Butyrylcholinesterase zwitterionic polymer conjugates
The present invention provides recombinant butyrylcholinesterase fusion proteins, including Fc fusion proteins and multi-armed high MW polymers containing hydrophilic groups conjugated to the fusion proteins, and methods of preparing such polymers.
1. A conjugate comprising a butyrylcholinesterase covalently bound to a zwitterionic polymer wherein the polymer comprises one or more types of monomer units and wherein at least one type of monomer unit comprises a zwitterionic group, wherein the butyrylcholinesterase is fused to an immunoglobulin (Ig) domain as fusion protein.
2. A conjugate according to claim 1 wherein the Ig domain is IgG-Fc (SEQ ID NO: 8).
3. A conjugate according to claim 2 wherein the Fc domain further comprises amino acid substitutions in one or more of the positions selected from the group consisting of E13, L14, L15, G16, G17, A107, A110, and P111 as numbered within SEQ ID NO: 8.
4. A conjugate according to claim 1 further comprising a linker between the butyrylcholinesterase and the Ig domain, wherein the linker is selected from the group consisting of G, GG, GGGGS (SEQ ID NO: 22), GGGS (SEQ ID NO: 23), and GGGES (SEQ ID NO: 24), and oligomers of the foregoing.
5. A conjugate according to claim 4 wherein the linker is selected from the group consisting of GGGSGGGSGGGS (SEQ ID NO: 21) and GGSGGGSGGGS (SEQ ID NO: 25).
6. A conjugate according to claim 1 wherein the butyrylcholinesterase enzyme comprises or consists of amino acids 29-562 of SEQ ID NO: 1.
7. A conjugate according to claim 1 wherein the butyrylcholinesterase enzyme comprises amino acids 29-562 of SEQ ID NO: 1, except that residue C94 of SEQ ID NO: 1 is Y.
8. A conjugate according to claim 1 , wherein the zwitterionic group comprises phosphorylcholine.
9. A conjugate according to claim 8 wherein the at least one type of monomer unit comprises 2-(methacryloyloxyethyl)-2′-(trimethylammoniumethyl) phosphate (HEMA-PC).
10. A conjugate according to claim 8 wherein the at least one type of monomer unit comprises 2-(acryloyloxyethyl)-2′-(trimethylammoniumethyl) phosphate.
11. A conjugate according to claim 10 wherein the polymer has 3 or more arms.
12. A conjugate according to claim 10 wherein the polymer has 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 arms.
13. A conjugate according to claim 12 wherein the polymer has 3, 6 or 9 arms.
14. A conjugate according to claim 13 wherein the polymer has 9 arms.
15. A conjugate according to claim 12 wherein the polymer has a peak molecular weight of between 300,000 and 1,750,000 Daltons.
16. A butyrylcholinesterase enzyme fusion protein comprising:
a butyrylcholinesterase enzyme;
a linker connected to the butyrylcholinesterase enzyme;
an immunoglobulin (Ig) domain connected to the linker,
wherein the butyrylcholinesterase enzyme has a mutation, wherein the residue C94 of SEQ ID NO: 1 is Y.
17. The butyrylcholinesterase enzyme fusion protein of claim 16 , wherein the Ig domain is IgG-Fc (SEQ ID NO: 8) and wherein the Fc domain has amino acid substitutions in one or more of the positions selected from the group consisting of E13, L14, L15, G16, G17, A107, A110, and P111 as numbered within SEQ ID NO: 8.
18. A method for effecting a long term prophylaxis against organophosphorous (OP) poisoning, comprising administering to a subject the conjugate according to claim 1 at least 7 days prior to expected exposure to an OP compound.
19. A method for effecting rapid prophylaxis against organophosphorous (OP) poisoning, comprising administering to a subject the conjugate according to claim 1 at least 12 hours prior to expected exposure to an organophosphorous (OP) compound.
20. A method for treatment or prophylaxis of organophosphorous (OP) poisoning, comprising administering an effective amount of the fusion protein according to claim 16 to a subject in need thereof.
21. The method of claim 20 , wherein the subject is or expected to be exposed to an OP compound.
22. The method of claim 21 , wherein the OP compound is an OP pesticide or OP nerve agent.
23. The method of claim 20 , wherein the fusion protein is administered intravenously.
24. The method of claim 20 , wherein the fusion protein is administered intramuscularly.
25. The method of claim 20 , comprising administering the effective amount of the fusion protein at least 7 days prior to expected exposure to an OP compound.
26. The method of claim 20 , comprising administering the effective amount of the fusion protein at least 12 hours prior to expected exposure to an OP compound.