, J. European society of molecular biology 10: 3655-59(1991) and Trautech et al, J. cancer Supply 7: 51-52(1992)) can be prepared using standard molecular biology techniques as taught in this application for selecting antibodies with the desired specificity. In other embodiments, bispecific or chimeric antibodies can be prepared that encompass the ligands of the present disclosure. For example, a chimeric antibody may comprise CDRs and framework regions from different antibodies, while a bispecific antibody may be generated that specifically binds to one or more FZD receptors via one binding domain and specifically binds to a second molecule via a second binding domain. These antibodies can be produced by recombinant molecular biology techniques or can be physically bound together.

Single chain fv (scfv) polypeptides are covalently linked VH expressed from a gene fusion: a VL heterodimer, the gene fusion comprising a VH-and VL-encoding genes linked by a peptide-encoding linker. Huston et al (1988) Proc. Natl. Acad. Sci. USA 85(16): 5879-. A number of methods have been described to identify the chemical structures used to convert the naturally aggregated but chemically separated light and heavy chain polypeptide chains of antibody V regions into scFv molecules that will fold into three-dimensional structures that are substantially similar to the structures of the antigen binding sites. See, e.g., U.S. Pat. nos. 5,091,513 and 5,132,405 to Huston et al; and U.S. patent No. 4,946,778 to Ladner et al.

In certain embodiments, an antibody as described herein is in the form of a bifunctional antibody. Bifunctional antibodies are multimers of polypeptides, each polypeptide comprising a first domain comprising a binding region of an immunoglobulin light chain and a second domain comprising a binding region of an immunoglobulin heavy chain, which are linked (e.g., by a peptide linker) but cannot associate with each other to form an antigen binding site: the antigen binding site is formed by association of a first domain of one polypeptide within the multimer with a second domain of another polypeptide within the multimer (WO 94/13804).

dAb fragments of antibodies consist of VH domains (Ward, E.S. et al, Nature 341, 544-546 (1989)).

Where bispecific antibodies are used, these may be conventional bispecific antibodies, which may be manufactured in various ways (Holliger, P. and Winter G., (Current Opinion Biotechnol.) -4, 446-449(1993)), e.g. chemically or by hybridomas, or may be any of the bispecific antibody fragments mentioned above. Bifunctional antibodies and scfvs can be constructed using only variable domains without an Fc region, potentially reducing the impact of anti-idiotypic responses.

Bispecific diabodies may also be particularly useful relative to bispecific holoantibodies, as they can be readily constructed and expressed in E.coli. Bifunctional antibodies (and many other polypeptides, such as antibody fragments) with appropriate binding specificity can be readily selected from libraries using phage display (WO 94/13804). If one arm of the bifunctional antibody is to be kept constant, e.g.with specificity for antigen X, a library can be prepared in which the other arm is altered and an antibody with the appropriate specificity is selected. Bispecific whole antibodies can be prepared by knob-into-hole engineering (J.B. Ridgeway et al, Protein engineering (Protein Eng.), 9, 616-.

In certain embodiments, the antibodies described herein canIs provided in the form of (1).Is an IgG4 antibody with The hinge region removed (see Utrecht, The Netherlands GenMab; see also e.g.US 20090226421). This proprietary antibody technology produces stable smaller antibody formats with a longer therapeutic window than current small antibody formats are expected. The IgG4 antibody is considered inert and therefore does not interact with the immune system. The fully human IgG4 antibody can be modified by eliminating the hinge region of the antibody to obtain a half-molecule fragment with unique stability properties relative to the corresponding intact IgG4 (genemab). Halving the IgG4 moleculeLeaving only one region that can bind to a cognate antigen (e.g., a disease target), and thusBinding only monovalently to one site on the target cell.

In certain embodiments, the antibodies and antigen-binding fragments thereof as described herein comprise sets of heavy and light chain CDRs inserted between sets of heavy and light chain Framework Regions (FRs), respectively, that provide support for the CDRs and define the spatial relationship of the CDRs relative to each other. As used herein, the term "set of CDRs" refers to the three hypervariable regions of the heavy or light chain V regions. These regions start from the N-terminus of the heavy or light chain

Denoted as "CDR 1", "CDR 2" and "CDR 3", respectively. Thus, the antigen binding site includes six CDRs, including a set of CDRs from each of the heavy and light chain V regions. Polypeptides comprising a single CDR (e.g., CDR1, CDR2, or CDR3) are referred to herein as "molecular recognition units". Crystallographic analysis of many antigen-antibody complexes has demonstrated that the amino acid residues of the CDRs form extensive contacts with the bound antigen, with the most extensive antigen contact being with the heavy chain CDR 3. Thus, the molecular recognition unit is primarily responsible for the specificity of the antigen binding site.

As used herein, the term "FR set" refers to the four flanking amino acid sequences of the CDRs of the set of CDRs that make up the heavy or light chain V regions. Some FR residues may contact the bound antigen; however, the FR is primarily responsible for folding the V region into the antigen binding site, particularly FR residues immediately adjacent to the CDRs. Within the FR, certain amino residues and certain structural features are extremely highly conserved. In this regard, all V region sequences contain an internal disulfide loop of about 90 amino acid residues. When the V-region is folded into the binding site, the CDRs appear as prominent loop motifs, forming an antigen binding surface. It is generally accepted that there are conserved structural regions of the FR that affect the circular folding of the CDR into certain "canonical" structures, regardless of the precise CDR amino acid sequence. In addition, certain FR residues are known to be involved in non-covalent interdomain contacts that stabilize antibody heavy and light chain interactions.

"monoclonal antibody" refers to a homogeneous population of antibodies, wherein the monoclonal antibodies are composed of amino acids (both naturally occurring and non-naturally occurring) that are involved in selective binding of an epitope. Monoclonal antibodies are highly specific, being directed against a single epitope. The term "monoclonal antibody" encompasses not only intact monoclonal antibodies and full-length monoclonal antibodies, but also fragments thereof (e.g., Fab ', F (ab')2, Fv), single chains (scFv),their variants, fusion proteins comprising antigen binding fragments of monoclonal antibodies, humanized monoclonal antibodies, chimeric monoclonal antibodies and any other modified configuration of an immunoglobulin molecule comprising an antigen binding fragment (epitope recognition site) with the desired specificity and ability to bind to an epitope, including the WNT replacement molecules disclosed herein. . There is no intention to limit the source of the antibody or the manner in which the antibody is prepared (e.g., by hybridoma, phage selection, recombinant expression, transgenic animal, etc.). The term includes whole immunoglobulins and fragments and the like as described above under the definition of "antibody".

The proteolytic enzyme papain preferentially cleaves IgG molecules to produce fragments, two of which (f (ab) fragments) each comprise a covalent heterodimer that includes an intact antigen binding site. The enzyme pepsin is capable of cleaving IgG molecules to provide several fragments, including F (ab')2 fragments, which comprise two antigen binding sites. Fv fragments according to certain embodiments of the present disclosure can be produced by IgM, and in rare cases by preferential proteolytic cleavage of IgG or IgA immunoglobulin molecules. However, Fv fragments are more commonly derived using recombinant techniques known in the art. Fv fragments include non-covalent VH:: VL heterodimers, which comprise an antigen binding site that retains most of the antigen recognition and binding ability of the native antibody molecule. Inbar et al (1972) Proc. Natl. Acad. Sci. USA 69: 2659-; hochman et al (1976) biochemistry (Biochem) 15: 2706-2710; and Ehrlich et al (1980) biochemistry 19: 4091-.

In certain embodiments, single chain Fv or scFV antibodies are contemplated. For example, Kappa antibodies (Ill et al, protein engineering (prot. eng.) 10: 949-57 (1997)); minibodies (Martin et al, J. Eur. Biotech (EMBO J) 13: 5305-9 (1994)); bifunctional antibodies (Holliger et al, Proc. Natl. Acad. Sci. USA 90: 6444-8 (1993)); or Janusins (Trautech et al, J. European society of molecular biology 10: 3655-59(1991) and Trautech et al, J. cancer Supply 7: 51-52(1992)) can be prepared using standard molecular biology techniques as taught in this application for selecting antibodies with the desired specificity. In other embodiments, bispecific or chimeric antibodies can be prepared that encompass the ligands of the present disclosure. For example, a chimeric antibody may comprise CDRs and framework regions from different antibodies, and may be generated by one

A bispecific antibody in which the binding domain specifically binds to one or more FZD receptors and specifically binds to a second molecule through a second binding domain. These antibodies can be produced by recombinant molecular biology techniques or can be physically bound together.

Single chain fv (scfv) polypeptides are covalently linked VH expressed from a gene fusion: VL heterodimers, the gene fusions comprising

VH-and VL-encoding genes linked by a peptide-encoding linker. Huston et al (1988) Proc. Natl. Acad. Sci. USA 85(16): 5879-. A number of methods have been described to identify the chemical structures used to convert the naturally aggregated but chemically separated light and heavy chain polypeptide chains of antibody V regions into scFv molecules that will fold into three-dimensional structures that are substantially similar to the structures of the antigen binding sites. See, e.g., U.S. Pat. nos. 5,091,513 and 5,132,405 to Huston et al; and U.S. patent No. 4,946,778 to Ladner et al.

In certain embodiments, an antibody as described herein is in the form of a bifunctional antibody. Bifunctional antibodies are multimers of polypeptides, each polypeptide comprising a first domain comprising a binding region of an immunoglobulin light chain and a second domain comprising a binding region of an immunoglobulin heavy chain, which are linked (e.g., by a peptide linker) but cannot associate with each other to form an antigen binding site: the antigen binding site is formed by association of a first domain of one polypeptide within the multimer with a second domain of another polypeptide within the multimer (WO 94/13804).

dAb fragments of antibodies consist of VH domains (Ward, E.S. et al, Nature 341, 544-546 (1989)). When bispecific antibodies are used, these can be conventional bispecific antibodies, which can be varied

Made in a manner (Holliger, P. and Winter G., (Current Opinion Biotechnol.)) 4, 446-449(1993)), e.g.chemically or from hybridomas, or can be any of the bispecific antibody fragments mentioned above. Bifunctional antibodies and scfvs can be constructed using only variable domains without an Fc region, potentially reducing the impact of anti-idiotypic responses.

Bispecific diabodies may also be particularly useful relative to bispecific holoantibodies, as they can be readily constructed and expressed in E.coli. Bifunctional antibodies (and many other polypeptides, such as antibody fragments) with appropriate binding specificity can be readily selected from libraries using phage display (WO 94/13804). If one arm of the bifunctional antibody is to be kept constant, e.g.with specificity for antigen X, a library can be prepared in which the other arm is altered and an antibody with the appropriate specificity is selected. Bispecific whole antibodies can be prepared by knob-into-hole engineering (J.B. Ridgeway et al, Protein engineering (Protein Eng.), 9, 616-.

In certain embodiments, the antibodies described herein canIs provided in the form of (1).Is an IgG4 antibody with The hinge region removed (see Utrecht, The Netherlands GenMab; see also e.g.US 20090226421). This proprietary antibody technology produces stable smaller antibody formats with a longer therapeutic window than current small antibody formats are expected. The IgG4 antibody is considered inert and therefore does not interact with the immune system. The fully human IgG4 antibody can be modified by eliminating the hinge region of the antibody to obtain a half-molecule fragment with unique stability properties relative to the corresponding intact IgG4 (genemab). Halving the IgG4 moleculeLeaving only one region that can bind to a cognate antigen (e.g., a disease target), and thusBinding only monovalently to one site on the target cell.

In certain embodiments, antibodies of the disclosure can employ a single domain (sdAb) or VHH antibody fragment (also referred to as a VHH antibody fragment)) In the form of (1). sdAb or VHH technology was originally developed after the discovery and identification of camelids (e.g. camels and alpacas) with fully functional antibodies consisting of only heavy chains and thus lacking light chains. These heavy chain-only antibodies contain a single variable domain (VHH) and two constant domains (CH2, CH 3). The cloned and isolated individual variable domains have full antigen binding capacity and are very stable. These single variable domain constructs with their unique structural and functional propertiesThe basis of (1). sdabs or VHHs are encoded by a single gene and are efficiently produced in almost all prokaryotic and eukaryotic hosts, such as escherichia coli (see, e.g., U.S. patent No. 6,765,087), molds (e.g., aspergillus or trichoderma), and yeasts (e.g., yeast (Saccharomyces), kluyveromyces (kluyveromyces), Hansenula (Hansenula), or Pichia (Pichia)) (see, e.g., U.S. patent No. 6,838,254). The production process is scalable and already produces several kilogramsThe sdAb or VHH can be formulated as a ready-to-use solution with a longer shelf life.The method (see, e.g., WO06/079372) is based on automated high-throughput selection of B cellsTo a desired targetThe special method of (1). sdAb or VHH is a single domain antigen-binding fragment of only camelid-specific heavy chain antibodies.

Another antibody fragment envisioned is the double variant domain immunoglobulin (DVD-Ig), an engineered protein that combines the functions and specificities of two monoclonal antibodies in one molecular entity. DVD-Ig is designed as an IgG-like molecule, except that each light and heavy chain contains two variable domains connected in series by a short peptide bond, rather than one variable domain in IgG. The fusion orientation of the two variable domains and the choice of linker sequence are crucial for the functional activity and efficient expression of the molecule. DVD-Ig can be produced as a single material for manufacture and purification by conventional mammalian expression systems. DVD-Ig has the specificity of the parent antibody, is stable in vivo, and exhibits IgG-like physicochemical and pharmacokinetic properties. DVD-Ig and methods for its preparation are described in Wu, C.et al, Nature Biotechnology (Nature Biotechnology), 25:1290-1297 (2007).

