Modified L-Asparaginase

Abstract

The disclosure provides a modified protein that is a combination of (i) an L-asparaginase and (ii) one or more (poly)peptide(s), wherein the (poly)peptide consists solely of proline and alanine amino acid residues, and methods of preparation and use thereof.

1 . A modified protein comprising (i) an L-asparaginase and (ii) one or more polypeptides, wherein the one or more polypeptides consists solely of proline and alanine amino acid residues. 
     
     
         2 . The modified protein according to  claim 1 , wherein the L-asparaginase has at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 1. 
     
     
         3 . The modified protein according to  claim 1 , wherein the L-asparaginase has at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1. 
     
     
         4 . The modified protein according to  claim 1 , wherein the L-asparaginase has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 1. 
     
     
         5 . The modified protein according to  claim 1 , wherein the L-asparaginase comprises the amino acid sequence of SEQ ID NO: 1. 
     
     
         6 . The modified protein according to  claim 1 , wherein the L-asparaginase consists of the amino acid sequence of SEQ ID NO: 1. 
     
     
         7 . The modified protein according to  claim 1 , wherein the modified protein is a fusion protein of the L-asparaginase and the polypeptide. 
     
     
         8 . The modified protein according to  claim 7 , wherein polypeptide consists of 100 to 600 proline and alanine amino acid residues. 
     
     
         9 . The modified protein according to  claim 7 , wherein the polypeptide consists of 200 to 400 proline and alanine amino acid residues, and the proline amino acid residues constitute more than 10% and less than 70% of the polypeptide. 
     
     
         10 . The modified protein according to  claim 7 , wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 5. 
     
     
         11 . The modified protein according to  claim 7 , wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 7. 
     
     
         12 . The modified protein according to  claim 7 , wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 9. 
     
     
         13 . The modified protein according to  claim 7 , wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 11. 
     
     
         14 . The modified protein according to  claim 7 , wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 13. 
     
     
         15 . The modified protein according to  claim 7 , wherein the L-asparaginase comprises the amino acid sequence of SEQ ID NO: 1 and the polypeptide comprises the amino acid sequence of SEQ ID NO: 5, 7, 9, 11 or 13. 
     
     
         16 . The modified protein according to  claim 1 , wherein each of the one or more polypeptides is independently a peptide R N -(P/A)-R c ,
 wherein (P/A) is an amino acid sequence consisting solely of proline and alanine amino acid residues,   R N  is a protecting group attached to the N-terminal amino group of the amino acid sequence, and   R C  is an amino acid residue bound via its amino group to the C-terminal carboxy group of the amino acid sequence,   the each of the one or more polypeptides is conjugated to the L-asparaginase via an amide linkage formed from the carboxy group of the C-terminal amino acid residue R c  of the peptide and a free amino group of the L-asparaginase, and   at least one of the free amino groups, which the one or more polypeptide(s) are conjugated to, is not an N-terminal α-amino group of the L-asparaginase.   
     
     
         17 . The modified protein according to  claim 16 , wherein the each of the one or more polypeptides independently consists of 15 to 45 proline and alanine amino acid residues. 
     
     
         18 . The modified protein according to  claim 16 , wherein the each of the one or more polypeptides independently consists of 20 to 40 proline and alanine amino acid residues, and the proline amino acid residues constitute more than 10% and less than 70% of the amino acid sequence. 
     
     
         19 . The modified protein according to  claim 16 , wherein at least one of the one or more polypeptides comprises the amino acid sequence of SEQ ID NO: 5. 
     
     
         20 . The modified protein according to  claim 16 , wherein at least one of the one or more polypeptides comprises the amino acid sequence of SEQ ID NO: 15. 
     
     
         21 . The modified protein according to  claim 16 , wherein
 R N  is pyroglutamoyl or acetyl, and/or   R C  is ε-aminohexanoic acid.   
     
     
         22 . The modified protein according to  claim 16 ,
 wherein (i) at least one of the free amino groups, which the one or more polypeptides are conjugated to, is an ε-amino group of a lysine residue of the L-asparaginase; or   (ii) the free amino groups, which the one or more polypeptides are conjugated to, are selected from the group consisting of the ε-amino group(s) of any lysine residue(s) of the L-asparaginase and the N-terminal α-amino group(s) of the L-asparaginase.   
     
     
         23 . The modified protein according to  claim 16 , wherein
 the L-asparaginase is composed of four subunits, and   9 to 13 polypeptides are conjugated to each subunit of the L-asparaginase.   
     
     
         24 . The modified protein according to  claim 16 , wherein the L-asparaginase comprises the amino acid sequence of SEQ ID NO: 1 and the one or more polypeptides comprise the amino acid sequence of SEQ ID NO: 5 or 15. 
     
     
         25 . A pharmaceutical composition comprising the modified protein according to  claim 1  and one or more pharmaceutical acceptable carriers or excipients. 
     
     
         26 . The pharmaceutical composition of  claim 25 , wherein the one or more polypeptides is present in an amount sufficient to mediate a decreased immunogenicity of the modified protein following administration to a human subject. 
     
     
         27 . The pharmaceutical composition of  claim 25 , wherein the one or more polypeptides is present in an amount sufficient to increase the plasma half-life of the L-asparaginase following administration to a human subject.