Analogs of cationic proteins

Abstract

The in vivo circulating life and/or absorption of cationic therapeutic proteins, including but not limited to basic proteins such as NT-3 and BDNF, can be increased by generating analogs that have a lower isoelectric point and, preferably, also a lower protein charge relative to the protein of native sequence.

1 . A polypeptide analog of a cationic polypeptide wherein the analog has an amino acid sequence that differs from the native sequence of the original polypeptide by one or more amino acid residues, or by chemical modification of one or more amino acid residues in the native sequence, such that the isoelectric point is lower and the in vivo circulating life and/or absorption is increased for the analog relative to those same properties in the unmodified cationic polypeptide.  
     
     
         2 . A polypeptide according to  claim 1 , which is also characterized by a lower charge under physiological conditions compared to the unmodified cationic polypeptide.  
     
     
         3 . A polypeptide according to  claim 1 , which is an analog of a cationic protein selected from the group consisting of neurotrophic factor-3 (NT-3), brain derived neurotrophic factor (BDNF), macrophage growth and differentiation factor (MGDF) and keratinocyte growth factor (KGF).  
     
     
         4 . A polypeptide according to  claim 3 , which is an analog of NT-3.  
     
     
         5 . A polypeptide according to  claim 4 , having the amino acid sequence of SEQ ID NO: 3.  
     
     
         6 . A polypeptide according to  claim 4 , having the amino acid sequence of SEQ ID NO: 5.  
     
     
         7 . A polypeptide according to  claim 4 , having the amino acid sequence of SEQ ID NO: 6.  
     
     
         8 . A polypeptide according to  claim 4 , having the amino acid sequence of SEQ ID NO: 7.  
     
     
         9 . A DNA molecule encoding a polypeptide analog of a cationic polypeptide wherein the analog has an amino acid sequence that differs from the native sequence of the original polypeptide by one or more amino acid residues, or by chemical modification of one or more amino acid residues in the native sequence, such that the isoelectric point is lower and the in vivo circulating life and/or absorption is increased for the analog relative to those same properties in the unmodified cationic polypeptide.  
     
     
         10 . A DNA molecule according to  claim 9  which encodes an analog of a cationic protein selected from the group consisting of NT-3, BDNF, MGDF and KGF.  
     
     
         11 . A DNA molecule according to  claim 10  which encodes a polypeptide analog of NT-3.  
     
     
         12 . A DNA molecule according to  claim 11  which encodes the polypeptide of SEQ ID NO: 3.  
     
     
         13 . A DNA molecule according to  claim 11  which encodes the polypeptide of SEQ ID NO: 5.  
     
     
         14 . A DNA molecule according to  claim 11  which has the nucleic acid sequence of SEQ ID NO: 2.  
     
     
         15 . A DNA molecule according to  claim 11  which has the nucleic acid sequence of SEQ ID NO: 4.  
     
     
         16 . A biologically functional expression vector which includes a DNA molecule according to  claim 9  operatively linked to expression regulatory sequences.  
     
     
         17 . A prokaryotic or eukaryotic host cell transformed or transfected with an expression vector according to  claim 16  in a manner allowing the host cell to express the polypeptide encoded by the DNA molecule.  
     
     
         18 . A transformed or transfected bacterial host cell according to  claim 17 .  
     
     
         19 . A transformed or transfected  E. coli  host cell according to  claim 18 .  
     
     
         20 . A transformed or transfected mammalian host cell according to  claim 17 .  
     
     
         21 . A transformed or transfected CHO cell according to  claim 20 .  
     
     
         22 . A transformed or transfected COS cell according to  claim 20 .  
     
     
         23 . A process for the production of a polypeptide analog of a cationic polypeptide, wherein the analog has an amino acid sequence that differs from the native sequence of the original polypeptide by one or more amino acid residues, such that the isoelectric point is lower and the in vivo circulating life and/or absorption is increased for the analog relative to those same properties in the unmodified cationic polypeptide, said process comprising culturing under suitable nutrient conditions a prokaryotic or eukaryotic host cell transformed or transfected with an expression vector comprising a DNA molecule encoding said polypeptide in a manner allowing the host cell to express the polypeptide, and optionally isolating the polypeptide product of the expression.  
     
     
         24 . A process according to  claim 23 , in which the polypeptide is an analog of NT-3.  
     
     
         25 . A process according to  claim 23  in which the DNA molecule has been prepared by site directed mutagenesis.  
     
     
         26 . A process according to  claim 23 , in which the analog has the amino acid sequence of SEQ ID NO: 3.  
     
     
         27 . A process according to  claim 23 , in which the analog has the amino acid sequence of SEQ ID NO: 5.  
     
     
         28 . A process according to  claim 23 , in which the analog has the amino acid sequence of SEQ ID NO: 6.  
     
     
         29 . A process according to  claim 23 , in which the analog has the amino acid sequence of SEQ ID NO: 7.  
     
     
         30 . A process according to  claim 23 , in which the host cell is bacterial.  
     
     
         31 . A process according to  claim 30 , in which the bacterial host cell is  E. coli.    
     
     
         32 . A polypeptide product of expression in a eukaryotic or prokaryotic host cell of a DNA molecule according to  claim 9 .  
     
     
         33 . An antibody against a polypeptide according to  claim 1 .  
     
     
         34 . An antibody according to  claim 33  which is polyclonal.  
     
     
         35 . An antibody according to  claim 34  which is monoclonal.  
     
     
         36 . A pegylated derivative of a polypeptide according to  claim 1 .  
     
     
         37 . A pharmaceutical composition comprising a therapeutically effective amount of a polypeptide according to  claim 1  and a pharmaceutically acceptable carrier or diluent.  
     
     
         38 . A method for the treatment of peripheral neuropathies, comprising administering to a patient having the disorder a therapeutically effective amount of a polypeptide according to  claim 4.