In certain embodiments, an antibody or antigen-binding fragment thereof as disclosed herein is humanized. This refers to a chimeric molecule, typically prepared using recombinant techniques, having an antigen binding site derived from an immunoglobulin of a non-human species and the remaining immunoglobulin structure of the molecule based on the structure and/or sequence of a human immunoglobulin. The antigen binding site may comprise the entire variable domain fused to a constant domain, or only the CDRs grafted onto the appropriate framework regions in the variable domain. The epitope binding site may be wild-type or modified by one or more amino acid substitutions. This eliminates the constant region as an immunogen in human individuals, but the possibility of an immune response to foreign variable regions still exists (LoBuglio, A.F. 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). For

Illustrative methods for humanizing the anti-FZD or LRP antibodies disclosed herein include the methods described in U.S. patent No. 7,462,697.

Another approach not only focuses on providing human-derived constant regions, but also modifies the variable regions in order to remodel them as much as possible into human form. It is known that the variable regions of both heavy and light chains contain three Complementarity Determining Regions (CDRs) that vary in response to the epitope in question and determine binding capacity, flanked by four Framework Regions (FRs) that are relatively conserved in a given species and are presumed to provide a scaffold for the CDRs. When a non-human antibody is prepared with respect to a specific epitope, the variable region can be "reshaped" or "humanized" by grafting CDRs derived from the non-human antibody onto FRs present in the human antibody to be modified. Application of this method to various antibodies has been reported: sato, k. et al, (1993) cancer research 53: 851-; riechmann, l. et al, (1988) nature 332: 323-327; verhoeyen, m. et al, (1988) science 239: 1534 — 1536; ketleborough, c.a. et al, (1991) protein engineering 4: 773-3783; maeda, H.et al, (1991) Human antibody hybridomas (Human Antibodies Hybridoma) 2: 124-134; gorman, s.d. et al, (1991) journal of the american academy of sciences "88: 4181-4185; tempest, P.R. et al, (1991) Biotechnology (Bio/Technology) 9: 266-271; co, m.s. et al, (1991) journal of the american academy of sciences "88: 2869 vs 2873; carter, p. et al, (1992) journal of the american academy of sciences 89: 4285-; and Co, m.s. et al, (1992) journal of immunology (J Immunol) 148: 1149-1154. In some embodiments, the humanized antibody retains all CDR sequences (e.g., a humanized mouse antibody that contains all six CDRs from a mouse antibody). In other embodiments, the humanized antibody has one or more CDRs (one, two, three, four, five, six) that are altered relative to the original antibody, also referred to as one or more CDRs that are "derived" from the one or more CDRs of the original antibody.

In certain embodiments, the antibodies of the present disclosure may be chimeric antibodies. In this regard, chimeric antibodies are composed of antigen-binding fragments of an antibody operably linked or otherwise fused to a heterologous Fc portion of a different antibody. In certain embodiments, the heterologous Fc domain is of human origin. In other embodiments, the heterologous Fc domain may be from 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 be comprised of CH2 and CH3 domains from one or more different Ig classes. As described above with respect to humanized antibodies, an antigen-binding fragment of a chimeric antibody may comprise only one or more CDRs of an antibody described herein (e.g., 1,2, 3, 4, 5, or 6 CDRs of an antibody described herein), or may comprise the entire variable domain (VL, VH, or both).

The structure and position of immunoglobulin CDRs and variable domains can be determined by reference to: kabat, E.A. et al, immunologically of protein Sequences (Sequences of Proteins of Immunological Interest), 4 th edition. The united states Department of Health and public Services (US Department of Health and Human Services), 1987, and newer versions thereof, are now available on the internet (immunology.

In some embodiments, the WNT-replacement molecule comprises one or more fabs or antigen-binding fragments thereof and one or more VHHs or sdabs or antigen-binding fragments thereof (or alternatively, one or more scfvs or antigen-binding fragments thereof). In certain embodiments, the Fab specifically binds to one or more Fzd receptors, and the VHH or sdAb (or scFv) specifically binds to LRP5 and/or LRP 6. In certain embodiments, the Fab specifically binds to LRP5 and/or LRP6, and the VHH or sdAb (or scFv) specifically binds to one or more Fzd receptors. In certain embodiments, the VHH or sdAb (or scFv) is fused to the N-terminus of the Fab, while in some embodiments, the VHH or sdAb (or scFv) is fused to the C-terminus of the Fab. In particular embodiments, the Fab is present as an intact IgG and the VHH or sdAb (or scFv) is fused to the N-terminus and/or C-terminus of the IgG light chain. In particular embodiments, the Fab is present as an intact IgG and the VHH or sdAb (or scFv) is fused to the N-terminus and/or C-terminus of the IgG heavy chain. In particular embodiments, two or more VHHs or sdabs (or scfvs) are fused to IgG in any combination of these positions.

Fab can be converted to an intact IgG format, including both Fab and Fc fragments, e.g., using genetic engineering, to produce a fusion polypeptide comprising Fab fused to an Fc region, i.e., Fab is present in an intact IgG format. The Fc region for the full IgG format can be derived from any of a variety of different Fc's, including but not limited to wild-type or modified IgG1, IgG2, IgG3, IgG4, or other isotypes, such as wild-type or modified human IgG1, human IgG2, human IgG3, human IgG4, human IgG4Pro (containing a core hinge region mutation that prevents IgG4 half-molecule formation), human IgA, human IgE, human IgM, or modified IgG1 known as IgG1 lapg. The L235A, P329G (LALA-PG) variants have been shown to abolish complement binding and fixation in murine IgG2a and human IgG1 and Fc-gamma dependent antibody dependent cell mediated cytotoxicity (ADCC). These LALA-PG substitutions allowed for more accurate translation of the results of "should not" antibody framework scaffold generation between mouse and primate. In particular embodiments of any of the iggs disclosed herein, the IgG comprises one or more of the following amino acid substitutions: N297G, N297A, N297E, L234A, L235A or P236G.

The present invention provides non-limiting examples of bivalent and bispecific WNT substitute molecules that are bivalent against one or more Fzd receptors and LRP5 and/or LRP 6. The VHH or sdAb (or scFv) can be fused to the N-termini of the two light chains, to the N-termini of the two heavy chains, to the C-termini of the two light chains, or to the C-termini of the two heavy chains. It is further contemplated that, for example, the VHH or sdAb (or scFv) can be fused to both the N-terminus and C-terminus of the heavy and/or light chain, to the light chain and N-terminus of the heavy chain, to the heavy chain and C-terminus of the light chain, to the N-terminus of the heavy chain and C-terminus of the light chain, or to the heavy chain and N-terminus of the light chain. In other related embodiments, two or more VHHs or sdabs (or scfvs) can be fused together, optionally via a linker moiety, and fused to a Fab or IgG at one or more of these positions. In a related embodiment, the WNT replacement molecule has a heterologous IgG format, while the Fab is present as a half-antibody, and one or more VHHs or sdabs (or scfvs) are fused to one or more of the N-terminus of the Fc, the N-terminus of the Fab, the C-terminus of the Fc, or the C-terminus of the Fab. In certain embodiments, the Fab or antigen binding fragment thereof (or IgG) is fused directly to the VHH or sdAb (or scFv) or antigen binding fragment thereof, while in other embodiments the binding region is fused via a linker moiety.

In various embodiments, the WNT-replacement molecule comprises one or more fabs or antigen-binding fragments thereof that bind to one or more FZD receptors and one or more fabs or antigen-binding fragments thereof that bind LRP5 and/or LRP 6. In certain embodiments, it comprises two fabs or antigen binding fragments thereof that bind to one or more FZD receptors and/or two fabs or antigen binding fragments thereof that bind LRP5 and/or LRP 6. In particular embodiments, one or more fabs are present in the intact IgG form, and in certain embodiments, two fabs are present in the intact IgG form. In certain embodiments, a Fab of the intact IgG form specifically binds to one or more FZD receptors, and another Fab specifically binds to LRP5 and/or LRP 6. In certain embodiments, the Fab specifically binds to one or more FZD receptors, and the Fab in an intact IgG form specifically binds to LRP5 and/or LRP 6. In certain embodiments, a Fab specifically binds to LRP5 and/or LRP6, and a Fab in the form of an intact IgG specifically binds to one or more FZD receptors. In certain embodiments, the Fab is fused to the N-terminus of the IgG, e.g., the heavy or light chain N-terminus, optionally via a linker. In certain embodiments, the Fab is fused to the N-terminus of the heavy chain of the IgG, and not to the light chain. In particular embodiments, the two heavy chains may be fused together directly or through a linker. Examples of such bispecific and bivalent versus two receptors are shown at the top of figure 1B. In other related embodiments, two or more VHHs or sdabs can be fused together, optionally via a linker moiety, and to a Fab or IgG at one or more of these positions. In related embodiments, the WNT replacement molecule has a heterologous IgG format, while one of the fabs is present as a half-antibody, and the other Fab is fused to one or more of the N-terminus of the Fc, the N-terminus of the Fab, or the C-terminus of the Fc. In certain embodiments, a Fab or antigen binding fragment thereof is fused directly to another Fab or IgG or antigen binding fragment thereof, while in other embodiments, the binding region is fused via a linker moiety.

In certain embodiments, a WNT agonist of the invention may have, comprise, or consist of any one of the sequences provided in table 2, table 4, table 5, table 6, or table 7, or a functional fragment or variant thereof.

In certain embodiments, the antagonist or agonist binding agent has a dissociation constant (K) of about 1 μ M or less, about 100nM or less, about 40nM or less, about 20nM or less, or about 10nM or less_D) And (4) combining. For example, in certain embodiments, a FZD-binding agent or antibody described herein that binds to more than one FZD is at a K of about l00nM or less, about 20nM or less, or about 10nM or less_DIn combination with those FZD. In certain embodiments, a binding agent binds to one or more of its target antigens with an EC50 of about 1 μ M or less, about 100nM or less, about 40nM or less, about 20nM or less, about 10nM or less, or about 1nM 20 or less.

The antibodies or other agents of the invention can 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 blots, radioimmunoassays, ELISA, "sandwich" immunoassays, immunoprecipitation assays, precipitation reactions, gel diffusion precipitation reactions, immunodiffusion assays, agglutination assays, complement fixation assays, immunoradiometric assays, fluorescent immunoassays, and protein a immunoassays. Such assays are conventional and well known in the art (see, e.g., Ausubel et al, eds., 1994, "Molecular Biology laboratory guidelines (Current Protocols in Molecular Biology)," Vol.1, N.Y. Wiley & Sons, Inc., New York, which is incorporated herein by reference in its entirety).

For example, specific binding of an antibody to a target antigen can be determined using ELISA. ELISA assays involve preparing an antigen, coating the wells of a 96-well microtiter plate with the antigen, adding an antibody or other binding agent that binds to a detectable compound such as an enzyme substrate (e.g., horseradish peroxidase or alkaline phosphatase) to the wells, incubating for a period of time, and detecting the presence of the antigen. In some embodiments, the antibody or agent does not bind to the detectable compound, but a second binding antibody that recognizes the first antibody or agent is added to the well. In some embodiments, instead of coating the wells with an antigen, an antibody or agent may be coated to the wells, and a second antibody that binds to a detectable compound may be added after the antigen is added to the coated wells. Those skilled in the art will appreciate which parameters can be modified to increase the detected signal and other variations of ELISAs known in the art (see, e.g., Ausubel et al, editors, 1994, molecular biology laboratory Manual, volume 1, new yohn williams father, 11.2.1).

The binding affinity of an antibody or another agent to a target antigen and the off-rate of antibody-antigen interaction can be determined by competitive binding assays. One example of a competitive binding assay is a radioimmunoassay comprising incubating a labeled antigen (e.g., FZD, LRP) or fragment or variant thereof with an antibody of interest in the presence of an increasing amount of unlabeled antigen, followed by detecting the antibody that binds to the labeled antigen. The affinity and the binding off-rate of the antibody can be determined from the data by scatchard (scatchard) plot analysis. In some embodiments, BIAcore kinetic analysis is used to determine the binding and dissociation rates of an antibody or agent. BIAcore kinetic analysis involves analysis of the binding and dissociation of antibodies to a chip on which the antigen is immobilized on its surface.

The WNT-replacement molecules of the invention bind to one or more FZD receptors and one or more of LRP5 and LRP6 and are biologically active in the activation of WNT signaling, i.e., the WNT-replacement molecules are WNT agonists. The term "WNT agonist activity" refers to the ability of an agonist to mimic the effect or activity of WNT protein binding to frizzled receptor protein and/or LRP5 or LRP 6. The ability of WNT-surrogate molecules and other WNT agonists disclosed herein to mimic WNT activity can be confirmed by a number of assays. WNT agonists typically elicit responses or activities similar to or identical to those elicited by the natural ligands of the receptors. In particular, the WNT agonists disclosed herein activate, enhance or increase the canonical WNT/β -catenin signaling pathway. As used herein, the term "potentiation" refers to a measurable increase in the level of WNT/β -catenin signaling compared to the level in the absence of a WNT agonist, e.g., in the absence of a WNT replacement molecule disclosed herein. In particular embodiments, the increase in WNT/β -catenin signaling level is at least 10%, at least 20%, at least 50%, at least two-fold, at least five-fold, at least 10-fold, at least 20-fold, at least 50-fold, or at least 100-fold, e.g., in the same cell type as compared to WNT/β -catenin signaling level in the absence of a WNT agonist. Methods of measuring WNT/β -catenin signaling are known in the art and include those described herein.

In particular embodiments, the WNT-replacement molecules disclosed herein are bispecific, i.e., they specifically bind two or more different epitopes, e.g., one or more FZD receptors, and LRP5 and/or LRP 6. In certain embodiments, the WNT-replacement molecule binds to FZD5 and/or FZD8, and LRP5 and/or LRP 6.

