IP Library › Granted Patent US 7,556,817
Granted Patent B2
US 7,556,817 · App. 11/533,223 · Granted Jul 7, 2009

Clostridial toxin activatable Clostridial toxins

Assignee: Allergan, Inc.
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Quick Facts
Patent No.
US 7,556,817
App. No.
11/533,223
Granted
Jul 7, 2009
Kind
B2
Abstract

The specification discloses modified Clostridial toxins comprising a Clostridial toxin substrate cleavage site located within the di-chain loop region; polynucleotide molecules encoding such modified Clostridial toxins comprising a Clostridial toxin substrate cleavage site located in the di-chain loop region; and method of producing modified Clostridial toxins comprising Clostridial toxin substrate cleavage site located within the di-chain loop region.

Claims (41)

1. A recombinantly modified Clostridial toxin comprising an enzymatic domain having a zinc-dependent endopeptidase activity which targets a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein, a translocation domain, a binding domain and a modified di-chain loop region, the modified di-chain loop region intervening between the enzymatic domain and the translocation domain;

wherein the di-chain loop region modification comprises a Clostridial toxin substrate cleavage site from a SNARE protein of at most 40 amino acids in length located within the di-chain loop region that can be proteolytically cleaved by the enzymatic domain of the recombinantly modified Clostridial toxin; and

wherein expression of the recombinantly modified Clostridial toxin within a cell results in single chain to di-chain conversion of the recombinantly modified Clostridial toxin due to proteolytic cleavage of the Clostridial toxin substrate cleavage site.

2. The modified Clostridial toxin according to claim 1 , wherein the Clostridial toxin substrate cleavage site from a SNARE protein is a Botulinum toxin substrate cleavage site from a SNARE protein of at most 40 amino acids in length.

3. The modified Clostridial toxin according to claim 2 , wherein the Botulinum toxin substrate cleavage site from a SNARE protein comprises a BoNT/A cleavage site from a 25 kilo Dalton synaptosomal associated protein SNAP-25) of at most 40 amino acids in length.

4. The modified Clostridial toxin according to claim 3 , wherein the BoNT/A substrate cleavage site from a SNAP-25 comprises at least six consecutive residues of a SNAP-25, said six consecutive residues comprising Gln-Arg.

5. The modified Clostridial toxin according to claim 3 , wherein the BoNT/A substrate cleavage site from a SNAP-25 comprises at least six consecutive residues of a SNAP-25, said six consecutive residues comprising Lys-His.

6. The modified Clostridial toxin according to claim 3 , wherein the BoNT/A substrate cleavage site from a SNAP-25 comprises SEQ ID NO: 104, SEQ ID NO: 105 or SEQ ID NO: 106.

7. The modified Clostridial toxin according to claim 2 , wherein the Botulinum toxin substrate cleavage site comprises a BoNT/B cleavage site from a vesicle associated membrane protein (VAMP) of at most 40 amino acids in length.

8. The modified Clostridial toxin according to claim 7 , wherein the BoNT/B substrate cleavage site comprises at least six consecutive residues of a VAMP, said six consecutive residues comprising Gln-Phe.

9. The modified Clostridial toxin according to claim 7 , wherein the BoNT/B substrate cleavage site comprises SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111 or SEQ ID NO: 112.

10. The modified Clostridial toxin according to claim 2 , wherein the Botulinum toxin substrate cleavage site comprises a BoNT/Ci cleavage site from a SNAP-25 or Syntaxin of at most 40 amino acids in length.

11. The modified Clostridial toxin according to claim 10 , wherein the BoNT/Ci substrate cleavage site comprises at least six consecutive residues of a SNAP-25, said six consecutive residues comprising Arg-Ala.

12. The modified Clostridial toxin according to claim 10 , wherein the BoNT/Ci substrate cleavage site comprises at least six consecutive residues of a Syntaxin, said six consecutive residues comprising Lys-Ala.

13. The modified Clostridial toxin according to claim 10 , wherein the BoNT/Ci substrate cleavage site comprises at least six consecutive residues of a Syntaxin, said six consecutive residues comprising Arg-Ala.

14. The modified Clostridial toxin according to claim 10 , wherein the BoNT/Ci substrate cleavage site comprises SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120 or SEQ ID NO: 121.

15. The modified Clostridial toxin according to claim 2 , wherein the Botulinum toxin substrate cleavage site comprises a BoNT/D cleavage site from a VAMP of at most 40 amino acids in length.

16. The modified Clostridial toxin according to claim 15 , wherein the BoNT/D substrate cleavage site comprises at least six consecutive residues of a VAMP, said six consecutive residues comprising Lys-Leu.

