Targeted therapeutic proteins

• US 7,629,309 B2
• Filed: 11/03/2004
• Issued: 12/08/2009
• Est. Priority Date: 05/29/2002
• Status: Expired due to Fees

• Current Assignee: Biomarin Pharmaceutical Incorporated (Amgen Inc.)
• Original Assignee: ZyStor Therapeutics, Inc. (Amgen Inc.)
• Inventors: Sly, William S., Beverley, Stephen M., LeBowitz, Jonathan H.
• Primary Examiner(s): Gamett; Daniel C
• Application Number:
  US10/981,267
• Publication Number:
  US 20050281805A1
• Time in Patent Office: 1,861 Days
• Field of Search: None
• US Class Current: 514/1.1
• 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: A61K 38/47 acting on glycosyl compounds (3.2), e.g. cellulases, lactases
• CPC Class Codes: A61K 47/551 one of the codrug's components being a vitamin, e.g. niacinamide, vitamin B3, cobalamin, vitamin B12, folate, vitamin A or retinoic acid
• CPC Class Codes: A61K 47/6425 the peptide or protein in the drug conjugate being a receptor, e.g. CD4, a cell surface antigen, i.e. not a peptide ligand targeting the antigen, or a cell surface determinant, i.e. a part of the surface of a cell
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07K 14/65 Insulin-like growth factors, i.e. somatomedins, e.g. IGF-1, IGF-2
• 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 2319/61 containing an enzyme fusion for detection (lacZ, luciferase)
• CPC Class Codes: C07K 2319/75 containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones
• CPC Class Codes: C12N 15/62 DNA sequences coding for fusion proteins
• CPC Class Codes: G01N 2333/924 acting on glycosyl compounds (3.2)

Abstract

Targeted therapeutics that localize to a specific subcellular compartment such as the lysosome are provided. The targeted therapeutics include a therapeutic agent and a targeting moiety that binds a receptor on an exterior surface of the cell, permitting proper subcellular localization of the targeted therapeutic upon internalization of the receptor. Nucleic acids, cells, and methods relating to the practice of the invention are also provided.

Claims

1. A deglycosylated targeted therapeutic fusion protein comprising:

a lysosomal enzyme; and

a lysosomal targeting domain that binds the human cation-independent mannose-6-phosphate receptor in a mannose-6-phosphate-independent manner;

wherein the lysosomal targeting domain is mature human IGF-II or a mutein of human IGF-II having an amino acid sequence at least 70% identical to mature human IGF-II.

2. The deglycosylated targeted therapeutic fusion protein of claim 1, wherein the lysosomal targeting domain is mature human IGF-II.

3. The deglycosylated targeted therapeutic fusion protein of claim 1, wherein the lysosomal targeting domain is a mutein of human IGF-II having an amino acid sequence at least 70% identical to mature human IGF-II.

4. The deglycosylated targeted therapeutic fusion protein of claim 3, wherein the mutein of human IGF-II comprises amino acids 48-55 of mature human IGF-II.

5. The deglycosylated targeted therapeutic fusion protein of claim 3, wherein the mutein of human IGF-II comprises at least three amino acids selected from the group consisting of amino acids 8, 48, 49, 50, 54, and 55 of mature human IGF-II, wherein the relative positions of the at least three amino acids correspond to their positions in mature human IGF-II.

6. The deglycosylated targeted therapeutic fusion protein of claim 1, wherein the mutein of human IGF-II comprises amino acid sequence of IGF-I or of a mutein of IGF-I in which

(i) amino acids 55 and 56 are changed,

(ii) amino acids 1-4 are deleted or changed, or

(iii) amino acids 55 and 56 are changed and amino acids 1-4 are deleted or changed.

7. The deglycosylated targeted therapeutic fusion protein of claim 3, wherein the mutein of human IGF-II comprises, at positions corresponding to positions 54 and 55 of mature human IGF-II, amino acids each of which are uncharged or negatively charged at pH 7.4.

8. The deglycosylated targeted therapeutic fusion protein of claim 3, wherein the mutein of human IGF-II comprises the amino acid sequence phenylalanine-arginine-serine.