In particular embodiments, WNT-replacement molecules disclosed herein are multivalent, e.g., they comprise two or more regions that each specifically bind to the same epitope, e.g., two or more regions that bind to an epitope within one or more FZD receptors and/or two or more regions that bind to an epitope within LRP5 and/or LRP 6. In particular embodiments, they comprise two or more regions that bind to an epitope within one or more FZD receptors, and two or more regions that bind to an epitope within LRP5 and/or LRP 6. In certain embodiments, the WNT-substitute molecule comprises a ratio of the number of regions that bind to one or more FZD receptors to the number of regions that bind LRP5 and/or LRP6 of at or about: 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 2:3, 2:5, 2:7, 7:2, 5:2, 3:4, 3:5, 3:7, 3:8, 8:3, 7:3, 5:3, 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 WNT-replacement molecule is bispecific and multivalent.

In certain aspects, the present disclosure provides novel tissue-specific WNT signaling enhancing molecules capable of enhancing WNT activity in a tissue or cell-specific manner. In certain embodiments, the tissue-specific WNT signaling enhancing molecule is a bifunctional molecule comprising a first domain that binds to one or more ZNRF3 and/or RNF43 ligase, and a second domain that binds to one or more targeted tissue or cell types in a tissue or cell-specific manner. Each of the first domain and the second domain may be any moiety capable of binding to a ligase complex or targeting a tissue or cell, respectively. For example, each of the first domain and the second domain may be, but is not limited to, a moiety selected from the group consisting of: a polypeptide (e.g., an antibody or antigen-binding fragment thereof or a peptide or polypeptide different from an antibody), a small molecule, and a natural ligand or a variant, fragment, or derivative thereof. In certain embodiments, the natural ligand is a polypeptide, small molecule, ion, amino acid, lipid, or sugar molecule. The first domain and the second domain may be the same type of moiety as each other, or they may be different types of moieties. In certain embodiments, the tissue-specific WNT signaling enhancing molecule binds to a tissue-or cell-specific cell surface receptor. In particular embodiments, the tissue-specific WNT signaling-enhancing molecule increases or enhances WNT signaling by at least 50%, at least two-fold, at least three-fold, at least five-fold, at least ten-fold, at least twenty-fold, at least thirty-fold, at least forty-fold, or at least fifty-fold as compared to a negative control.

The tissue-specific WNT signaling enhancer molecules may have different forms. In particular embodiments, the tissue-specific WNT signaling enhancing molecule is a fusion protein comprising a first polypeptide sequence that binds to ZNRF3/RNF43 and a second polypeptide sequence that binds in a tissue or cell-specific manner to one or more targeted tissue or cell types. In certain embodiments, the two polypeptide sequences may be fused directly or through a linker. In certain embodiments, the tissue-specific WNT signal-enhancing molecule comprises two or more polypeptides, e.g., a dimer or multimer comprising two or more fusion proteins, each comprising a first domain and a second domain, wherein the two or more polypeptides are linked, e.g., by a linker moiety or by a bond between amino acid residues in each of the two or more polypeptides, e.g., an intermolecular disulfide bond between cysteine residues.

In particular embodiments, the tissue-specific WNT signaling enhancing molecule is an antibody comprising an antibody heavy chain and light chain (or antigen-binding fragment thereof) that make up the first domain or the second domain, wherein the other domain (i.e., the second domain or the first domain) is linked to the antibody heavy chain or light chain as a fusion protein or via a linker moiety. In particular embodiments, the other domain is linked to the N-terminus of the heavy chain, the C-terminus of the heavy chain, the N-terminus of the light chain, or the C-terminus of the light chain. Such structures may be referred to herein as attached IgG scaffolds or formats. For example, the tissue-specific WNT signaling enhancing molecule may be an antibody that binds to ZNRF3/RNF43, wherein the binding domain that binds to a tissue-specific receptor or a cell-specific receptor is fused or attached to the heavy or light chain of the antibody that binds to ZNRF3/RNF 43. In another example, the tissue-specific WNT signaling enhancing molecule may be an antibody that binds to a tissue-or cell-specific receptor, wherein the binding domain that binds ZNRF3/RNF43 is fused or attached to the heavy or light chain of the antibody that binds to the tissue-or cell-specific receptor.

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

In certain embodiments, the tissue-specific WNT signaling enhancing molecule comprises a first domain that binds ZNRF3/RNF43 ("action module") and a second domain that binds a tissue or cell specific receptor, e.g., with high affinity ("targeting module"). In certain embodiments, each of these two domains has substantially reduced activity, or does not function in itself in enhancing WNT signaling. However, when the tissue-specific WNT signaling enhancing molecule is engaged with target tissues expressing tissue-specific receptors, the E3 ligase ZNRF3/RNF43 is recruited to the ternary complex with the tissue-specific receptors, sequestered, and/or cleared from the cell surface via receptor-mediated endocytosis. The end result is an enhancement of WNT signaling in a tissue-specific manner.

In certain embodiments, the action module is a binder for ZNRF3/RNF 43E 3 ligase and can be designed based on R-spondins, e.g., R-spondins-1-4, including but not limited to human R-spondins-1-4. In certain embodiments, the action module is an R-spondin, e.g., wild-type R-spondin-1-4, optionally human R-spondin-1-4, or variant or fragment thereof. In particular embodiments, it is a variant of any of R-spondins-1-4 having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the corresponding wild-type R-spondins-1-4 sequence. In certain embodiments, the action module comprises or consists of furin domain 1 of R-spondin, e.g., any one of R-spondin 1-4 that binds ZNRF3/RNF 43. Expanded forms of furin domain 1 (including but not limited to those having a mutated furin domain 2 that no longer binds to LGR4-6 or has reduced binding to LGR4-6) or engineered antibodies other than antibodies capable of specifically binding to ZNRF3/RNF43 or any other derivative or any engineered polypeptide may also be used. In certain embodiments, the action module comprises one or more furin domain 1 of R-spondin.

In certain embodiments, the action module does not comprise furin domain 2 of R-spondin, or it comprises a modified or variant furin domain 2 of R-spondin, e.g., furin domain 2 with reduced activity, as compared to wild-type furin domain 2. In certain embodiments, the action module comprises furin domain 1, but not furin domain 2, of the R-spondin. In certain embodiments, the action module comprises two or more furin domain 1 or multimers of furin domain 1. The action domain may comprise one or more wild-type furin domain 1 of R-spondin. In particular embodiments, the action module comprises a modified or variant furin domain 1 having increased activity, e.g., R-spondin binding to ZNRF3/RNF43, as compared to wild-type furin domain 1. Variants with increased binding to ZNRF3/RNF43 can be identified, for example, by screening phage or yeast display libraries comprising variants of R-spondin furin domain 1. Peptides or polypeptides that are not related to domain 1 of R-spondin furin but have increased binding to ZNRF3/RNF43 can also be identified by screening. The action module may further comprise additional portions or polypeptide sequences, such as additional amino acid residues, to stabilize the structure of the action module or the tissue-specific WNT signal-enhancing molecule in which the module is present.

In other embodiments, the module of action comprises another inhibitory moiety, e.g., a nucleic acid molecule, e.g., an antisense oligonucleotide, that reduces or prevents ZNRF3/RNF43 activity or expression; small interfering rna (sirna); short hairpin rna (shrna); micro rna (mirna); or a ribonuclease. As used herein, "antisense" refers to a nucleic acid sequence that is 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 a single cell, tissue, or individual and results in reduced expression of a target gene by a mechanism that does not necessarily rely on the endogenous gene silencing pathway. Antisense nucleic acids can contain modified backbones, such as phosphorothioate, phosphorodithioate, or other backbones known in the art, or can contain non-natural internucleoside linkages. The antisense nucleic acid can comprise, for example, Locked Nucleic Acid (LNA). In particular embodiments, the additional inhibitor 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/RNF 43. In certain embodiments, the inhibitory moiety is a nucleic acid molecule that binds to the ZNRF3/RNF43 gene or mRNA or complement thereof.

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

Tissue-specific and cell-specific cell surface receptors are known in the art. Examples of tissue and cell specific surface receptors include, but are not limited to, GPA33, CDH17, and MUC-13. In certain embodiments, the targeting module comprises antibodies or antigen-binding fragments thereof that specifically bind to these gut-specific receptors.

In certain embodiments, the components of the WNT surrogate and WNT signal-enhancing molecule may be combined to confer more tissue specificity.

Pharmaceutical composition

Also disclosed are pharmaceutical compositions comprising a WNT agonist molecule described herein and one or more pharmaceutically acceptable diluents, carriers, or excipients.

In additional embodiments, pharmaceutical compositions comprising a polynucleotide comprising a nucleic acid sequence encoding a WNT agonist molecule described herein and one or more pharmaceutically acceptable diluents, carriers, or excipients are also disclosed. In certain embodiments, the polynucleotide is DNA or mRNA, e.g., a modified mRNA. In particular embodiments, the polynucleotide is a modified mRNA further comprising a5 '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 linked to a coding sequence.

In some embodiments, WNT agonists are engineered recombinant polypeptides incorporating various epitope-binding fragments that bind to various molecules in the WNT signaling pathway. For example, FZD and LRP antibody fragments (e.g., Fab, scFv, sdAb, VHH, etc.) can be joined together on one molecule, either directly or with linkers of various sizes. Similarly, polypeptides such as RSPO can be engineered to contain antibodies or fragments thereof against tissue-specific cell surface antigens, such as MUC-13. RSPO can also be administered simultaneously or sequentially with an enhancer of E3 ligase, ZNRF3/RNF 43. The E3 ligase enhancer may be an agonist antibody or fragment that binds ZNRF3/RNF43 and enhances the activity of E3 ligase.

Conversely, an engineered WNT agonist may also be a recombinant polypeptide incorporating epitope-binding fragments that bind to various molecules in the WNT signaling pathway and enhance WNT signaling. For example, the WNT antagonist may be an antibody or fragment thereof that binds to FZD receptors and/or LRP receptors and enhances WNT signaling. FZD and LRP antibody fragments (e.g., Fab, scFv, sdAb, VHH, etc.) can be joined together directly or with linkers of various sizes on one molecule.

In additional embodiments, pharmaceutical compositions comprising an expression vector, e.g., a viral vector, comprising a polynucleotide comprising a nucleic acid sequence encoding a WNT agonist molecule described herein and one or more pharmaceutically acceptable diluents, carriers, or excipients are also disclosed.

The present disclosure further encompasses a pharmaceutical composition comprising a cell comprising an expression vector comprising a polynucleotide comprising a promoter operably linked to a nucleic acid encoding a WNT agonist molecule and one or more pharmaceutically acceptable diluents, carriers, or excipients. In particular embodiments, the pharmaceutical composition further includes a cell comprising an expression vector comprising a polynucleotide comprising a promoter operably linked to a nucleic acid sequence encoding a WNT agonist. In particular embodiments, the cells are allogeneic or autologous cells obtained from the individual to be treated.

The subject molecules, alone or in combination, can be combined with pharmaceutically acceptable carriers, diluents, excipients, and agents useful in preparing formulations that are generally safe, non-toxic, and desirable and include excipients acceptable for use in mammals, such as humans or primates. Such excipients may be solid, liquid, semi-solid or gaseous (in the case of aerosol compositions). Examples of such carriers, diluents and excipients include, but are not limited to, water, saline, Ringer's solution, dextrose solution and 5% human serum albumin. Supplementary active compounds may also be incorporated into the formulation. Solutions or suspensions for the formulation may include: sterile diluents such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial compounds such as benzyl alcohol or methyl paraben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating compounds such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetate, citrate or phosphate; detergents such as Tween (Tween)20 for preventing aggregation; and compounds for regulating tonicity, such as sodium chloride or dextrose. The pH can be adjusted with an acid or base, such as hydrochloric acid or sodium hydroxide. In particular embodiments, the pharmaceutical composition is sterile.

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

Sterile solutions can be prepared by incorporating a WNT agonist antibody or antigen-binding fragment thereof (or encoding polynucleotide or cells comprising the encoding polynucleotide) in the required amount in an appropriate solvent with one or a combination of the ingredients enumerated above, as required, followed by filter sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

In one embodiment, the pharmaceutical composition is prepared with a carrier that will protect the antibody or antigen-binding fragment thereof from rapid elimination from the body, such as controlled release formulations, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid may be used. Methods for preparing such formulations will be apparent to those skilled in the art. The materials are also commercially available. Liposomal suspensions may also be used as pharmaceutically acceptable carriers. These suspensions may be prepared according to methods known to those skilled in the art.

It may be advantageous to formulate the pharmaceutical composition in unit dosage form for ease of administration and uniformity of dosage. Unit dosage form as used herein refers to physically discrete units suitable as unitary dosages for the individual to be treated; each unit containing a predetermined quantity of active antibody or antigen-binding fragment thereof calculated to produce the desired therapeutic effect, in association with the required pharmaceutical carrier. The specification of a unit dosage form is indicated by and directly depends on: the unique characteristics of an antibody or antigen-binding fragment thereof and the specific therapeutic effects to be achieved, as well as the limitations inherent in the art of compounding such active antibodies or antigen-binding fragments thereof to treat individuals.

The pharmaceutical composition may be contained in a container, package, or dispenser (e.g., a syringe, such as a pre-filled syringe) with instructions for administration.

The pharmaceutical compositions of the present disclosure encompass any pharmaceutically acceptable salt, ester, or salt of such ester, or any other compound capable of providing (directly or indirectly) a biologically active antibody or antigen-binding fragment thereof upon administration to an animal comprising a human.

The present disclosure includes pharmaceutically acceptable salts of WNT agonist molecules described herein. The term "pharmaceutically acceptable salt" refers to physiologically and pharmaceutically acceptable salts of the compounds of the present disclosure: that is, salts that retain the desired biological activity of the parent compound and do not produce undesirable toxicological effects to the parent compound. Various pharmaceutically acceptable salts are known in the art and are described, for example, in "Remington's Pharmaceutical Sciences" of Remington, edition 17, Alfonso r.gennaro (eds.), Mark Publishing Company of Easton, PA, USA (and more recent versions thereof), "Encyclopedia of Pharmaceutical Technology" (3 rd edition, James swartbrick (eds.), american healthcare Company of new york, USA) (Inc.), 2007, and journal of Pharmaceutical science (j.pharm.sci.) -66: 2(1977). For a review of suitable salts, see, for example, the handbook of pharmaceutical salts: properties, Selection and Use (Handbook of Pharmaceutical Salts: Properties, Selection, and Use) ", written by 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.

