Pegylated L-asparaginase

Abstract

Disclosed is a conjugate of a protein having substantial L-asparagine aminohydrolase activity and polyethylene glycol. In particular, the polyethylene glycol has a molecular weight less than or equal to about 5000 Da and the protein is an L-asparaginase from Erwinia. The conjugate of the invention has shown superior properties such as maintenance of a high level of in vitro activity and an unexpected increase in half-life in vivo. Also disclosed are methods of producing the conjugate and use of the conjugate in therapy. In particular, a method is disclosed for use of the conjugate in the treatment of cancer, particularly Acute Lymphoblastic Leukemia (ALL). More specifically, a method is disclosed for use of the conjugate as a second line therapy for patients who have developed hypersensitivity or have had a disease relapse after treatment with other L-asparaginase preparations.

What is claimed is: 
     
       1. A conjugate comprising an L-asparaginase monomer from Erwinia chrysanthemi having at least 90% 95% sequence identity to the amino acid of SEQ ID NO:1 and conjugated to at least one polyethylene glycol (PEG) molecule, wherein the at least one PEG molecule has a molecular weight less than 5000, 4000, 3000, or 2500 Da, wherein the at least one PEG molecule is covalently linked to at least about 60% of the accessible amino groups of said L-asparaginase monomer, and the conjugate has at least fifty twenty times more in vivo plasma L-asparaginase activity compared to an Erwinia chrysanthemi L-asparaginase not conjugated to PEG. 
     
     
       2. The conjugate of  claim 1 , wherein said L-asparaginase has at least 95% to 99% sequence identity to the amino acid of SEQ ID NO: 1. 
     
     
       3. The conjugate of  claim 1 , wherein said L-asparaginase comprises the amino acid sequence of SEQ ID NO: 1. 
     
     
       4. The conjugate of  claim 1 , wherein the PEG is covalently linked to one or more amino groups of said L-asparaginase. 
     
     
       5. The conjugate of  claim 4 , wherein the PEG molecules are covalently linked to at least from about 40% to about 100% of the accessible amino groups of said L-asparaginase. 
     
     
       6. The conjugate of  claim 1  having the formula:
   Asp-[NH—CO—(CH 2 )x-CO—NH-PEG]n
 
 wherein Asp is the L-asparaginase, NH is one or more of the NH groups of the lysine residues and/or the N-terminus in the Asp, PEG is a polyethylene glycol moiety, n is a number that represents at least 40% to 100% 60% of the accessible amino groups in the Asp, and x is an integer ranging from 1 to 8. 
 
     
     
       7. The conjugate of  claim 1 , wherein said at least one PEG is molecule comprises monomethoxy-polyethylene glycol. 
     
     
       8. The conjugate of  claim 1 , wherein said conjugate has at least sixty, seventy, eighty, ninety or one-hundred thirty times more in vivo plasma L-asparaginase activity compared to an Erwinia chrysanthemi L-asparaginase not conjugated to PEG. 
     
     
       9. The conjugate of  claim 1 , wherein the conjugate has increased residual enzymatic activity in vivo when compared to Erwinia chrysanthemi L-asparaginase conjugated to at least one PEG molecule with a molecular weight of 10,000 Da. 
     
     
       10. The conjugate of  claim 1 , wherein the L-asparaginase does not cross-react with antibodies which react with Escherichia coli L-asparaginase. 
     
     
       11. The conjugate of  claim 1  comprising an L-asparaginase monomer from Erwinia chrysanthemi consisting of the amino acid of SEQ ID NO: 1 and the conjugate has reduced immunogenicity when administered to a human subject hypersensitive to Escherichia coli L-asparaginase. 
     
     
       12. The conjugate of  claim 1 , wherein the conjugate has an in vitro L-asparaginase activity of at least 60%, 70%, 80%, or 87% of an L-asparaginase not conjugated to PEG. 
     
     
       13. The conjugate of  claim 1 , wherein said PEG has a molecular weight of less than 5000 Da. 
     
     
       14. The conjugate of  claim 1 , wherein the PEG molecules are covalently linked to at least 60%, 70%, 80%, 90% or 100% of the accessible amino groups of said L-asparaginase. 
     
     
       15. The conjugate of claim 1, wherein the at least one PEG molecule is covalently linked to at least about 70% of the accessible amino groups of said L-asparaginase monomer. 
     
     
       16. The conjugate of claim 1, wherein the at least one PEG molecule is covalently linked to at least about 80% of the accessible amino groups of said L-asparaginase monomer. 
     
     
       17. The conjugate of claim 1, wherein the at least one PEG molecule is covalently linked to at least about 90% of the accessible amino groups of said L-asparaginase monomer. 
     
     
       18. The conjugate of claim 1, wherein the at least one PEG molecule is covalently linked to about 100% of the accessible amino groups of said L-asparaginase monomer. 
     
     
       19. The conjugate of claim 1, wherein said L-asparaginase monomer has 100% sequence identity to the amino acid sequence of SEO ID NO: 1. 
     
     
       20. The conjugate of claim 1, wherein said conjugate has at least forty times more in vivo L-asparaginase activity compared to an Erwinia chrysanthemi L-asparaginase not conjugated to PEG. 
     
     
       21. The conjugate of claim 1, wherein said conjugate has at least fifty times more in vivo L-asparaginase activity compared to an Erwinia chrysanthemi L-asparaginase not conjugated to PEG. 
     
     
       22. The conjugate of claim 1, wherein the conjugate has an in vitro L-asparaginase activity of at least 70% of an L-asparaginase not conjugated to PEG. 
     
     
       23. The conjugate of claim 1, wherein the conjugate has an in vitro L-asparaginase activity of at least 80% of an L-asparaginase not conjugated to PEG. 
     
     
       24. The conjugate of claim 1, wherein the conjugate has an in vitro L-asparaginase activity of at least 87% of an L-asparaginase not conjugated to PEG. 
     
     
       25. The conjugate of claim 1, wherein said at least one PEG molecule has a molecular weight of between about 500 Da and about 3500 Da. 
     
     
       26. The conjugate of claim 1, wherein the at least one PEG molecule has a molecular weight of between about 500 Da and about 2500 Da. 
     
     
       27. The conjugate of claim 1, wherein the conjugate is a homotetramer comprising four L-asparaginase monomers. 
     
     
       28. The conjugate of claim 1, wherein the at least one PEG molecule has a molecular weight less than 4000, 3000, or 2500 Da.