Pegylated L-Asparaginase
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)