9. The deglycosylated targeted therapeutic fusion protein of claim 3, wherein the mutein of human IGF-II comprises amino acids 8-28 and 41-61 of mature human IGF-II.

10. The deglycosylated targeted therapeutic fusion protein of claim 3, wherein the mutein comprises a deletion or a replacement of amino acids 1-7 of mature human IGF-II.

11. The deglycosylated targeted therapeutic fusion protein of claim 1, wherein the mutein binds to the IGF-I receptor with diminished affinity relative to the affinity of naturally-occurring human IGF-II for the IGF-I receptor.

12. The deglycosylated therapeutic fusion protein of claim 1, wherein a cellular or subcellular deficiency in the enzymatic activity is associated with a human disease.

13. The deglycosylated therapeutic fusion protein of claim 12, wherein the human disease is a lysosomal storage disease.

14. The deglycosylated therapeutic fusion protein of claim 13, wherein the lysosomal storage disease is Pompe Disease.

15. The deglycosylated therapeutic fusion protein of claim 13, wherein the lysosomal storage disease is Fabry Disease.

16. The deglycosylated therapeutic fusion protein of claim 13, wherein the lysosomal storage disease is Gaucher Disease.

17. The deglycosylated targeted therapeutic fusion protein of claim 1, wherein the targeted therapeutic fusion protein is deglycosylated by periodate treatment.

18. A deglycosylated targeted therapeutic fusion protein comprising:

a lysosomal enzyme, and

a mutein of mature human IGF-II that binds human cation-independent mannose-6-phosphate receptor in a mannose-6-phosphate-independent manner, wherein the mutein differs from mature human IGF-II only by a deletion or a replacement of amino acids 1-7.

19. The deglycosylated targeted therapeutic fusion protein of claim 18, wherein a cellular or subcellular deficiency in the lysosomal enzyme is associated with a lysosomal storage disease.

20. The deglycosylated targeted therapeutic fusion protein of claim 19, wherein the lysosomal storage disease is Pompe Disease.

21. The deglycosylated targeted therapeutic fusion protein of claim 19, wherein the lysosomal storage disease is Fabry Disease.

22. The deglycosylated targeted therapeutic fusion protein of claim 19, wherein the lysosomal storage disease is Gaucher Disease.

23. A deglycosylated targeted therapeutic fusion protein comprising:

a lysosomal enzyme, and

a mutein of mature human IGF-II that binds human cation-independent mannose-6-phosphate receptor in a mannose-6-phosphate-independent manner, wherein the mutein differs from mature human IGF-II only by a deletion or a replacement of amino acids 62-67.

24. A deglycosylated targeted therapeutic fusion protein comprising:

a lysosomal enzyme, and

a mutein of mature human IGF-II that binds human cation-independent mannose-6-phosphate receptor in a mannose-6-phosphate-independent manner, wherein the mutein differs from mature human IGF-II only by a deletion or a replacement of amino acids 29-40.

25. A deglycosylated targeted therapeutic fusion protein comprising:

a lysosomal enzyme, and

a mutein of mature human IGF-II that binds human cation-independent mannose-6-phosphate receptor in a mannose-6-phosphate-independent manner, wherein the mutein differs from mature human IGF-II only by an amino acid substitution selected from the group consisting of Tyr27Leu, Leu43Val, and Ser26Phe.

26. A deglycosylated targeted therapeutic fusion protein comprising:

a lysosomal enzyme, and

a mutein of mature human IGF-II that binds human cation-independent mannose-6-phosphate receptor in a mannose-6-phosphate-independent manner, wherein the mutein differs from mature human IGF-II only by a deletion or a replacement of amino acids 1-7 and a substitution of Tyr27Leu.

27. A deglycosylated targeted therapeutic fusion protein comprising:

a lysosomal enzyme, and

a mutein of mature human IGF-II that binds human cation-independent mannose-6-phosphate receptor in a mannose-6-phosphate-independent manner, wherein the mutein differs from mature human IGF-II only at a position selected from the group consisting of amino acid 9, amino acid 19, amino acid 26, and amino acid 27.