The metal used as the cation includes sodium, potassium, magnesium, calcium, and the like. Amines include N-N' -dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine (see, e.g., Berge et al, "pharmaceutically acceptable Salts", in the journal of pharmacy (j.pharma Sci), 1977,66, 119). The base addition salts of the acidic compounds are prepared by contacting the free acid form with a sufficient amount of the desired base to produce the salt in a conventional manner. The free acid form may be regenerated by contacting the salt form with an acid and isolating the free acid in a conventional manner. The free acid form differs slightly from its corresponding salt form in certain physical properties (such as solubility in polar solvents), but additionally, for the purposes of this disclosure, the salt is equivalent to its corresponding free acid.

In some embodiments, the pharmaceutical compositions provided herein comprise a therapeutically effective amount of a WNT agonist molecule or a pharmaceutically acceptable salt thereof in admixture with pharmaceutically acceptable carriers, diluents, and/or excipients, such as saline, phosphate buffered saline, phosphate and amino acids, polymers, polyols, sugars, buffers, preservatives, and other proteins. Exemplary amino acids, polymers, and sugars and the like are octylphenoxy polyethoxyethanol 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 Hank's solution, cysteine, arginine, carnitine, alanine, glycine, lysine, valine, leucine, polyvinylpyrrolidone, polyethylene, and glycols. Preferably, this formulation is stable at 4 ℃ for at least six months.

In some embodiments, the pharmaceutical compositions provided herein comprise a buffer, such as Phosphate Buffered Saline (PBS) or sodium phosphate/sulfate, tris buffer, glycine buffer, sterile water, and other buffers known to those of ordinary skill in the art, such as those disclosed by Good et al (1966) Biochemistry 5: 467 to those described. 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.

Method of use

The present disclosure also provides methods for using WNT agonist molecules and/or tissue-specific WNT signaling enhancing molecules, e.g., modulating WNT signaling pathways, e.g., increasing WNT signaling, and administering WNT agonist molecules and/or tissue-specific WNT signaling enhancing molecules in various therapeutic settings. Provided herein are therapeutic methods using WNT agonist molecules and/or tissue-specific WNT signaling enhancing molecules. Any of the methods disclosed herein can also be practiced using a combination of WNT agonist molecules and tissue-specific WNT signaling enhancing molecules or a combination molecule (combination molecule) comprising a WNT agonist molecule and tissue-specific WNT signaling enhancing, e.g., as described herein. In one embodiment, WNT agonist molecules and/or tissue-specific WNT signaling enhancing molecules or combinatorial molecules are provided to individuals with diseases involving inappropriate or deregulated WNT signaling. In certain embodiments, the methods disclosed herein comprise providing to an individual in need thereof a WNT agonist molecule and/or a tissue-specific WNT signaling enhancing molecule or combination molecule, alone or in combination. In certain embodiments, the WNT agonist molecule and the tissue-specific WNT signaling enhancing molecule are provided to the individual in the same or different pharmaceutical compositions. In some embodiments, the WNT agonist molecule and the tissue-specific WNT signaling enhancing molecule are provided to the individual at the same time or at different times, e.g., one before or after the other. In some embodiments, the methods comprise providing an effective amount of a WNT agonist molecule and/or a tissue-specific WNT signaling enhancing molecule to the individual. In some embodiments, an effective amount of a WNT agonist molecule and a tissue-specific WNT signaling enhancing molecule are present in an individual during an overlapping time period, e.g., one day, two days, or one week. In certain embodiments, the methods disclosed herein comprise providing a combination molecule (combinatorial molecule) comprising a WNT agonist molecule and/or a tissue-specific WNT signaling enhancing molecule to an individual in need thereof.

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

In certain embodiments, WNT agonist molecules or tissue-specific WNT signaling enhancing molecules or combinatorial molecules may be used to enhance WNT signaling pathways in tissues or cells. Agonizing the WNT signaling pathway may include, for example, increasing WNT signaling or enhancing WNT signaling in a tissue or cell. Thus, in some aspects, the present disclosure provides a method for agonizing a WNT signaling pathway in a cell comprising contacting the tissue or cell with an effective amount of a WNT agonist molecule and/or a tissue-specific WNT signaling enhancing molecule or combination molecule disclosed herein, or a pharmaceutically acceptable salt thereof, wherein the WNT agonist molecule and/or tissue-specific WNT signaling enhancing molecule or combination molecule is an agonist of the WNT signaling pathway. In some embodiments, the contacting occurs in vitro, ex vivo, or in vivo. In particular embodiments, the cell is a cultured cell, and the contacting occurs in vitro.

WNT agonists and/or tissue-specific WNT signaling enhancing molecules or combinatorial molecules may be used to treat gastrointestinal disorders, including but not limited to inflammatory bowel disease, including but not limited to crohn's disease, crohn's disease with fistula formation, and ulcerative colitis. In particular, the invention provides WNT/β -catenin signaling WNT/β -catenin agonists for use in enhancing the regeneration of intestinal epithelium due to injury from these conditions.

In another embodiment, the agonist molecule may also be incorporated into a tissue targeting moiety, such as an antibody or fragment thereof that recognizes a lung tissue specific receptor or cell surface molecule.

The invention also provides for the treatment of gastrointestinal disorders, particularly Inflammatory Bowel Disease (IBD), with known combinations of therapeutic agents. For example, a WNT agonist and/or a tissue-specific WNT signaling enhancing molecule or combination molecule may be combined with several known therapies for IBD, including but not limited to 5-aminosalicylate (5-ASA); immunosuppressants such as corticosteroids, azathioprine or 6-mercaptopurine, methotrexate and cyclosporin-a or tacrolimus; TNF inhibitors such as infliximab, adalimumab, and golimumab; anti-integrins, such as vedolizumab; inflammatory cytokine antagonists, such as eutigumab; janus kinase (JAK) inhibitors such as tofacitinib; SMAD 7 inhibitors, such as monersen; and S1P modulators, such as azanimod (ozanimod) and itramod (etrasimod). The above therapeutic agents may be administered sequentially or simultaneously with the molecules of the invention.

Therapeutic agents (e.g., WNT agonists and/or tissue-specific WNT signaling enhancing molecules or combinatorial molecules) may be administered before, during, or after onset of disease or injury. Treatment of developing diseases is of great interest, where treatment stabilizes or reduces undesirable clinical symptoms in the patient. It is desirable to perform such treatment before the affected tissue is completely lost of function. It is desirable to administer the subject therapy during and in some cases after the symptomatic phase of the disease. In some embodiments, the methods of the invention result in a therapeutic benefit, such as preventing the development of a disorder, interrupting the progression of a disorder, reversing the progression of a disorder, and the like. In some embodiments, the methods of the invention comprise the step of detecting the therapeutic benefit achieved. One of ordinary skill in the art will appreciate that measures of such treatment efficacy will apply to the particular disease being altered, and will recognize appropriate detection methods for measuring treatment efficacy.

All of the above U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification and/or listed in the application data sheet, are incorporated herein by reference, in their entirety.

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

The broad scope of the present invention is best understood with reference to the following examples, which are not intended to limit the invention to the specific embodiments described.

Examples of the invention

I. General procedure

Standard methods in molecular biology are described. Maniatis et al (1982) molecular cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; sambrook and Russell (2001) Molecular Cloning, 3 rd edition, Cold spring harbor laboratory Press, Cold spring harbor, N.Y.; wu (1993) recombinant DNA (recombinant DNA), vol.217, academic Press, san Diego, Calif. Standard methods also appear in Ausbel et al (2001) molecular biology laboratory Manual, volumes 1-4, Wiinterest father, N.Y., which describe cloning and DNA mutagenesis in bacterial cells (volume 1), cloning in mammalian cells and yeast (volume 2), glycoconjugates and protein expression (volume 3), and bioinformatics (volume 4).

Methods of protein purification are described, including immunoprecipitation, chromatography, electrophoresis, centrifugation, and crystallization. Coligan et al (2000) guidelines for Protein Science in Protein Science, Vol.1, N.Y., John Wiley father, Inc. Chemical analysis, chemical modification, post-translational modification, fusion protein production, glycosylation of proteins are described. See, e.g., Coligan et al (2000) guide to protein science, Vol.2, N.Y. John Willi, parent-son; ausubel et al (2001) molecular biology laboratory Manual, Vol.3, new york, John Willi father, N.Y., pp. 16.0.5-16.22.17; Sigma-Aldrich (Sigma-Aldrich, Co.) (2001) Products of Life Science Research (Products for Life Science Research), st. Pages 45-89; amersham Pharmacia Biotech (2001) BioDirectory, Piscataway, N.J.), pp.384-391. The production, purification and fragmentation of polyclonal and monoclonal antibodies is described. Coligan et al (2001) immunological protocols, Vol.1, N.Y. John Wiley father, Inc.; harlow and Lane (1999) use Antibodies (Using Antibodies), Cold spring harbor laboratory Press, Cold spring harbor, N.Y.; harlow and Lane, supra. Standard techniques are available for characterizing ligand/receptor interactions. See, e.g., Coligan et al (2001) immunological protocols, Vol.4, N.Y. John Willi.

Methods are available for flow cytometry, including fluorescence activated cell sorting detection systemsSee, e.g., Owens et al (1994) Flow Cytometry Principles for Clinical Laboratory Practice, Inc., John Willi, Hoboken, N.J.; givan (2001) Flow Cytometry, 2 nd edition; Wiley-Liss, Hobock, N.J.; shapiro (2003) Practical Flow Cytometry (John Willi's father and son, Hobock, N.J.). Fluorescent reagents are available that are suitable for modifying nucleic acids, including nucleic acid primers and probes, polypeptides, and antibodies, for use, for example, as diagnostic reagents. [ solution ] AMolecular Probes (Molecular Probes) (2003) catalog (Catalogue), Molecular Probes, Inc (Molecular Probes, Inc.), eugold, Oreg, oregon; the sigma-aldrich (2003) catalog, st louis, missouri.

Standard methods of histology of the immune system are described. See, e.g., Muller-Harmelink (eds.) (1986) human thymus: histopathology and Pathology (Human Thymus: Histopathology and Pathology), Schpringer (Springer Verlag), New York, N.Y.; hiatt et al (2000) Color Atlas of Histology, Lippincott Williams Wilkins publishing company (Lippincott, Williams, and Wilkins), Phla, Pa.; louis et al (2002) basic histology: text and Atlas (Basic history: Text and Atlas), McGraw-Hill, New York, N.Y..

Software packages and databases are available for determining, for example, antigen fragments, leader sequences, protein folds, functional domains, glycosylation sites, and sequence alignments. See, for example, GenBank for example,suite (Informatx, Inc., Bethesda, Md.) Bessedla, Maryland; GGC Wisconsin Package (Accelrys, Inc., san Diego, Calif.);(TimeLogic, Crystal Bay, Nevada (Crystal Bay, Nev.))); one et al (2000) Bioinformatics (Bioinformatics) 16: 741-742; menne et al (2000) Bioinformatics Applications notes (Bioinformatics Applications notes) 16: 741-742; wren et al (2002) computer methods and Programs in biomedical science (Comput. methods Programs Biomed.) 68: 177-181; von Heijne (1983) 133 (J.European Union of biochemistry) (Eur.J.biochem): 17-21; von Heijne (1986) Nucleic Acids research (Nucleic Acids Res.) 14: 4683-4690.

Expression of frizzled receptors in mouse small intestine and in mouse and human colon.

To determine the expression pattern of each frizzled receptor in mouse small intestine and colon epithelium by(ACD) detection of mRNA for individual frizzled receptors. The results used are shown in Table 1And (3) a probe. Compliance standard2.5HD Assay-Red protocol.

Table 1:

FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, and FZD9 were expressed at different levels in the mouse intestinal epithelium (fig. 1). FZD5 was expressed at the highest level in the intestinal crypts and villi. In the crypts, FZD5 expression is much higher near the apical compartment containing transport-amplifying (TA) cells. Low levels of FZD1 were detected in both the intestinal epithelium and the lamina propria immediately surrounding the intestinal crypt. FZD4, FZD6, and FZD7 are expressed at low levels and are evenly distributed in both intestinal villi and crypts. The expression of FZD2, FZD3, FZD8, FZD9, and FZD10 is very low and is mainly detected in intestinal crypts.

FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, and FZD9 are also expressed at different levels in the colon of mice. FZD5 was expressed at the highest level in the colonic crypts, and expression was higher towards the luminal side. FFZD1 and FZD7 were detected at lower levels in the colonic epithelium. FZD2, FZD3, FZD4, FZD6, FZD8, and FZD9 are expressed at low levels and are uniformly distributed in the colon crypt. There was no detectable expression of FZD10 in the intestine. FZD expression levels are affected in the colon of mouse DSS colitis IBD models and in the colon of human ulcerative colitis patients.

To determine the expression pattern of each frizzled receptor in human colonic epitheliumBy passing(ACD) detection of mRNA for individual frizzled receptors. Compliance standard2.5HD Assay-Red protocol. FZD5 was expressed at the highest level in the colon crypt. FZD7 was detected at lower levels in the colonic epithelium and stromal cells that encompass the colonic crypts.

Activity of engineered soluble WNT agonists

Three frizzled receptor biased WNT agonists, R2M3-26(FZD1,2, 5, 7, 8 and LRP6 binders), 1RC07-03(FZD1,2,7 and LRP5 binders) and R2M13-03(FZD5, 8 and LRP5 binders), were examined in human hepatocyte cell line Huh7 cells for their activity to determine their ability to activate WNT signaling. Three WNT agonists were previously described in WO 2019126398. Table 2 provides the sequences of the LC and HC chains of the three WNT agonists used.

