Neublastin variants

Abstract

Variant Neublastin polypeptides having substitutions at selected amino acid residues are disclosed. Substitution at one or more selected amino acid residues decreases heparin binding and increases serum exposure of variant Neublastin polypeptides. Also disclosed are methods of using variant Neublastin polypeptides to treat disorders and activate the RET receptor in a mammal.

What is claimed is: 
     
         1 . A polypeptide comprising an amino acid sequence at least 80% identical to amino acids 15-113 of SEQ ID NO:1, wherein the amino acid sequence comprises at least one amino acid substitution, relative to SEQ ID NO:1, selected from the group consisting of:
 an amino acid other than arginine at the position corresponding to position 48 of SEQ ID NO:1;   an amino acid other than arginine at the position corresponding to position 49 of SEQ ID NO:1; and   an amino acid other than arginine at the position corresponding to position 51 of SEQ ID NO:1,   wherein the polypeptide, when dimerized, binds to a complex containing GFRalpha3 and RET.   
     
     
         2 - 16 . (canceled) 
     
     
         17 . A polypeptide comprising amino acids 15-113 of SEQ ID NO:2, amino acids 15-113 of SEQ ID NO:3, amino acids 15-113 of SEQ ID NO:4, amino acids 15-113 of SEQ ID NO:5, amino acids 15-113 of SEQ ID NO:8, or amino acids 15-113 of SEQ ID NO:9. 
     
     
         18 - 19 . (canceled) 
     
     
         20 . A dimer comprising two polypeptides according to  claim 1 . 
     
     
         21 . A conjugate comprising the polypeptide of  claim 1  conjugated to a non-naturally occurring polymer. 
     
     
         22 . A fusion protein comprising the polypeptide of  claim 1  and a heterologous amino acid sequence. 
     
     
         23 . A pharmaceutical composition comprising the polypeptide of  claim 1  a pharmaceutically acceptable carrier or excipient. 
     
     
         24 . A nucleic acid comprising a sequence that encodes the polypeptide of  claim 1 . 
     
     
         25 . An expression vector comprising the nucleic acid of  claim 24 . 
     
     
         26 . A cell comprising the expression vector of  claim 25 . 
     
     
         27 . A method of making a polypeptide, the method comprising:
 providing the cell of  claim 26 , and   culturing the cell under conditions that permit expression of the nucleic acid.   
     
     
         28 . A method of treating or preventing a nervous system disorder in a mammal, the method comprising administering to the mammal a therapeutically effective amount of the pharmaceutical composition of  claim 23 . 
     
     
         29 . A method of treating neuropathic pain in a mammal, the method comprising administering to the mammal a therapeutically effective amount of the pharmaceutical composition of  claim 23 . 
     
     
         30 . A method of activating the RET receptor in a mammal, the method comprising administering to the mammal an effective amount of the polypeptide of  claim 1 . 
     
     
         31 . A polypeptide comprising an amino acid sequence at least 80% identical to amino acids 15-113 of SEQ ID NO:1, wherein the amino acid sequence comprises at least one amino acid substitution, relative to SEQ ID NO:1, selected from the group consisting of:
 an amino acid other than serine at the position corresponding to position 20 of SEQ ID NO:1;   an amino acid other than glutamine at the position corresponding to position 21 of SEQ ID NO:1;   an amino acid other than histidine at the position corresponding to position 32 of SEQ ID NO:1;   an amino acid other than arginine at the position corresponding to position 33 of SEQ ID NO:1;   an amino acid other than arginine at the position corresponding to position 39 of SEQ ID NO:1;   an amino acid other than serine at the position corresponding to position 46 of SEQ ID NO:1;   an amino acid other than arginine at the position corresponding to position 68 of SEQ ID NO:1;   an amino acid other than glycine at the position corresponding to position 72 of SEQ ID NO:1;   an amino acid other than serine at the position corresponding to position 73 of SEQ ID NO:1; and   an amino acid other than valine at the position corresponding to position 94 of SEQ ID NO:1,   wherein the polypeptide, when dimerized, binds to a complex containing GFRalpha3 and RET.   
     
     
         32 - 34 . (canceled) 
     
     
         35 . A polypeptide comprising an amino acid sequence at least 80% identical to SEQ ID NO:1, wherein the amino acid sequence comprises at least one amino acid substitution, relative to SEQ ID NO:1, selected from the group consisting of:
 an amino acid other than arginine at the position corresponding to position 7 of SEQ ID NO:1;   an amino acid other than arginine at the position corresponding to position 9 of SEQ ID NO:1; and   an amino acid other than arginine at the position corresponding to position 14 of SEQ ID NO:1,   wherein the polypeptide, when dimerized, binds to a complex containing GFRalpha3 and RET.   
     
     
         36 - 47 . (canceled) 
     
     
         48 . A dimer comprising two polypeptides according to  claim 31 . 
     
     
         49 . A conjugate comprising the polypeptide of  claim 31  conjugated to a non-naturally occurring polymer. 
     
     
         50 . A fusion protein comprising the polypeptide of  claim 31  and a heterologous amino acid sequence. 
     
     
         51 . A pharmaceutical composition comprising the polypeptide of  claim 31  and a pharmaceutically acceptable carrier or excipient. 
     
     
         52 . A nucleic acid comprising a sequence that encodes the polypeptide of  claim 31 . 
     
     
         53 . An expression vector comprising the nucleic acid of  claim 52 . 
     
     
         54 . A cell comprising the expression vector of  claim 53 . 
     
     
         55 . A method of making a polypeptide, the method comprising:
 providing the cell of  claim 54 , and   culturing the cell under conditions that permit expression of the nucleic acid.   
     
     
         56 . A method of treating or preventing a nervous system disorder in a mammal, the method comprising administering to the mammal a therapeutically effective amount of the pharmaceutical composition of  claim 51 . 
     
     
         57 . A method of treating neuropathic pain in a mammal, the method comprising administering to the mammal a therapeutically effective amount of the pharmaceutical composition of  claim 51 . 
     
     
         58 . A method of activating the RET receptor in a mammal, the method comprising administering to the mammal an effective amount of the polypeptide of  claim 31 .