Granted Patent B2

US 7,897,157 · App. 11/845,252 · Granted Mar 1, 2011

Assignee: Allergan, Inc.

Abstract

Claims (35)

1. A recombinant single-chain polypeptide comprising:

a) a first amino acid sequence region comprising

i) a first domain comprising a binding element comprising a granin peptide able to preferentially interact with a granin peptide receptor under physiological conditions; and

ii) a second domain comprising a translocation element comprising a Clostridial neurotoxin translocation domain able to facilitate the transfer of a Clostridial toxin light chain across a vesicular membrane;

b) a second amino acid sequence region comprising a therapeutic element comprising a Clostridial neurotoxin light chain having biological activity when released into the cytoplasm of said target cell;

c) a third amino acid sequence region comprising an exogenous protease cleavage site;

wherein said first and second amino acid sequence regions are separated by said third amino acid sequence region.

2. The polypeptide of claim 1 , wherein said granin peptide comprises a chromogranin A, a chromogranin B, or a chromogranin C.

3. The polypeptide of claim 2 , wherein said chromogranin A comprises a β-granin, a vasostatin, a chromostatin, a pancreastatin, a WE-14, a catestatin, a parastatin or a GE-25.

4. The polypeptide of claim 2 , wherein said chromogranin A comprises SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99 or SEQ ID NO: 100.

5. The polypeptide of claim 2 , wherein said chromogranin B comprises a GAWK peptide, an adrenomedullary peptide or a secretolytin.

6. The polypeptide of claim 2 , wherein said chromogranin B comprises SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104 or SEQ ID NO: 105.

7. The polypeptide of claim 2 , wherein said chromogranin C comprises a secretoneurin.

8. The polypeptide of claim 2 , wherein said chromogranin C comprises SEQ ID NO: 106.

9. The polypeptide of claim 1 , wherein said translocation element comprises a Clostridium botulinum neurotoxin translocation domain.

10. The polypeptide of claim 9 , wherein said Clostridium botulinum neurotoxin translocation domain is selected from the group consisting of a Clostridium botulinum serotype A neurotoxin translocation domain, a Clostridium botulinum serotype B neurotoxin translocation domain, a Clostridium botulinum serotype C1 neurotoxin translocation domain, a Clostridium botulinum serotype D neurotoxin translocation domain, a Clostridium botulinum serotype E neurotoxin translocation domain, a Clostridium botulinum serotype F neurotoxin translocation domain and a Clostridium botulinum serotype G neurotoxin translocation domain.

11. The polypeptide of claim 1 , wherein said translocation element comprises a Clostridium tetani neurotoxin translocation domain.

12. The polypeptide of claim 1 , wherein said therapeutic element comprises a Clostridium botulinum neurotoxin light chain.

13. The polypeptide of claim 12 , wherein said Clostridium botulinum neurotoxin light chain therapeutic element is selected from the group consisting of a Clostridium botulinum serotype A neurotoxin light chain, a Clostridium botulinum serotype B neurotoxin light chain, a Clostridium botulinum serotype C1 neurotoxin light chain, a Clostridium botulinum serotype D neurotoxin light chain, a Clostridium botulinum serotype E neurotoxin light chain, a Clostridium botulinum serotype F neurotoxin light chain and a Clostridium botulinum serotype G neurotoxin light chain.

14. The polypeptide of claim 1 , wherein said therapeutic element comprises a Clostridium tetani neurotoxin light chain.

15. The polypeptide of claim 1 , wherein said exogenous protease cleavage site comprises a non-human enterokinase cleavage site, a tobacco etch virus protease cleavage site, a tobacco vein mottling virus protease cleavage site, a human rhinovirus 3C protease cleavage site, a human enterovirus 3C protease cleavage site, a subtilisin cleavage site, a SUMO/ULP-1 protease cleavage site, or a non-human Caspase 3 protease cleavage site.

16. The polypeptide of claim 15 , wherein said non-human enterokinase cleavage site comprises SEQ ID NO: 21.

17. The polypeptide of claim 15 , wherein said tobacco etch virus protease cleavage site comprises SEQ ID NO: 22 or SEQ ID NO: 23.

18. The polypeptide of claim 15 , wherein said tobacco etch virus protease cleavage site comprises SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32 or SEQ ID NO: 33.

19. The polypeptide of claim 15 , wherein said tobacco vein mottling virus protease cleavage site comprises SEQ ID NO: 34 or SEQ ID NO: 35.

20. The polypeptide of claim 15 , wherein said tobacco vein mottling virus protease cleavage site comprises SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, or SEQ ID NO: 39.

21. The polypeptide of claim 15 , wherein said human rhinovirus 3C protease cleavage site comprises SEQ ID NO: 40.

22. The polypeptide of claim 15 , wherein said human rhinovirus 3C protease cleavage site comprises SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45 or SEQ ID NO: 46.

23. The polypeptide of claim 15 , wherein said subtilisin cleavage site comprises SEQ ID NO: 47 or SEQ ID NO: 48.

24. The polypeptide of claim 15 , wherein said subtilisin cleavage site comprises SEQ ID NO: 49, SEQ ID NO: 50, or SEQ ID NO: 51.

25. The polypeptide of claim 15 , wherein said non-human Caspase 3 protease cleavage site comprises SEQ ID NO: 57.

26. The polypeptide of claim 15 , wherein said non-human Caspase 3 protease cleavage site comprises SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62 or SEQ ID NO: 63.

27. The polypeptide of claim 1 , wherein said polypeptide further comprises a fourth amino acid sequence region comprising a target-binding portion of a binding tag.

28. A method of activating a single-chain polypeptide, the method comprising the step of incubating the single-chain polypeptide according to claim 1 with an exogenous protease; wherein the exogenous protease cleaves the exogenous protease cleavage site; and wherein cleavage of the single-chain polypeptide by the exogenous protease converts the single-chain polypeptide into a di-chain form, thereby activating the single-chain polypeptide.

29. A pharmaceutical composition comprising a carrier and a di-chain polypeptide obtained by the method according to claim 28 .

Assignments (1)

Continuity (8)