Table 2: WNT agonist sequences

On day 1, cells were seeded at 100 million per 96-well plate and grown overnight. Proteins were added to cells in triplicate at 10-fold dilutions starting at 100nM in the presence of 20nM R-spondin. Luciferase reporter activity in wells was measured with the luciferase assay system (Promega) and read on a SpectraMax plate reader (Molecular Devices). Mean absolute RLU values are shown for each protein dilution in triplicate (figure 2). R2M3-26(FZD1,2, 5, 7, 8) showed the highest reporter activity. R2M13-03(FZD5, 8) showed the lowest activity in the STF assay.

Stimulation of mouse small intestinal organoid proliferation by specific activation of WNT signaling by FZD5 and FZD8

Mouse intesticus maintained in mouse intestinal organoid^TMOrganoid growth medium (stem cell technology, ltd). To determine organoid proliferation, the organoids were dissociated for 10 minutes with gentle cell dissociation reagents (stem cell technology limited), washed twice in cold pbs (gibco), and 1:1 are resuspended on ice cubes of Matrigel (Corning). A resuspension of 25 microliters of cells in Matrigel was seeded into the center of each well in a pre-warmed 48-well tissue culture plate and solidified at 37 ℃ for 5 minutes. 300 microliters of basal medium (table 3), basal medium + IWP2, or basal medium + IWP2+ in place of WNT agonist was administered to the wells. Each condition included 5-6 replicates. Media and treatment were changed once on day 4 post plating. Images of 3D cultured organoids obtained at day 7 are shown in figure 3. Cell titers Glow 3D (promegate) were performed on the treated organoids on day 7.

TABLE 3 basal Medium composition

DMEM/F12K	Life technologies (Life technologies)
			HEPES	LIFE TECHNOLOGIES Corp.	10mM
Penicillin/streptomycin	LIFE TECHNOLOGIES Corp.	1X
			GlutaMAX	LIFE TECHNOLOGIES Corp.	1X
N2 supplement 100	LIFE TECHNOLOGIES Corp.	1X
			B27 supplement 50x	LIFE TECHNOLOGIES Corp.	1X
N-acetylcysteine	Sigma-Aldrich (Sigma-Aldrich)	1.25mM
			Recombinant human EGF	Pai Proro technology (Peprotech)	50ng/mL
Recombinant human Nojin element	Papiro technology	50ng/mL
			Recombinant human Rspondin-1	R&D Systems	500ng/mL

In the presence of WNT agonist treatment, the intestinal organoids proliferate and become morphologically round. Endogenous WNT expression was inhibited in assays with porcupine inhibitor IWP-2. Both R2M3-26(FZD1,2, 5, 7, 8) and R2M13-03(FZD5, 8) effectively stimulated organoid proliferation, which was reflected in increased organoid numbers and an increase in individual organoids (fig. 3A and 3C). 1RC07-03(FZD1,2,7) also stimulated organoid proliferation, but to a much lesser extent. All WNT agonists exhibited higher activity than 18R5-Dkk1c (the structures of which are described in Janda et al (2017) Nature 545: 234-237). FZD antagonists can be similarly tested in organoid cultures.

IHC analysis of mouse organoid cultures

The activity of R2M3-26 on mouse small intestine organoids was confirmed by staining with proliferation marker Ki 67. Mouse small intestine organoids grown in media-submerged Matrigel in 8-well chamber slides (Lab-Tek II, 154534) were treated with 100nM R2M3-26 for 7 days as described above. Organoids were then fixed in 4% PFA, permeabilized in PBS + 0.2% Triton for 20 min, and blocked in blocking buffer (PBS + 0.2% Triton + 3% BSA). The primary antibodies rabbit anti-Ki 67(Abcam ab15580, 1: 1000) and goat anti-E-cadherin (R)&D AF748, 1: 2000) mixed in blocking buffer and added to organoids. After 1 hour incubation with primary antibody at room temperature, organoids were washed with 3 times PBS + 0.2% Triton, then incubated with 1: the 1000 diluted secondary antibodies donkey anti-rabbit Alexa568(Abcam) and donkey anti-goat Alexa488(Abcam) were incubated for 30 min. The organoids were then washed 3 times with PBS + 0.1% Tween and mounted in ProLong^TMGoldd (Gold) anti-fluorescence decay fixative (Thermo Fisher). Z-stack signal channel images were taken using a Zeiss (Zeiss) DMi8 fluorescence microscope, deconvoluted digitally, projected on 2D, and the two channels were combined for illustration. WNT agonist treatment stimulated proliferation of mouse small intestinal organoids. Mouse intestinal organoids after treatment with 100nM R2M3-26 were stained with anti-Ki 67 (red) and anti-E-cadherin (green), showing cell proliferation after WNT agonist treatment (FIG. 4).

In vivo dextran sodium sulfate ("DSS") IBD mouse model

C57Bl/6J female mice were obtained from Jackson Laboratories (Jackson Laboratories, Barhar, ME, USA, Myon, USA) at 6 weeks of age and were housed 4 per cage. All animal experiments met the "laboratory animal Care and use guide" guidelines compiled by the national academy of sciences. Protocols for animal experiments were approved by the Surrozen institutional animal care and use committee. Mice were acclimated for at least two days prior to starting the experiment. The mice were kept in a humidity environment of 30% to 70% and at room temperature in the range of 20 ℃ to 26 ℃ for 12/12 hours on a light/dark cycle.

To induce acute colitis, female mice of 7 to 8 weeks of age were given free drinking water containing 4.0% (w/v) dextran sodium sulfate (DSS, MP bio-medical company (MP bio-medical company), MFCD00081551) for 7 days and drinking water containing 1.0% (w/v) DSS for 2 days (fig. 5A). Mice undergoing DSS developed severe colitis characterized by profound and sustained weight loss (fig. 5B) and bloody diarrhea, resulting in an increased disease index as indicated by the fecal score (fig. 5C). RSPO2-Fc (R-Spondin 2-Fc; SEQ ID NO:24) plus R2M3-26 combination therapy, twice weekly or daily, the Disease Activity Index (DAI) at day 9 was significantly improved compared to negative controls. Treatment with R2M3-26 and R2M3-26 alone plus RSPO2-Fc significantly improved body weight at day 10.

Histological evaluation of the transverse colon of DSS model mice showed inflammation, crypt hyperplasia, goblet cell loss, and ulceration extending from the mucosa into the serous fluid (fig. 6A-6E); in contrast, the colon of WNT agonist treated mice was nearly normal with the lowest histological score in all treatment groups (fig. 6C). RSPO2-Fc had no significant positive effect on histological scores of colon tissues. The RSPO2-Fc plus R2M3-26 combination group had a lower histological score of colon tissue (P) compared to the control anti-GFP group<0.0, fig. 7A). R2M3-26 did not appear to affect the small intestine crypts or villi (FIG. 8C), while RSPO2-Fc and combination induced proliferation of villi and crypts (FIGS. 8D-8E). Such asThe aboveHistological scores were assessed as described in Geboes et al (2000).

Serum inflammatory cytokines were analyzed by the pro-inflammatory panel 1 kit (Meiso Scale Diagnostics, Inc.), K15048D, and treatment of R2M3-26, RSPO2-Fc, and R2M3-26 plus RSPO2-Fc, all of which reduced cytokine levels of IFN-. gamma., TNF-. alpha.and IL-1. beta. (FIGS. 9A-9J, specifically, FIGS. 9A, 9J and 9B, respectively).

RSPO2-Fc alone induced small intestine hyperplasia with no significant benefit for weight loss and DAI. WNT agonist/RSPO 2-Fc combined treatment reduced disease activity, repaired damaged colonic epithelium, and induced proliferation of the small intestine. R2M3-26 alone: a) improving body weight; b) repair of damaged colonic epithelium; c) reducing serum inflammatory cytokine markers; and d) does not cause proliferation of the small intestine, thus demonstrating that WNT agonists alone can treat acute colitis by improving the epithelial barrier, thereby reducing inflammation.

Amelioration of intestinal inflammation and epithelial tissue repair

Previous studies demonstrated that the multispecific WNT agonist R2M3-26 is able to improve intestinal inflammation and repair epithelial injury in a DSS colitis mouse model. FZD5, 8-specific WNT agonist R2M13-26 and FZD1,2, 7-specific WNT agonist 1RC07-26 were tested to determine whether one or both could ameliorate DSS-induced colitis in a mouse model, taking into account the selective expression of FZD5 and FZD8 in the colon.

6-week-old C57Bl/6J female mice were obtained from Jackson laboratories (Balport, Maine, USA) and housed 5 mice per cage. All animal experiments met the "laboratory animal Care and use guide" guidelines compiled by the national academy of sciences. Protocols for animal experiments were approved by the internal institutional animal care and use committee.

To induce acute colitis, female mice of 7 to 8 weeks old were given free drinking water containing 4.0% (w/v) dextran sodium sulfate (DSS, MP bio-medical company, MFCD00081551) for 7 days and drinking water containing 1.0% (w/v) DSS for 3 days. Mice undergoing DSS developed severe colitis characterized by profound and sustained weight loss (fig. 10A) and bloody diarrhea, resulting in increased stool scores (fig. 10B) and disease activity index. R2M3-26, R2M13-26, and 1RC07-26, respectively, treated twice weekly significantly improved body weight (fig. 10A) and stool score (fig. 10B) on day 10 compared to negative controls (PBS or anti-GFP). Histological evaluation of the transverse colon of DSS model mice showed neutrophil infiltration, crypt hyperplasia, goblet cell loss, and ulceration (fig. 11B and 11C). Treatment with R2M3-26, R2M13-26, and 1RC07-26 restored colonic tissue structures, showing improvement in epithelial erosion, goblet cell loss, and neutrophil migration (FIGS. 11D-H). R2M3-26, R2M13-26, or C07-3 did not cause small intestine hyperplasia (FIGS. 11B and 11C), while R2M3-26/RSPO combined treatment induced small intestine hyperplasia (FIGS. 12D-H). Inflammatory cytokines were analyzed in serum and colon tissues using the pro-inflammatory panel 1 kit (McSockey Gault diagnostics, K15048D) and the results showed that R2M3-26 and R2M13-26 treatment significantly reduced TNF- α and IL-8 levels in serum (FIGS. 13A and 13C), and IL-6 and IL-8 levels in colon tissues (FIGS. 13E and 13F).

As mentioned in example IV above, R2M3-26 reduced intestinal inflammation and repaired epithelial injury in DSS colitis mice. The present study further demonstrates that FZD5, an 8-specific WNT agonist, R2M13-26 and FZD1, a2, 7-specific WNT agonist, 1RC07-26, are able to improve DAI, repair damaged colonic epithelium without small intestine hyperplasia, and reduce inflammatory cytokine levels in the colon and serum.

IX. dose response analysis of R2M13-26 in mouse DSS model

To determine the optimal dose of R2M13-26(FZD5, 8 binder) in a DSS mouse model of IBD, six week old C57Bl/6J female mice were obtained from jackson laboratories (balport, maine, usa) and were housed 5 mice per cage. All animal experiments met the "laboratory animal Care and use guide" guidelines compiled by the national academy of sciences. Protocols for animal experiments were approved by the Surrozen institutional animal care and use committee.

To induce acute colitis, female mice of 7 to 8 weeks old were given free drinking water containing 4.0% (w/v) dextran sodium sulfate (DSS, MP bio-medical company, MFCD00081551) for 7 days and drinking water containing 1.0% (w/v) DSS for 3 days. Control mice subjected to DSS developed severe colitis characterized by profound and sustained weight loss and bloody diarrhea, resulting in an increased disease activity index (DAI, fig. 14A-14B).

R2M13-26 treatment at 0.3, 1, 3, 10mpk, twice weekly improved DAI in a dose-response pattern (fig. 14A). Treatment with R2M13-26 at other concentrations of 1, 3, 10, 30mpk, once weekly, improved DAI in a dose-response pattern (fig. 14B). Histological evaluation of the transverse colon of DSS model mice showed neutrophil infiltration, edema, crypt hyperplasia, goblet cell loss, and ulceration. R2M13-26 treatments at different doses and frequency all showed improvement with respect to epithelial erosion, goblet cell loss and neutrophil migration in DSS colitis mice (fig. 15A-15J). The results of analysis of inflammatory cytokines in serum and colon tissues using the pro-inflammatory panel 1 kit (McOsgelr diagnostics GmbH, K15048D) showed that R2M13-26 treatment at different doses and frequency all significantly reduced TNF- α, IL-6 and IL-8 levels in serum figures 16A-16C) and colon tissues (figures 17A-17C).

In a DSS colitis mouse model, a broad dose range of RR2M13-26 reduced intestinal inflammation and repaired epithelial damage, further validating that FZD5, 8-specific molecule (R2M13-26) treats acute colitis by improving the intestinal epithelial barrier.

Efficacy of different FZD5,8 specific WNT agonists in acute DSS models

The activity of four FZD5, 8-specific WNT agonists, 57SE8-26, 57SB8-26, 174R-E01-26 and 57SA10-26, were examined in human hepatocyte cell line Huh7 cells to determine their ability to activate WNT signaling. Table 4 provides the sequences of the components of FZD5, 8WNT agonist. These WNT agonists comprise a FZD binding domain that is a Fab containing Heavy Chains (HC) and Light Chains (LC) and an LRP binding domain that is a VHH linked via a linker to a FZD Fab at the N-terminus of the LC. WNT agonists include two of the indicated LC chains and two of the indicated HC chains.

