Polypeptides that bound to IL-23 receptor and inhibit binding of IL-23 and cell signaling thereof

Abstract

The present invention relates to novel linear and cyclic polypeptides that bind to IL-23 receptor and inhibit the binding of IL-23 to its corresponding receptor and cell signaling thereof. The novel polypeptides of the present invention has a core structure of WX1X2X3W, where W is tryptophan, and X1, X2 and X3 are amino acids, with the proviso that when one of X1, X2 or X3 is W, the remaining two of X1, X2 or X3 cannot be W. Preferred core structures include WVDYW or WQDYW. The present invention relates a composition containing the novel polypeptides (linear or cyclic), and use of same in inhibiting cell functions including production of IL-22 and IL-17F from immune cells as well as in treating IL-23 associated human diseases including, for example, inflammatory bowel diseases, psoriasis and Crohn's disease, ulcerative colitis and multiple sclerosis.

What is claimed is: 
     
       1. An isolated cyclic polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 177, SEQ ID NO: 178 and SEQ ID NO: 179, wherein said isolated cyclic polypeptide inhibits binding of IL-23 to IL-23 receptor and inhibits IL-23-mediated cell signaling. 
     
     
       2. The isolated cyclic polypeptide of  claim 1 , wherein said isolated cyclic polypeptide consisting of SEQ ID NO: 177. 
     
     
       3. The isolated cyclic polypeptide of  claim 1 , wherein said isolated cyclic polypeptide consisting of SEQ ID NO: 178. 
     
     
       4. The isolated cyclic polypeptide of  claim 1 , wherein said isolated cyclic polypeptide consisting of SEQ ID NO: 179. 
     
     
       5. A method of inhibiting production of IL-17F in a Th17 cell in a human, comprising the steps of:
 a) providing a human Th17 cell in need of inhibiting production of IL-17F; and 
 b) exposing said Th17 cell to said isolated cyclic polypeptide of  claim 1 . 
 
     
     
       6. The method of  claim 5 , wherein said IL-17F is IL-17F protein. 
     
     
       7. The method of  claim 5 , wherein said production of IL-17F is assayed by an ELISA. 
     
     
       8. A method of inhibiting production of IL-17F in a splenocyte in a human, comprising the steps of:
 a) providing a human splenocyte in need of inhibiting production of IL-17F from said splenocyte; and 
 b) exposing said splenocyte to said isolated cyclic polypeptide of  claim 1 . 
 
     
     
       9. The method of  claim 8 , wherein said IL-17F is IL-17F mRNA. 
     
     
       10. The method of  claim 8 , wherein said production of IL-17F is assayed by RT-PCR. 
     
     
       11. A method of inhibiting production of IL-22 from a mononuclear cell in a human, comprising the steps of:
 a) providing a human mononuclear cell in need of inhibiting production of IL-22 from said mononuclear cell; and 
 b) exposing said mononuclear cell to said isolated cyclic polypeptide of  claim 1 . 
 
     
     
       12. The method of  claim 11 , wherein said IL-22 is IL-22 protein. 
     
     
       13. The method of  claim 11 , wherein said production of IL-22 is assayed by an ELISA. 
     
     
       14. The method of claim 11, wherein said isolated cyclic polypeptide is SEQ ID NO: 177. 
     
     
       15. The method of claim 11, wherein said isolated cyclic polypeptide is SEQ ID NO: 178. 
     
     
       16. The method of claim 11, wherein said isolated cyclic polypeptide is SEQ ID NO: 179. 
     
     
       17. A method of identifying a polypeptides that inhibits binding of IL-23 to IL-23 receptor on a cell, comprising
 screening a phage display library of phage displaying candidate polypeptides for ability to bind a protein comprising a fragment of Δ9 IL-23 receptor, wherein the fragment consists of an amino acid sequence selected from amino acids 1-250 of Δ9 IL-23 receptor and amino acids 24-250 of Δ9 IL-23 receptor sequence,   selecting a candidate polypeptide that was determined to have an ability to bind the protein by said screening; and   determining the ability of the candidate polypeptide from said selecting to inhibit binding of IL-23 to IL-23 receptor.   
     
     
       18. The method according to claim 17, wherein said candidate polypeptides comprise 12 to 18 amino acids. 
     
     
       19. The method according to claim 17, wherein said protein further comprises a Flag protein. 
     
     
       20. The method according to claim 19, wherein said Flag protein has the sequence of SEO ID NO: 175. 
     
     
       21. The method according to claim 19, wherein said method further comprises capturing the polypeptide that interacts with the protein with an anti-Flag affinity gel. 
     
     
       22. The method according to claim 17, wherein said protein further comprises an Fc chimera. 
     
     
       23. The method according to claim 22, wherein the Fc chimera comprises an Fc region of human IgG 1 . 
     
     
       24. The method according to claim 22, wherein said method further comprises capturing the polypeptide that interacts with the protein with protein A Sepharose. 
     
     
       25. The method according to claim 17, further comprising conducting a binding assay to detect binding between candidate polypeptides from said selecting to verify said candidate polypeptide binds the IL-23 receptor. 
     
     
       26. The method according to claim 25, wherein said binding assay is by immunoprecipitation. 
     
     
       27. The method according to claim 25, wherein said binding assay is an ELISA assay. 
     
     
       28. The method according to claim 17, wherein said determining the ability of the candidate polypeptide from said selecting to inhibit the binding of IL-23 to IL-23 receptor is by competitive ELISA. 
     
     
       29. The method according to claim 17, further comprising sequencing candidate polypeptide determined to inhibit the binding of IL-23 to IL-23 receptor. 
     
     
       30. The method according to claim 17, wherein the binding of IL-23 is via the p19 subunit of IL-23 cytokine. 
     
     
       31. The method of claim 5, wherein said isolated cyclic polypeptide is SEQ ID NO: 177. 
     
     
       32. The method of claim 5, wherein said isolated cyclic polypeptide is SEQ ID NO: 178. 
     
     
       33. The method of claim 5, wherein said isolated cyclic polypeptide is SEQ ID NO: 179. 
     
     
       34. The method of claim 8, wherein said isolated cyclic polypeptide is SEQ ID NO: 177. 
     
     
       35. The method of claim 8, wherein said isolated cyclic polypeptide is SEQ ID NO: 178. 
     
     
       36. The method of claim 8, wherein said isolated cyclic polypeptide is SEQ ID NO: 179.