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.

1 . A conjugate comprising an L-asparaginase from  Erwinia  having at least 80% identity to the amino acid of SEQ ID NO: 1 and polyethylene glycol (PEG), wherein the PEG has a molecular weight less than or equal to about 5000 Da. 
     
     
         2 . The conjugate of  claim 1 , wherein said L-asparaginase has at least from 90% to 99% identity to the amino acid of SEQ ID NO: 1. 
     
     
         3 . (canceled) 
     
     
         4 . The conjugate of  claim 1 , wherein said L-asparaginase comprises the amino acid sequence of SEQ ID NO: 1. 
     
     
         5 . The conjugate of  claim 1 , wherein said PEG has a molecular weight of less than or equal to about 5000 Da. 
     
     
         6 - 30 . (canceled) 
     
     
         31 . The conjugate of  claim 1  wherein the PEG is covalently linked to one or more amino of said L-asparaginase. 
     
     
         32 . The conjugate of  claim 31 , wherein the PEG is covalently linked to said one or more amino groups by an amide bond. 
     
     
         33 . The conjugate of  claim 31 , wherein the PEG is covalently linked to at least from about 40% to about 100% of the accessible amino groups. 
     
     
         34 . (canceled) 
     
     
         35 . The conjugate of  claim 1  having the formula:
   Asp-[NH—CO—(CH2) 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 about 100% of the accessible amino groups in the Asp, and x is an integer ranging from 1 to 8. 
 
     
     
         36 - 38 . (canceled) 
     
     
         39 . The conjugate of  claim 1 , wherein said PEG is monomethoxy-polyethylene glycol. 
     
     
         40 . A method of making the conjugate of  claim 1 , said method comprising combining an amount of said PEG with an amount of said L-asparaginase in a buffered solution for a time period sufficient to covalently link said PEG to said L-asparaginase. 
     
     
         41 . The method of  claim 40 , wherein said buffered solution has a pH value of between about 7.0 and about 9.0. 
     
     
         42 . (canceled) 
     
     
         43 . The method of  claim 40 , wherein the amount of said L-asparaginase is a protein concentration of between about 0.5 mg/mL and about 25 mg/mL. 
     
     
         44 - 45 . (canceled) 
     
     
         46 . The method of  claim 40 , wherein the amount of said PEG is a molar excess of polymer over amino groups in said L-asparaginase of less than about 20:1. 
     
     
         47 - 48 . (canceled) 
     
     
         49 . The method of  claim 40 , wherein said PEG is monomethoxy-polyethylene glycol. 
     
     
         50 . A method of treating a disease treatable by L-asparagine depletion in a patient, said method comprising administering to said patient an effective amount of the conjugate of  claim 1 . 
     
     
         51 . The method of  claim 50 , wherein said disease treatable by L-asparagine depletion is a cancer. 
     
     
         52 . The method of  claim 51 , wherein said cancer is selected from the group consisting of Acute Lymphoblastic Leukemia (ALL), non-Hodgkin's lymphoma, NK lymphoma, and pancreatic cancer. 
     
     
         53 . The method of  claim 52 , wherein said cancer is ALL. 
     
     
         54 . The method of  claim 53 , wherein said conjugate is administered at an amount of about 5 U/kg to about 25 U/kg. 
     
     
         55 . (canceled) 
     
     
         56 . The method of  claim 53 , wherein said conjugate is administered in a dose that depletes L-asparagine to undetectable levels for a period of about 3 days to about 10 days. 
     
     
         57 - 59 . (canceled) 
     
     
         60 . The method of  claim 50 , wherein said conjugate is administered intravenously or intramuscularly. 
     
     
         61 . (canceled) 
     
     
         62 . The method of  claim 50 , wherein said conjugate is administered once or twice per week. 
     
     
         63 . (canceled) 
     
     
         64 . The method of  claim 50 , wherein said conjugate is administered less than once per week. 
     
     
         65 . The method of  claim 50 , wherein said conjugate is administered as monotherapy. 
     
     
         66 . The method of  claim 65 , wherein said conjugate is not administered with an asparagine synthetase inhibitor. 
     
     
         67 . The method of  claim 50 , wherein said patient has had a previous hypersensitivity to an L-asparaginase selected from the group consisting of an  E. coli  L-asparaginase,  Erwinia  L-asparaginase and PEGylated form thereof. 
     
     
         68 . (canceled) 
     
     
         69 . The method of  claim 67 , wherein said hypersensitivity is selected from the group consisting of allergic reaction, anaphylactic shock, and silent hypersensitivity. 
     
     
         70 . The method of  claim 50 , wherein said patient has had a disease relapse. 
     
     
         71 . The method of  claim 70 , wherein said disease relapse occurs after treatment with an  E. coli  L-asparaginase or PEGylated form thereof. 
     
     
         72 . A pharmaceutical composition comprising the conjugate of  claim 1 . 
     
     
         73 - 94 . (canceled)