Table 4: fZD5,8 specific WNT agonists

The FZD-VH sequence is indicated in bold; the FZD-CH1 sequence is indicated in italics; the hinge sequence is indicated in bold italics; the CH2 sequence is indicated in underlined italics; the CH3 sequence is indicated in bold underlining; LRP (26) VHH sequence, linked to the N-terminus of VL by a linker, indicated in bold italic underlining; the linker sequence is underlined only; FZD-VL is shaded gray; and FZD-CL is shaded gray and underlined.

FZD5/8 specific binding domains that specifically bind FZD5 and FZD8 (and do not significantly bind other FZD) are shown in table 5.

Table 5: fZD5,8 specific binding domain

In certain embodiments, the disclosure provides a polypeptide comprising a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to any one of SEQ ID NOs 7-14 or 33-40. In certain embodiments, the present disclosure provides a WNT agonist comprising a FZD binding domain comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to any one of SEQ ID NOs 7-14 or 33-40. In certain embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof comprising a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to any one of SEQ ID NOs 33-40. In certain embodiments, the present disclosure provides a combination molecule comprising a FZD binding domain comprising a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to any one of SEQ ID NOs 7-14 or 33-40.

Table 6 provides the CDR sequences of VH and VL of the FZD5,8 binding domains described above. In certain embodiments, the present disclosure provides polypeptides comprising one or both of the VH and/or VL CDR sequences of either of FZD5,8 binding domains. In certain embodiments, the present disclosure provides a WNT agonist comprising a FZD binding domain comprising FZD5,8 binding domain identified herein, e.g., one or both of the heavy chain (CDRH1-3) and/or light chain (CDRL1-3) CDR sequences of any one of 57SE8, 57SB8174RE, or 57SA 10. In certain embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof comprising one or both of the VH and/or VL CDR sequences of either of the FZD5,8 binding domains. In certain embodiments, the present disclosure provides a WNT agonist comprising one or both of the VH and/or VL CDR sequences of either of FZD5,8 binding domains. In certain embodiments, the present disclosure provides a combination molecule comprising a FZD binding domain comprising one or both of the VH and/or VL CDR sequences of either of FZD5,8 binding domains. In other embodiments, the polypeptide, antibody or binding fragment thereof, WNT agonist, or combinatorial molecule synthesizes at least 5 of the six CDRs present in any binding domain. In other embodiments, the polypeptide, antibody or binding fragment thereof, WNT agonist, or combination molecule comprises six CDRs present in any binding domain, wherein the CDRs collectively comprise one or more, e.g., one, two, three, four, five, six, or more amino acid modifications as compared to the native CDRs. In particular embodiments, a WNT agonist or combination molecule comprises two heavy chains and two light chains that share either of the disclosed CDRs or variants thereof.

TABLE 6 CDR sequences of FZD5,8 binding domain

On day 1, cells were seeded at 100 million per 96-well plate and grown overnight. Proteins were added to cells in triplicate at 10-fold dilutions starting at 100nM in the presence of 20nM R-spondin. Luciferase reporter activity in wells was measured with the luciferase assay system (Promega) and read on a SpectraMax plate reader (molecular instrumentation). Mean absolute RLU values are shown for each protein dilution in triplicate (figure 18). R2M13-26 was included in the same assay for comparison.

To determine the efficacy of the additional FZD5, 8WNT agonist in a DSS mouse model of IBD, six week old C57Bl/6J female mice were obtained from jackson laboratories (port balk, maine, usa) and were housed 5 mice per cage. All animal experiments met the "laboratory animal Care and use guide" guidelines compiled by the national academy of sciences. Protocols for animal experiments were approved by the Surrozen institutional animal care and use committee.

To induce acute colitis, female mice of 7 to 8 weeks old were given free drinking water containing 4.0% (w/v) dextran sodium sulfate (DSS, MP bio-medical company, MFCD00081551) for 7 days and drinking water containing 1.0% (w/v) DSS for 3 days. Control mice subjected to DSS developed severe colitis characterized by profound and sustained weight loss and bloody diarrhea, resulting in an increased disease activity index (DAI, fig. 19A).

FZD5, 8-specific WNT agonist twice weekly at 10mpk improved DAI, similar to R2M13-26 (fig. 19A). Analysis of inflammatory cytokines in serum using the pro-inflammatory panel 1 kit (McSockey Gault diagnostics, K15048D) showed that FZD5, an 8-specific WNT agonist, except 58SE8-26, all significantly reduced TNF- α, IL-6 and IL-8 levels in serological FIGS. 16A-16C) and colon tissue (FIGS. 19B-19D).

FZD5, 8-specific WNT agonists reduced intestinal inflammation and repaired epithelial damage in a DSS colitis mouse model, leading to improved DAI, further validating that FZD5, 8-specific WNT agonist molecules described herein treat acute colitis by improving the intestinal epithelial barrier.

Tissue-specific WNT signaling enhancing molecules are effective in activating WNT signaling and stimulating gut organoids in vitro Growth of

MUC-13 binding agents C4, C7 and C14 (see e.g. WO2016168607a1) were cloned and produced as full length iggs and their binding capacity to MUC13 was determined by FACS analysis of HT29 cells expressing MUC 13. They were also analyzed for potential binding to HEK293 cells that do not express MUC-13 as a negative control. Cells were harvested and FACS buffer (PBS (-Ca))²⁺，-Mg²⁺) 0.1% BSA, 0.5% sodium azide) 2 times and 10 times⁶Individual cells/ml were resuspended in FACS buffer. 60 microliters of cell suspension was aliquoted into each well of a 96-well v-bottom plate, and the plate was rotated at 1500rpm for 3 minutes to remove the FACS buffer, then 10nM of the corresponding MUC-13 antibody or anti-GFP control antibody diluted in FACS buffer and incubated at 4C for 1 hour was added. The plate was then spun to remove primary antibodies and washed 1 time with FACS buffer, followed by addition of Alexa488 goat anti-human secondary antibody (seimer feishell technology) and incubated at 4C for 30 minutes. This medium was then removed after spinning and the plates were washed 1 time in FACS buffer. The cells were then resuspended in 150FACS buffer and at 10,000 events under BD Accuri^TMThe analysis was performed on a cell analyzer (Becton-Dickinson, Becton Dickinson). Comparing FACS plots of HT29 (fig. 20A-20C) and HEK293 cells (fig. 20D-20F), only one C14 of the tested MUC-13 binding agents showed specific FACS shift in HT29 cells, indicating MUC-13 specific binding activity of C14. Table 7 provides the sequences of MUC-13 binding agents tested.

Table 7: tissue targeting WNT enhancer

To examine whether the above-described MUC-13 binding agents could be used to drive the tissue specificity of gut-specific WNT signaling enhancer molecules, mutant (F105R/F109A) RSPO2(mutRSPO2, with amino acid mutations in the Furin2 binding domain, thus reducing binding to LGR1-4 (see, e.g., WO2020014271) was fused to the C-terminus of the heavy chain of MUC-13IgG antibody (or GFP antibody as a negative control) with a 15 amino acid GS linker-these mutRSPO2 (mutrpro) targeted luciferase reporter molecules were tested for signaling activity by Super TOPFlash luciferase reporter (STF) assay in HT29 cells or HEK293 cells as described above-the dose response curves for C4-mutRSPO2, C7-mutRSPO2 and C36-mutRSPO 2 reporter activities were measured (figure 21) for the dose response curves for the luciferase reporter genes in mutRSPO 3638 cells but only the left-t 14 targeted mutRSPO molecules showed no shift in HEK 14 cells, in which EC50 is comparable to wild-type Fc-RSPO2(SEQ ID NO: 24). This is consistent with the MUC-13 binding activity of C-14 as an IgG, suggesting that WNT-enhancing molecules lacking Lgr4-6 binding ability may act like native RSPO2 to modulate WNT signaling in cells when targeted by MUC-13 binding.

The signaling activity of MUC-13 targeted WNT signaling enhancing molecules in human small intestine organoids was also examined. Growth and maintenance of organoid cultures is described above. Human small intestine organoid growth was maintained when wild-type RSPO in the medium was replaced with C14-mutRSPO 2. Human small intestine organoids were grown in basal medium with RSPO-1 replaced by non-intestinal epithelial cell-targeted mutRSPO1(ASGR1-mutRSPO 1; see e.g. WO2020014271) (fig. 21A-21C) in the concentration dilution series shown or by C14-mutRSPO2 (fig. 21D-21F) at the same concentration. While organoids grown in ASGR1-mutRSPO1 stopped growing and began to denature, similar to the results observed when grown in basal medium without any RSPO (fig. 21G), C14-mutRSPO was able to maintain organoid growth similar to intesticult (stem cell Technologies) organoid growth medium with wild-type RSPO (fig. 21H). This assay demonstrates that MUC-13 targeted WNT signaling enhancing molecules can act on intact epithelium in human small intestine microtissue.

The various embodiments described above can be combined to provide further embodiments. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification and/or listed in the application data sheet, are incorporated herein by reference, in their entirety. Aspects of the embodiments can be modified, if necessary, to employ aspects of the various patents, applications, and publications to provide yet further embodiments. These and other changes can be made to the embodiments in light of the above detailed description.

In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the disclosure.

Sequence listing

<110> Surrozen, Inc.

Li, Yang

Lu, Chenggang

Baribault, Helene

Yeh, Wen-Chen

Xie, Liqin

Wang, I-Chieh

Meng, Weixu

<120> modulation of WNT signaling in gastrointestinal disorders

<130> SRZN-014/02WO 328202-2067

<150> US 62/816,720

<151> 2019-03-11

<150> US 62/888,749

<151> 2019-08-19

<160> 64

<170> PatentIn version 3.5

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Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

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Gly Trp Ile Ser Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Leu

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Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

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Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

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Ala Ser Ser Lys Glu Lys Ala Thr Tyr Tyr Tyr Gly Met Asp Val Trp

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Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

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Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

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Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

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Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

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Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

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Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

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His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

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Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala

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Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

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Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

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His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

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Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

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Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

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Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro

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Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

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Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val

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Ala Met Ile Arg Pro Val Val Thr Glu Ile Asp Tyr Ala Asp Ser Val

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Lys Gly Arg Phe Thr Ile Ser Arg Asn Asn Ala Met Lys Thr Val Tyr

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Leu Gln Met Asn Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

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Asn Ala Lys Arg Pro Trp Gly Ser Arg Asp Glu Tyr Trp Gly Gln Gly

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Thr Gln Val Thr Val Ser Ser Gly Ser Gly Ser Gly Gln Ala Val Val

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Leu Gln Glu Pro Ser Leu Ser Val Ser Pro Gly Gly Thr Val Thr Leu

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Thr Cys Gly Leu Ser Ser Gly Ser Val Ser Thr Asn Tyr Tyr Pro Ser

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Trp Tyr Gln Gln Thr Pro Gly Gln Ala Pro Arg Thr Leu Ile Tyr Tyr

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Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys Ala Ala

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Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln Ala Asn

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Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly Ala Val

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Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly Val Glu

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Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser Ser

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Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser Tyr Ser

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Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val Ala Pro

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Thr Glu Cys Ser

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Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Thr Tyr Arg

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Tyr Leu His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

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Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

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Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

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Ala Ser Ser Met Val Arg Val Pro Tyr Tyr Tyr Gly Met Asp Val Trp

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Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

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Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

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Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

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Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

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Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

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His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

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Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala

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Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

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Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

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His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

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Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

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Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro

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Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

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Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

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Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

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Ser Pro Gly Lys

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Gly Arg Phe Thr Met Ser Thr Glu Ser Ala Lys Asn Thr Met Tyr Leu

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Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Tyr

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Val Lys Leu Arg Asp Asp Asp Tyr Val Tyr Arg Gly Gln Gly Thr Gln

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Val Thr Val Ser Ser Gly Gly Ser Gly Ser Gly Ser Gly Asp Ile Gln

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Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val

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Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn Trp

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Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala

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Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser

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Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe

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Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu Thr Phe Gly

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Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val

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Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser

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Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln

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Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val

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Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu

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Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu

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Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg

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Gly Glu Cys

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Tyr Trp Thr Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

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Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

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Asn Leu Lys Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

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Arg Gly Gly Gln Gly Gly Tyr Asp Trp Gly His Tyr His Gly Leu Asp

100 105 110

Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys

115 120 125

Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly

130 135 140

Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

145 150 155 160

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

165 170 175

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

180 185 190

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn

195 200 205

Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro

210 215 220

Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

225 230 235 240

Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

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Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

260 265 270

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

275 280 285

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn

290 295 300

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

305 310 315 320

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly

325 330 335

Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

340 345 350

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn

355 360 365

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

370 375 380

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

385 390 395 400

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

405 410 415

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

420 425 430

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

435 440 445

Ser Leu Ser Pro Gly Lys

450

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Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ser Ser Ala Asn Ile Asn Ser Ile Glu

20 25 30

Thr Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Ile

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Ala Asn Met Arg Gly Gly Gly Tyr Met Lys Tyr Ala Gly Ser Leu Lys

50 55 60

Gly Arg Phe Thr Met Ser Thr Glu Ser Ala Lys Asn Thr Met Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Tyr

85 90 95

Val Lys Leu Arg Asp Asp Asp Tyr Val Tyr Arg Gly Gln Gly Thr Gln

100 105 110

Val Thr Val Ser Ser Gly Ser Gly Ser Gly Ser Tyr Val Leu Thr Gln

115 120 125

Pro Pro Ser Val Ser Val Ser Pro Gly Gln Thr Ala Ser Ile Thr Cys

130 135 140

Ser Gly Asp Lys Val Gly His Lys Tyr Ala Ser Trp Tyr Gln Gln Lys

145 150 155 160

Pro Gly Gln Ser Pro Val Leu Val Ile Tyr Glu Asp Ser Gln Arg Pro

165 170 175

Ser Gly Ile Pro Val Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala

180 185 190

Thr Leu Thr Ile Ser Gly Thr Gln Ala Met Asp Glu Ala Asp Tyr Tyr

195 200 205

Cys Gln Ala Trp Asp Ser Ser Thr Asp Val Val Phe Gly Gly Gly Thr

210 215 220

Lys Leu Thr Val Leu Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu

225 230 235 240

Phe Pro Pro Ser Ser Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val

245 250 255

Cys Leu Ile Ser Asp Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys

260 265 270

Ala Asp Ser Ser Pro Val Lys Ala Gly Val Glu Thr Thr Thr Pro Ser

275 280 285

Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr

290 295 300

Pro Glu Gln Trp Lys Ser His Arg Ser Tyr Ser Cys Gln Val Thr His

305 310 315 320

Glu Gly Ser Thr Val Glu Lys Thr Val Ala Pro Thr Glu Cys Ser

325 330 335

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Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly His Trp Tyr Phe Asp Leu Trp Gly Arg Gly Thr Leu Val

