Methods and Compositions for Targeting Proteins Across the Blood Brain Barrier

• US 20080241118A1
• Filed: 02/19/2008
• Published: 10/02/2008
• Est. Priority Date: 10/16/2001
• Status: Active Grant

• Current Assignee: Biomarin Pharmaceutical Incorporated (Amgen Inc.)
• Original Assignee: ZyStor Therapeutics, Inc. (Amgen Inc.)
• Inventors: LeBowitz, Jonathan
• Granted Patent: US 7,981,864 B2
• Time in Patent Office: Days
• US Class Current: 424/94.1
• CPC Class Codes: A61K 2300/00 Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
• CPC Class Codes: A61K 38/30 Insulin-like growth factors, i.e. somatomedins, e.g. IGF-1, IGF-2 insulin-like growth factor binding protein A61K38/1754
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/02 containing a signal sequence
• CPC Class Codes: C07K 2319/50 containing protease site
• CPC Class Codes: C07K 2319/74 containing a fusion for binding to a cell surface receptor

Abstract

Disclosed are methods and compositions for targeting therapeutic proteins to the brain. Methods and compositions of the invention involve associating an IGF moiety with a therapeutic protein in order to target the therapeutic protein to the brain. Soluble fusion proteins that include an IGF targeting moiety are transported to neural tissue in the brain from blood. Methods and compositions of the invention include therapeutic applications for treating lysosomal storage diseases. The invention also provides nucleic acids and cells for expressing IGF fusion proteins.

Claims

1-6. -6. (canceled)

7. A targeted therapeutic fusion protein comprising:

a lysosomal enzyme; and

a mutein of human IGF-I having an amino acid sequence at least 70% identical to human IGF-I;

wherein the mutein binds to the human cation-independent mannose-6-phosphate receptor in a mannose-6-phosphate-independent manner, and has a diminished binding affinity to the IGF-I receptor relative to wild-type human IGF-I.

8. The targeted therapeutic fusion protein of claim 7, wherein the mutein of human IGF-I comprises a mutation diminishing binding to IGF binding proteins.

9. The targeted therapeutic fusion protein of claim 7, wherein the mutein of human IGF-I comprises an amino acid substitution at a position corresponding to Leu⁶⁰ of human IGF-I.

10. The targeted therapeutic fusion protein of claim 7, wherein the mutein of human IGF-I comprises an amino acid substitution at a position corresponding to Leu²⁴ of human IGF-I.

11. The targeted therapeutic fusion protein of claim 7, wherein the mutein of human IGF-I comprises a substitution or deletion corresponding to amino acids 1-3 of human IGF-I.

12. The targeted therapeutic fusion protein of claim 7, wherein the mutein of human IGF-I comprises an amino acid substitution of serine at a position corresponding to Phe¹⁶ of human IGF-I

13. The targeted therapeutic fusion protein of claim 7, wherein the mutein of human IGF-I comprises an amino acid substitution of lysine at a position corresponding to Glu⁹ of human IGF-I.

14. The targeted therapeutic fusion protein of claim 7, wherein the mutein of human IGF-1 comprises amino acid substitutions at positions corresponding to Arg⁵⁵ and Arg⁵⁶ of human IGF-I.

15. The targeted therapeutic fusion protein of claim 7, wherein the fusion protein is underglycosylated.

16. The targeted therapeutic fusion protein of claim 7, wherein the fusion protein is deglycosylated.

17. The targeted therapeutic fusion protein of claim 16, wherein the fusion protein is deglycosylated by periodate treatment.

18. A nucleic acid encoding the targeted therapeutic fusion protein of any of claim 7.

19. A cell comprising the nucleic acid of claim 18.

20. A method for producing a targeted lysosomal enzyme, the method comprising a step of expressing a fusion protein comprising a lysosomal enzyme and a mutein of human IGF-I having an amino acid sequence at least 70% identical to human IGF-I;

wherein the mutein binds to the human cation-independent mannose-6-phosphate receptor in a mannose-6-phosphate-independent manner, and has diminished binding affinity to the IGF-I receptor relative to the wild-type human IGF-I.

21. A method for targeting blood brain barrier in a subject, the method comprising a step of administering to the subject a fusion protein comprising

a lysosomal enzyme;

and

a mutein of human IGF-I having an amino acid sequence at least 70% identical to human IGF-I;

wherein the mutein binds to the human cation-independent mannose-6-phosphate receptor in a mannose-6-phosphate-independent manner, and has diminished binding affinity to the IGF-I receptor relative to the wild-type human IGF-I.