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 fusion protein comprising: a butyrylcholinesterase enzyme or carboxy terminal truncation fragment thereof and an immunoglobulin (Ig) domain or fragment thereof, wherein a linker is positioned between and connecting the butyrylcholinesterase enzyme or fragment thereof and the Ig domain of fragment, wherein the Ig domain or fragment thereof comprises substitutions L234A, L235E, and G237A per EU numbering which corresponds to substitutions L14A, L15E, and G17A as numbered within SEQ ID NO: 8, wherein the linker is selected from the group consisting of at least one of Q, GOG, GGGGS (SEQ ID NO: 22), GGGS (SEQ ID NO: 23), GGGES (SEQ II) NO: 24), GGGSGGGSGGGS (SEQ ID NO: 21), and GGSGGGSGGGS (SEQ ID NO: 25).
2. The fusion protein according to claim 1 , wherein the butyrylcholinesterase enzyme or fragment thereof comprises amino acids 29-561 of SEQ ID NO: 1.
3. The fusion protein according to claim 1 , wherein the butyrylcholinesterase enzyme or fragment thereof comprises amino acids 29-562 of SEQ ID NO: I.
4. The fusion protein according to claim 1 , wherein the butyrylcholinesterase enzyme fragment thereof comprises amino acids 29-561 of SEQ ID NO: I, except that residue C94 of SEQ ID NO: I is a Y.
5. The fusion protein according to claim 1 , wherein the butyrylcholinesterase enzyme or fragment thereof comprises amino acids 29-562 of SEQ ID NO: 1, except that residue C94 of SEQ ID NO: I is a Y.
6. The fusion protein according to claim 1 , wherein the fusion protein comprises SEQ ID NO: 2.
7. The fusion protein according to claim 1 , wherein the fusion protein comprises SEQ ID NO: 2 except that residue C672 of SEQ ID NO: 2 is a Q.
8. A fusion protein that shares at least 85% sequence identity to a protein comprising residues 29-561 of SEQ ID NO: I linked to residues 546-772 of SEQ ID NO: 2, wherein the protein comprises amino acid substitution C672Q in SEQ ID NO: 2, wherein a linker is positioned between and connecting the residues of SEQ ID NO: 1 and the residues of SEQ ID NO: 2, wherein the protein comprises an Ig domain or fragment that comprises substitutions L234A, L235E, and G237A per EU numbering which corresponds to substitutions L14A, L15E, and G17A as numbered within SEQ ID NO: 8, wherein the linker is selected from the group consisting of at least one of G, GG, GGGGS (SEQ ID NO: 22), GGGS (SEQ ID NO: 23), GGGES (SEQ ID NO: 24), GGGSGGGSGGGS (SEQ ID NO: 21), and GGSGGGSGGGS (SEQ I1) NO: 25).
9. The fusion protein of claim 8 , wherein the protein shares at least 98% sequence identify to a protein comprising residues 29-561 of SEQ ID NO: 1 linked to residues 546-772 of SEQ ID NO: 2, wherein the protein comprises amino acid substitution C672Q in SEQ ID NO: 2.
10. The fusion protein of claim 8 , wherein the protein shares at least 99% sequence identity to a protein comprising residues 29-561 of SEQ ID NO: 1 linked to residues 546-772 of SEQ ID NO: 2, wherein the protein comprises a amino acid substitution C672Q in SEQ ID NO: 2.
11. The fusion protein of claim 8 comprising residues 29-561 of SEQ ID NO: 1 linked to residues 546-772 of SEQ ID NO: 2, wherein the protein comprises amino acid substitution C672Q in SEQ ID NO: 2.
12. A protein that shares at least 98% sequence identity to the amino acids in SEQ ID NO: 2, wherein the protein comprises A559, E560, and A562.
13. A protein comprising the amino acids in SEQ ID NO: 2, wherein there is one or more amino acid substitutions within SEQ ID NO: 2 at position E560, S655, S656, and C672.
14. The protein of claim 13 , wherein the one or more amino acid substitutions includes: S655A.
15. The protein of claim 14 , wherein the one or amino acid substitutions includes E560, S655, and S656 of SEQ ID NO: 2.
16. A fusion protein comprising: a butyrylcholinesterase truncation; and an immunoglobulin (Ig) domain or fragment thereof; and a linker, wherein the linker is selected from the group consisting of at least one of G, GG, GGGGS (SEQ ID NO: 22), GGGS (SEQ ID NO: 23), GGGES (SEQ ID NO: 24), GGGSGGGSGGGS (SEQ ID NO: 21), and GGSGGGSGGGS (SEQ ID NO: 25), wherein the Ig domain or fragment comprises substitutions L234A, L235E, and G237A per EU numbering which corresponds to substitutions L14A, L15E, and G17A as numbered within SEQ ID NO: 8.
17. A fusion protein comprising: a butyrylcholinesterase enzyme carboxy terminal truncation fragment and an immunoglobulin (Ig) domain or fragment thereof that comprises substitutions L234A, L235E, and G237A per EU numbering which corresponds to substitutions L14A, L15E, and G17A as numbered within SEQ ID NO: 8, wherein the butyrylcholinesterase fragment consists of amino acids 29-561 of SEQ ID NO.1.
18. A fusion protein comprising: a butyrylcholinesterase enzyme carboxy terminal truncation fragment and an immunoglobulin (Ig) domain or fragment thereof, wherein a linker is positioned between and connecting the butyrylcholinesterase enzyme or truncation and the Ig domain of fragment, wherein the linker is selected from the group consisting of at least one of G, GG, GGGS (SEQ ID NO: 23), GGGES (SEQ ID NO: 24), GGGSGGGSGGGS (SEQ ID NO: 21), and GGSGGGSGGGS (SEQ ID NO: 25), and wherein the butyrylcholinesterase enzyme or truncation is a carboxyl truncate, wherein the fusion protein shares at least 85% sequence identity to a protein comprising residues 29-561 of SEQ ID NO: 1 linked to residues 546-772 of SEQ ID NO: 2, wherein the protein comprises C672Q in SEQ ID NO: 2.
19. A fusion protein comprising: a butyrylcholinesterase enzyme or carboxy terminal truncation fragment thereof and an immunoglobulin (Ig) domain or fragment thereof, wherein a linker is positioned between and connecting the butyrylcholinesterase enzyme or truncation and the Ig domain of fragment, wherein the Ig domain or fragment comprises substitutions L14A, L15E, and G17A of SEQ ID NO: 8, wherein the linker is selected from the group consisting of at least one of G, G, GGGS (SEQ ID NO: 23), CGOES (SEQ ID NO: 24), GGGSGGGSGGGS (SEQ ID NO: 21), and GGSGGGSG(GS (SEQ ID NO: 25).