100 105 110

Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

115 120 125

Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu

130 135 140

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

145 150 155 160

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser

165 170 175

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

180 185 190

Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr

195 200 205

Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr

210 215 220

Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe

225 230 235 240

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

245 250 255

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

260 265 270

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

275 280 285

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

290 295 300

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

305 310 315 320

Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser

325 330 335

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

340 345 350

Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

355 360 365

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

370 375 380

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

385 390 395 400

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

405 410 415

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

420 425 430

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 8

<211> 338

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<220>

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Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ala Cys Ala Gly Ser Gly Arg Ile Phe Ala Ile Tyr

20 25 30

Asp Ile Ala Trp Tyr Arg His Pro Pro Gly Asn Gln Arg Glu Leu Val

35 40 45

Ala Met Ile Arg Pro Val Val Thr Glu Ile Asp Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asn Asn Ala Met Lys Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Asn Ala Lys Arg Pro Trp Gly Ser Arg Asp Glu Tyr Trp Gly Gln Gly

100 105 110

Thr Gln Val Thr Val Ser Ser Gly Gly Gly Gly Ser Asp Ile Gln Met

115 120 125

Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr

130 135 140

Ile Thr Cys Arg Ala Ser Glu Ser Ile Arg Ser Trp Leu Ala Trp Tyr

145 150 155 160

Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Gly Ala Ser

165 170 175

Arg Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly

180 185 190

Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala

195 200 205

Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Trp Thr Phe Gly Gln

210 215 220

Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe

225 230 235 240

Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val

245 250 255

Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp

260 265 270

Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr

275 280 285

Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr

290 295 300

Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val

305 310 315 320

Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly

325 330 335

Glu Cys

<210> 9

<211> 453

<212> PRT

<213> Artificial sequence

<220>

<223> laboratory manufacturing-synthetic wnt agonists

<400> 9

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Lys Asp

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Arg Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Leu Pro Pro Ala Ala Gly Gly Gly Gly Tyr Phe Gln His

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly

115 120 125

Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly

130 135 140

Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

145 150 155 160

Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

165 170 175

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

180 185 190

Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val

195 200 205

Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys

210 215 220

Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala

225 230 235 240

Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

245 250 255

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

260 265 270

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

275 280 285

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

290 295 300

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

305 310 315 320

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala

325 330 335

Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

340 345 350

Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln

355 360 365

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

370 375 380

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

385 390 395 400

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

405 410 415

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

420 425 430

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

435 440 445

Leu Ser Pro Gly Lys

450

<210> 10

<211> 338

<212> PRT

<213> Artificial sequence

<220>

<223> laboratory manufacturing-synthetic wnt agonists

<400> 10

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ala Cys Ala Gly Ser Gly Arg Ile Phe Ala Ile Tyr

20 25 30

Asp Ile Ala Trp Tyr Arg His Pro Pro Gly Asn Gln Arg Glu Leu Val

35 40 45

Ala Met Ile Arg Pro Val Val Thr Glu Ile Asp Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asn Asn Ala Met Lys Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Asn Ala Lys Arg Pro Trp Gly Ser Arg Asp Glu Tyr Trp Gly Gln Gly

100 105 110

Thr Gln Val Thr Val Ser Ser Gly Gly Gly Gly Ser Asp Ile Gln Met

115 120 125

Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr

130 135 140

Ile Thr Cys Arg Ala Ser Gln Asn Val Asn Asp Trp Leu Ala Trp Tyr

145 150 155 160

Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser

165 170 175

Asn Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly

180 185 190

Thr Asp Phe Thr Leu Thr Ile Arg Ser Leu Gln Pro Glu Asp Phe Ala

195 200 205

Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Phe Thr Phe Gly Pro

210 215 220

Gly Thr Lys Val Asp Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe

225 230 235 240

Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val

245 250 255

Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp

260 265 270

Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr

275 280 285

Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr

290 295 300

Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val

305 310 315 320

Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly

325 330 335

Glu Cys

<210> 11

<211> 448

<212> PRT

<213> Artificial sequence

<220>

<223> laboratory manufacturing-synthetic wnt agonists

<400> 11

Glu Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Gly Asp Thr Phe Gly Val Gly His Phe Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 12

<211> 343

<212> PRT

<213> Artificial sequence

<220>

<223> laboratory manufacturing-synthetic wnt agonists

<400> 12

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ala Cys Ala Gly Ser Gly Arg Ile Phe Ala Ile Tyr

20 25 30

Asp Ile Ala Trp Tyr Arg His Pro Pro Gly Asn Gln Arg Glu Leu Val

35 40 45

Ala Met Ile Arg Pro Val Val Thr Glu Ile Asp Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asn Asn Ala Met Lys Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Asn Ala Lys Arg Pro Trp Gly Ser Arg Asp Glu Tyr Trp Gly Gln Gly

100 105 110

Thr Gln Val Thr Val Ser Ser Gly Gly Ser Gly Ser Asp Val Val Met

115 120 125

Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly Glu Pro Ala Ser

130 135 140

Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser Asn Gly Tyr Asn

145 150 155 160

Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu

165 170 175

Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro Asp Arg Phe Ser

180 185 190

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Gln Ile Ser Arg Val Glu

195 200 205

Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Gly Leu His Thr Pro

210 215 220

Val Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala

225 230 235 240

Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser

245 250 255

Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu

260 265 270

Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser

275 280 285

Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu

290 295 300

Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val

305 310 315 320

Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys

325 330 335

Ser Phe Asn Arg Gly Glu Cys

340

<210> 13

<211> 452

<212> PRT

<213> Artificial sequence

<220>

<223> laboratory manufacturing-synthetic wnt agonists

<400> 13

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Ser

20 25 30

Val Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Ser Val Tyr Asn Gly Asn Thr Asn Tyr Ala Glu Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Phe Ala Met Val Arg Gly Gly Val Tyr Tyr Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro Gly Lys

450

<210> 14

<211> 338

<212> PRT

<213> Artificial sequence

<220>

<223> laboratory manufacturing-synthetic wnt agonists

<400> 14

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ala Cys Ala Gly Ser Gly Arg Ile Phe Ala Ile Tyr

20 25 30

Asp Ile Ala Trp Tyr Arg His Pro Pro Gly Asn Gln Arg Glu Leu Val

35 40 45

Ala Met Ile Arg Pro Val Val Thr Glu Ile Asp Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asn Asn Ala Met Lys Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Asn Ala Lys Arg Pro Trp Gly Ser Arg Asp Glu Tyr Trp Gly Gln Gly

100 105 110

Thr Gln Val Thr Val Ser Ser Gly Gly Gly Gly Ser Asp Ile Gln Met

115 120 125

Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr

130 135 140

Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Tyr Leu Asn Trp Tyr

145 150 155 160

Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser

165 170 175

Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly

180 185 190

Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala

195 200 205

Thr Tyr Tyr Cys Gln His Tyr Tyr Asn Leu Pro Leu Thr Phe Gly Gln

210 215 220

Gly Thr Arg Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe

225 230 235 240

Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val

245 250 255

Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp

260 265 270

Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr

275 280 285

Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr

290 295 300

Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val

305 310 315 320

Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly

325 330 335

Glu Cys

<210> 15

<211> 451

<212> PRT

<213> Artificial sequence

<220>

<223> laboratory fabricated-synthesized wnt enhancer

<400> 15

Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Ser Leu Ser Leu Thr Cys Ser Val Thr Gly Tyr Ser Ile Thr Ser Gly

20 25 30

Tyr Tyr Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp

35 40 45

Met Gly Tyr Ile Ser Tyr Asp Gly Ser Asn Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Asn Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe

65 70 75 80

Leu Lys Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys

85 90 95

Val Arg Val Pro Thr Met Ile Thr Ser Tyr Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

355 360 365

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Lys

450

<210> 16

<211> 216

<212> PRT

<213> Artificial sequence

<220>

<223> laboratory fabricated-synthesized wnt enhancer

<400> 16

Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Ile Ser Cys Ser Ala Ser Ser Ser Val Gly Tyr Ile

20 25 30

Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr

35 40 45

Arg Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Tyr His Ser Tyr Pro Pro Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Arg Thr Val

100 105 110

Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys

115 120 125

Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg

130 135 140

Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn

145 150 155 160

Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser

165 170 175

Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys

180 185 190

Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr

195 200 205

Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 17

<211> 573

<212> PRT

<213> Artificial sequence

<220>

<223> laboratory fabricated-synthesized wnt enhancer

<400> 17

Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Ser Leu Ser Leu Thr Cys Ser Val Thr Gly Tyr Ser Ile Thr Ser Gly

20 25 30

Tyr Tyr Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp

35 40 45

Met Gly Tyr Ile Ser Tyr Asp Gly Ser Asn Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Asn Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe

65 70 75 80

Leu Lys Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys

85 90 95

Val Arg Val Pro Thr Met Ile Thr Ser Tyr Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

355 360 365

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Lys Gly Gly Gly Gly Ser Gly Ser Gly Gly Ser Gly Gly Gly

450 455 460

Gly Ser Asn Pro Ile Cys Lys Gly Cys Leu Ser Cys Ser Lys Asp Asn

465 470 475 480

Gly Cys Ser Arg Cys Gln Gln Lys Leu Phe Phe Phe Leu Arg Arg Glu

485 490 495

Gly Met Arg Gln Tyr Gly Glu Cys Leu His Ser Cys Pro Ser Gly Tyr

500 505 510

Tyr Gly His Arg Ala Pro Asp Met Asn Arg Cys Ala Arg Cys Arg Ile

515 520 525

Glu Asn Cys Asp Ser Cys Arg Ser Lys Asp Ala Cys Thr Lys Cys Lys

530 535 540

Val Gly Phe Tyr Leu His Arg Gly Arg Cys Phe Asp Glu Cys Pro Asp

545 550 555 560

Gly Phe Ala Pro Leu Glu Glu Thr Met Glu Cys Val Glu

565 570

<210> 18

<211> 443

<212> PRT

<213> Artificial sequence

<220>

<223> laboratory fabricated-synthesized wnt enhancer

<400> 18

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ala

1 5 10 15

Ser Val Thr Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe His Asp Tyr

20 25 30

Glu Ile His Trp Val Lys Gln Thr Pro Val Tyr Gly Leu Glu Trp Ile

35 40 45

Gly Ala Ile Asp Pro Glu Thr Gly Gly Thr Ala Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Lys Ala Tyr

65 70 75 80

Val Glu Phe Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Thr Ile Val Arg Gly Phe Trp Gly Gln Gly Thr Leu Val Thr Val Ser

100 105 110

Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser

115 120 125

Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp

130 135 140

Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr

145 150 155 160

Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr

165 170 175

Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln

180 185 190

Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp

195 200 205

Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro

210 215 220

Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro

225 230 235 240

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

245 250 255

Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn

260 265 270

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

275 280 285

Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

290 295 300

Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

305 310 315 320

Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys

325 330 335

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu

340 345 350

Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

355 360 365

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

370 375 380

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

385 390 395 400

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly

405 410 415

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

420 425 430

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440

<210> 19

<211> 222

<212> PRT

<213> Artificial sequence

<220>

<223> laboratory fabricated-synthesized wnt enhancer

<400> 19

Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Arg Ser Gly Gln Thr Ile Val His Ser

20 25 30

Asp Gly Asn Ile Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Ala Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Ile Pro Phe Thr Phe Gly Gly Gly Thr Glu Leu Glu Ile Lys

100 105 110

Arg Ala Asp Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro

115 120 125

Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu

130 135 140

Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn

145 150 155 160

Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser

165 170 175

Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala

180 185 190

Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly

195 200 205

Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<210> 20

<211> 565

<212> PRT

<213> Artificial sequence

<220>

<223> laboratory fabricated-synthesized wnt enhancer

<400> 20

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ala

1 5 10 15

Ser Val Thr Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe His Asp Tyr

20 25 30

Glu Ile His Trp Val Lys Gln Thr Pro Val Tyr Gly Leu Glu Trp Ile

35 40 45

Gly Ala Ile Asp Pro Glu Thr Gly Gly Thr Ala Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Lys Ala Tyr

65 70 75 80

Val Glu Phe Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Thr Ile Val Arg Gly Phe Trp Gly Gln Gly Thr Leu Val Thr Val Ser

100 105 110

Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser

115 120 125

Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp

130 135 140

Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr

145 150 155 160

Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr

165 170 175

Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln

180 185 190

Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp

195 200 205

Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro

210 215 220

Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro

225 230 235 240

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

245 250 255

Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn

260 265 270

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

275 280 285

Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

290 295 300

Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

305 310 315 320

Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys

325 330 335

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu

340 345 350

Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

355 360 365

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

370 375 380

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

385 390 395 400

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly

405 410 415

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

420 425 430

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser

435 440 445

Gly Ser Gly Gly Ser Gly Gly Gly Gly Ser Asn Pro Ile Cys Lys Gly

450 455 460

Cys Leu Ser Cys Ser Lys Asp Asn Gly Cys Ser Arg Cys Gln Gln Lys

465 470 475 480

Leu Phe Phe Phe Leu Arg Arg Glu Gly Met Arg Gln Tyr Gly Glu Cys

485 490 495

Leu His Ser Cys Pro Ser Gly Tyr Tyr Gly His Arg Ala Pro Asp Met

500 505 510

Asn Arg Cys Ala Arg Cys Arg Ile Glu Asn Cys Asp Ser Cys Arg Ser

515 520 525

Lys Asp Ala Cys Thr Lys Cys Lys Val Gly Phe Tyr Leu His Arg Gly

530 535 540

Arg Cys Phe Asp Glu Cys Pro Asp Gly Phe Ala Pro Leu Glu Glu Thr

545 550 555 560

Met Glu Cys Val Glu

565

<210> 21

<211> 444

<212> PRT

<213> Artificial sequence

<220>

<223> laboratory fabricated-synthesized wnt enhancer

<400> 21

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ser

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Thr Tyr

20 25 30

Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Gln Ile Tyr Pro Gly Asp Gly Asp Thr Tyr Tyr Asn Gly Asn Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Val Phe Trp Asp Gly Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val