17. The modified Clostridial toxin according to claim 15 , wherein the BoNT/D substrate cleavage site comprises SEQ ID NO: 122 or SEQ ID NO: 123.

18. The modified Clostridial toxin according to claim 2 , wherein the Botulinum toxin substrate cleavage site comprises a BoNT/E cleavage site from a SNAP-25 of at most 40 amino acids in length.

19. The modified Clostridial toxin according to claim 18 , wherein the BoNT/E substrate cleavage site comprises at least six consecutive residues of a SNAP-25, said six consecutive residues comprising Arg-11e.

20. The modified Clostridial toxin according to claim 18 , wherein the BoNT/E substrate cleavage site comprises at least six consecutive residues of a SNAP-25, said six consecutive residues comprising Lys-Ile.

21. The modified Clostridial toxin according to claim 18 , wherein the BoNT/E substrate cleavage site comprises SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127 or SEQ ID NO: 128.

22. The modified Clostridial toxin according to claim 2 , wherein the Botulinum toxin substrate cleavage site comprises a BoNT/F cleavage site from a VAMP of at most 40 amino acids in length.

23. The modified Clostridial toxin according to claim 22 , wherein the BoNT/F substrate cleavage site comprises at least six consecutive residues of a VAMP, said six consecutive residues comprising Gln-Lys.

24. The modified Clostridial toxin according to claim 22 , wherein the BoNT/F substrate cleavage site comprises SEQ ID NO: 129 or SEQ ID NO: 130.

25. The modified Clostridial toxin according to claim 2 , wherein the Botulinum toxin substrate cleavage site comprises a BoNT/G cleavage site from a VAMP of at most 40 amino acids in length.

26. The modified Clostridial toxin according to claim 25 , wherein the BoNT/G substrate cleavage site comprises at least six consecutive residues of a VAMP, said six consecutive residues comprising Ala-Ala.

27. The modified Clostridial toxin according to claim 25 , wherein the BoNT/G substrate cleavage site comprises SEQ ID NO: 131 or SEQ ID NO: 132.

28. The modified Clostridial toxin according to claim 1 , wherein the Clostridial toxin substrate cleavage site is a Tetanus toxin substrate cleavage site from a VAMP of at most 40 amino acids in length.

29. The modified Clostridial toxin according to claim 28 , wherein the Tetanus toxin substrate cleavage site comprises at least six consecutive residues of a VAMP, said six consecutive residues comprising Gln-Phe.

30. The modified Clostridial toxin of claim 1 , wherein the modification further comprises a flexible region comprising one or more flexible spacers located within the di-chain loop region.

31. The modified Clostridial toxin of claim 30 , wherein the one or more flexible spacer are G-spacer, A-spacers or any combination thereof.

32. The modified BoNT/A according to claim 30 , wherein the BoNT/A substrate cleavage site comprises at least six consecutive residues of a SNAP-25, said six consecutive residues comprising Gln-Arg.

33. The modified BoNT/A according to claim 30 , wherein the BoNT/A substrate cleavage site comprises at least six consecutive residues of a SNAP-25, said six consecutive residues comprising Lys-His.

34. The modified BoNT/A according to claim 30 , wherein the BoNT/A substrate cleavage site comprises SEQ ID NO: 104, SEQ ID NO: 105 or SEQ ID NO: 106.

35. A recombinantly modified BoNT/A comprising an enzymatic domain having a zinc-dependent endopeptidase activity which targets a SNAP-25, a translocation domain, a binding domain and a modified di-chain loop region, the modified di-chain loop region-intervening between the enzymatic domain and the translocation domain;

wherein the di-chain loon region modification comprises a BoNT/A substrate cleavage site from a SNAP-25 of at most 40 amino acids in length located within the di-chain loop region that is proteolytically cleaved by the enzymatic domain of the recombinantly modified BoNT/A: and

wherein expression of the recombinantly modified BoNT/A within a cell results in single chain to di-chain conversion of the recombinantly modified BoNT/A due to proteolytic cleavage of the BoNT/A substrate cleavage site.

36. The modified Clostridial toxin of claim 35 , wherein the modification further comprises a flexible region comprising one or more flexible spacers located within the di-chain loop region.

37. The modified Clostridial toxin of claim 36 , wherein the one or more flexible spacer are G-spacer, A-spacers or any combination thereof.

Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Nov 29, 2006
From: STEWARD, LANCE E.; OKA, MELVIN S.
To: ALLERGAN, INC.
Reel/Frame 018560/0273 →
Continuity (2)
Provisional Application 6071861600 · Sep 19, 2005
Related Publication 20070166332A1 · Jul 19, 2007