100 105 110

Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser

115 120 125

Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys

130 135 140

Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu

145 150 155 160

Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu

165 170 175

Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr

180 185 190

Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val

195 200 205

Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

210 215 220

Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe

225 230 235 240

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

245 250 255

Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe

260 265 270

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

275 280 285

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

290 295 300

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

305 310 315 320

Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

325 330 335

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

340 345 350

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

355 360 365

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

370 375 380

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

385 390 395 400

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

405 410 415

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

420 425 430

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440

<210> 22

<211> 216

<212> PRT

<213> Artificial sequence

<220>

<223> laboratory fabricated-synthesized wnt enhancer

<400> 22

Gln Ile Val Leu Thr Gln Ser Pro Thr Ile Met Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Thr Tyr Ile

20 25 30

His Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr

35 40 45

Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala Arg Phe Gly Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Asn Ser Met Glu Thr Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Phe Thr

85 90 95

Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Arg Thr Val

100 105 110

Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys

115 120 125

Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg

130 135 140

Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn

145 150 155 160

Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser

165 170 175

Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys

180 185 190

Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr

195 200 205

Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 23

<211> 566

<212> PRT

<213> Artificial sequence

<220>

<223> laboratory fabricated-synthesized wnt enhancer

<400> 23

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ser

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Thr Tyr

20 25 30

Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Gln Ile Tyr Pro Gly Asp Gly Asp Thr Tyr Tyr Asn Gly Asn Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Val Phe Trp Asp Gly Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val

100 105 110

Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser

115 120 125

Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys

130 135 140

Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu

145 150 155 160

Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu

165 170 175

Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr

180 185 190

Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val

195 200 205

Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

210 215 220

Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe

225 230 235 240

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

245 250 255

Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe

260 265 270

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

275 280 285

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

290 295 300

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

305 310 315 320

Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

325 330 335

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

340 345 350

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

355 360 365

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

370 375 380

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

385 390 395 400

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

405 410 415

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

420 425 430

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly

435 440 445

Ser Gly Ser Gly Gly Ser Gly Gly Gly Gly Ser Asn Pro Ile Cys Lys

450 455 460

Gly Cys Leu Ser Cys Ser Lys Asp Asn Gly Cys Ser Arg Cys Gln Gln

465 470 475 480

Lys Leu Phe Phe Phe Leu Arg Arg Glu Gly Met Arg Gln Tyr Gly Glu

485 490 495

Cys Leu His Ser Cys Pro Ser Gly Tyr Tyr Gly His Arg Ala Pro Asp

500 505 510

Met Asn Arg Cys Ala Arg Cys Arg Ile Glu Asn Cys Asp Ser Cys Arg

515 520 525

Ser Lys Asp Ala Cys Thr Lys Cys Lys Val Gly Phe Tyr Leu His Arg

530 535 540

Gly Arg Cys Phe Asp Glu Cys Pro Asp Gly Phe Ala Pro Leu Glu Glu

545 550 555 560

Thr Met Glu Cys Val Glu

565

<210> 24

<211> 349

<212> PRT

<213> Artificial sequence

<220>

<223> laboratory fabricated-synthesized wnt enhancer

<400> 24

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

225 230 235 240

Gly Ser Asn Pro Ile Cys Lys Gly Cys Leu Ser Cys Ser Lys Asp Asn

245 250 255

Gly Cys Ser Arg Cys Gln Gln Lys Leu Phe Phe Phe Leu Arg Arg Glu

260 265 270

Gly Met Arg Gln Tyr Gly Glu Cys Leu His Ser Cys Pro Ser Gly Tyr

275 280 285

Tyr Gly His Arg Ala Pro Asp Met Asn Arg Cys Ala Arg Cys Arg Ile

290 295 300

Glu Asn Cys Asp Ser Cys Phe Ser Lys Asp Phe Cys Thr Lys Cys Lys

305 310 315 320

Val Gly Phe Tyr Leu His Arg Gly Arg Cys Phe Asp Glu Cys Pro Asp

325 330 335

Gly Phe Ala Pro Leu Glu Glu Thr Met Glu Cys Val Glu

340 345

<210> 25

<211> 348

<212> DNA

<213> Artificial sequence

<220>

<223> synthetic polynucleotide

<220>

<221> misc_feature

<222> (48)..(48)

<223> n is a, c, g or t

<220>

<221> misc_feature

<222> (63)..(63)

<223> n is a, c, g or t

<220>

<221> misc_feature

<222> (84)..(84)

<223> n is a, c, g or t

<400> 25

caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctggggcntc agtgaaggtt 60

tcntgcaagg catctggata cacnttcacc aactactata tgcactgggt gcgtcaggcc 120

cctggacaag ggcttgagtg gatgggatgg atcaacccta acagtggtgg cacaaattat 180

gcacagaagt ttcagggccg tgtcaccatg acccgcgaca cgtccacgag cacagtctac 240

atggagctga gcagcctgcg ttctgaggac acggccgtgt attactgtgc gagagggcac 300

tggtacttcg atctctgggg ccgtggcacc ctggtcaccg tctcctca 348

<210> 26

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> synthetic polynucleotide

<400> 26

gacatccgga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggccagtga gagtattagg agctggttgg cctggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctatggt gcatcgcgtt tgcaaagtgg ggtcccatca 180

aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240

gaagattttg caacttacta ctgtcaacag agttacagta ccccttggac gttcggccaa 300

ggtaccaagg tggaaatcaa a 321

<210> 27

<211> 369

<212> DNA

<213> Artificial sequence

<220>

<223> synthetic polynucleotide

<400> 27

gaggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc 60

tcctgcaagg cttctggata caccttcacc aaagactata tgcactgggt gcggcaggcc 120

cctggacaag ggcttgagtg gatgggaggg atcatcccta tatttggtac agcaaactac 180

gcacagaggt tccagggccg ggtcacgatt accgcggacg aatccacgag cacagcctac 240

atggagctga gcagcctgcg gtctgaggac acggccgtgt attactgtgc gagaggactc 300

ccaccagcag ctggtggcgg cggatacttc cagcactggg gccagggcac cctggtcacc 360

gtctcctca 369

<210> 28

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> synthetic polynucleotide

<400> 28

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggccagtca gaatgttaat gactggttgg cctggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctatagt gcatccaatt tgcaatctgg ggtcccatca 180

aggttcagtg gcagtggatc tgggacagat ttcactctca ccatccgcag tctgcaacct 240

gaagattttg caacttacta ctgtcaacag agctacagta ccccattcac tttcggccct 300

ggtaccaaag tggatatcaa a 321

<210> 29

<211> 354

<212> DNA

<213> Artificial sequence

<220>

<223> synthetic polynucleotide

<400> 29

gaggtccagc tggtgcagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60

tcctgtgcag cctctggatt caccttcagt agctatggca tgcactgggt ccgccaggct 120

ccaggcaagg ggctggagtg ggtggcagtt atatcatatg atggaagtaa taaatactat 180

gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctttat 240

ctgcaaatga acagcctcag agccgaggac acggccgtgt attactgtgc gggggacacc 300

tttggagtgg gacacttcta ctggggccag ggaaccctgg tcaccgtctc aagc 354

<210> 30

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> synthetic polynucleotide

<400> 30

gatgttgtga tgactcagtc tccactctcc ctgcccgtca cccctggaga gccggcctcc 60

atctcctgca ggtctagtca gagcctcctg catagtaatg gatacaacta tttggattgg 120

tacctgcaga agccagggca gtctccacag ctcctgatct atttgggttc taatcgggcc 180

tccggggtcc ctgacaggtt cagtggcagt ggatcaggca cagactttac actgcaaatc 240

agcagagtgg aggctgagga tgttggggtc tattactgca tgcaaggact tcacactccg 300

gtcactttcg gcggagggac caaggtggag atcaaa 336

<210> 31

<211> 366

<212> DNA

<213> Artificial sequence

<220>

<223> synthetic polynucleotide

<400> 31

caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc 60

tcctgcaagg cttctggagg caccttcagc agctctgtta tcagctgggt gcggcaggcc 120

cctggacaag ggcttgagtg gatgggatgg atcagtgttt acaatggtaa cacaaactat 180

gcagagaagt tccagggccg ggtcacgatt accgcggacg aatccacgag cacagcctac 240

atggagctga gcagcctgcg gtctgaggac acggccgtgt attactgtgc gagatttgct 300

atggttcggg gaggggtcta ctactttgac tactggggcc agggaaccct ggtcaccgtc 360

tcctca 366

<210> 32

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> synthetic polynucleotide

<400> 32

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcgagtca gggcattagc agttatttaa attggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240

gaagattttg caacttacta ctgtcaacat tattataatc tcccgctcac cttcggccaa 300

ggtacccgac tggagattaa a 321

<210> 33

<211> 116

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide

<400> 33

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly His Trp Tyr Phe Asp Leu Trp Gly Arg Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210> 34

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide

<400> 34

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Ile Arg Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Gly Ala Ser Arg Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 35

<211> 123

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide

<400> 35

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Lys Asp

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Arg Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Leu Pro Pro Ala Ala Gly Gly Gly Gly Tyr Phe Gln His

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 36

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide

<400> 36

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asn Val Asn Asp Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Asn Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Arg Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Phe

85 90 95

Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys

100 105

<210> 37

<211> 118

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide

<400> 37

Glu Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Gly Asp Thr Phe Gly Val Gly His Phe Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 38

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide

<400> 38

Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser

20 25 30

Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Gln Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Gly

85 90 95

Leu His Thr Pro Val Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 39

<211> 122

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide

<400> 39

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Ser

20 25 30

Val Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Ser Val Tyr Asn Gly Asn Thr Asn Tyr Ala Glu Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Phe Ala Met Val Arg Gly Gly Val Tyr Tyr Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 40

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide

<400> 40

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Tyr Tyr Asn Leu Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105

<210> 41

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide-CDRH 1

<400> 41

Tyr Thr Phe Thr Asn Tyr Tyr Met His

1 5

<210> 42

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide-CDRH 1

<400> 42

Tyr Thr Phe Thr Lys Asp Tyr Met His

1 5

<210> 43

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide-CDRH 1

<400> 43

Phe Thr Phe Ser Ser Tyr Gly Met His

1 5

<210> 44

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide-CDRH 1

<400> 44

Gly Thr Phe Ser Ser Ser Val Ile Ser

1 5

<210> 45

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide-CDRH 2

<400> 45

Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala

1 5 10

<210> 46

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide-CDRH 2

<400> 46

Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala

1 5 10

<210> 47

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide-CDRH 2

<400> 47

Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala

1 5 10

<210> 48

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide-CDRH 2

<400> 48

Gly Trp Ile Ser Val Tyr Asn Gly Asn Thr Asn Tyr Ala

1 5 10

<210> 49

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide-CDRH 3

<400> 49

Cys Ala Arg Gly His Trp Tyr Phe Asp Leu Trp

1 5 10

<210> 50

<211> 18

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide-CDRH 3

<400> 50

Cys Ala Arg Gly Leu Pro Pro Ala Ala Gly Gly Gly Gly Tyr Phe Gln

1 5 10 15

His Trp

<210> 51

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide-CDRH 3

<400> 51

Cys Ala Gly Asp Thr Phe Gly Val Gly His Phe Tyr Trp

1 5 10

<210> 52

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide-CDRH 3

<400> 52

Cys Ala Arg Phe Ala Met Val Arg Gly Gly Val Tyr Tyr Phe Asp Tyr

1 5 10 15

Trp

<210> 53

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide-CDRL 1

<400> 53

Arg Ala Ser Glu Ser Ile Arg Ser Trp Leu Ala

1 5 10

<210> 54

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide-CDRL 1

<400> 54

Arg Ala Ser Gln Asn Val Asn Asp Trp Leu Ala

1 5 10

<210> 55

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide-CDRL 1

<400> 55

Arg Ser Ser Gln Ser Leu Leu His Ser Asn Gly Tyr Asn Tyr Leu Asp

1 5 10 15

<210> 56

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide-CDRL 1

<400> 56

Arg Ala Ser Gln Gly Ile Ser Ser Tyr Leu Asn

1 5 10

<210> 57

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide-CDRL 2

<400> 57

Gly Ala Ser Arg Leu Gln Ser

1 5

<210> 58

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide-CDRL 2

<400> 58

Ser Ala Ser Asn Leu Gln Ser

1 5

<210> 59

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide-CDRL 2

<400> 59

Leu Gly Ser Asn Arg Ala Ser

1 5

<210> 60

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide-CDRL 2

<400> 60

Ala Ala Ser Ser Leu Gln Ser

1 5

<210> 61

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide-CDRL 3

<400> 61

Cys Gln Gln Ser Tyr Ser Thr Pro Trp Thr Phe

1 5 10

<210> 62

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide-CDRL 3

<400> 62

Cys Gln Gln Ser Tyr Ser Thr Pro Phe Thr Phe

1 5 10

<210> 63

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide-CDRL 3

<400> 63

Cys Met Gln Gly Leu His Thr Pro Val Thr Phe

1 5 10

<210> 64

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide-CDRL 3

<400> 64

Gln His Tyr Tyr Asn Leu Pro Leu Thr Phe

1 5 10