Subcellular targeting of therapeutic proteins

• US 7,396,811 B2
• Filed: 04/30/2002
• Issued: 07/08/2008
• Est. Priority Date: 04/30/2001
• Status: Expired due to Term

• Current Assignee: Biomarin Pharmaceutical Incorporated (Amgen Inc.)
• Original Assignee: ZyStor Therapeutics, Inc. (Amgen Inc.)
• Inventors: Beverley, Stephen M., LeBowitz, Jonathan H.
• Primary Examiner(s): Stucker; Jeffrey
• Assistant Examiner(s): Standley; Steven H.
• Application Number:
  US10/136,841
• Publication Number:
  US 20030082176A1
• Time in Patent Office: 2,261 Days
• Field of Search: 514/2, 514/8, 424/198.1, 424/192.1, 424/143.1
• US Class Current: 514/1.2
• 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 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 47/64 Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent peptidic linkers A61K47/65
• CPC Class Codes: A61K 47/642 the peptide or protein in the drug conjugate being a cytokine, e.g. IL2, chemokine, growth factors or interferons being the inactive part of the conjugate
• CPC Class Codes: A61P 3/00 Drugs for disorders of the metabolism of the blood or the extracellular fluid A61P7/00
• 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 2319/06 containing a lysosomal/endosomal localisation signal
• CPC Class Codes: C07K 2319/21 containing a His-tag
• 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
• 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

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 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,

wherein the mutein of mature human IGF-II is fused N-terminally to the lysosomal enzyme and wherein the therapeutic fusion protein is produced in a mammalian expression system using an IGF-II signal peptide and is therapeutically active in vivo.

2. A 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,

wherein the mutein of mature human IGF-II is fused N-terminally to the lysosomal enzyme and wherein the therapeutic fusion protein is produced in a mammalian expression system using an IGF-II signal peptide and is therapeutically active in vivo.

6. The therapeutic fusion protein of claim 2, wherein a cellular or subcellular deficiency in the lysosomal enzyme is associated with a lysosomal storage disease.

7. The therapeutic fusion protein of claim 6, wherein the lysosomal storage disease is Pompe Disease.

8. The therapeutic fusion protein of claim 6, wherein the lysosomal storage disease is Fabry Disease.

9. The therapeutic fusion protein of claim 6, wherein the lysosomal storage disease is Gaucher Disease.

3. A 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,

wherein the mutein of mature human IGF-II is fused N-terminally to the lysosomal enzyme and wherein the therapeutic fusion protein is produced in a mammalian expression system using an IGF-II signal peptide and is therapeutically active in vivo.

4. A 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,

wherein the mutein of mature human IGF-II is fused N-terminally to the lysosomal enzyme and wherein the therapeutic fusion protein is produced in a mammalian expression system using an IGF-II signal peptide and is therapeutically active in vivo.

5. A 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,

wherein the mutein of mature human IGF-II is fused N-terminally to the lysosomal enzyme and wherein the therapeutic fusion protein is produced in a mammalian expression system using an IGF-II signal peptide and is therapeutically active in vivo.

10. A 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,

wherein the mutein of mature human IGF-II is fused N-terminally to the lysosomal enzyme and wherein the therapeutic fusion protein is produced in a mammalian expression system using an IGF-II signal peptide and is therapeutically active in vivo.

11. The therapeutic fusion protein of claim 10, wherein a cellular or subcellular deficiency in the lysosomal enzyme is associated with a lysosomal storage disease.

12. The therapeutic fusion protein of claim 11, wherein the lysosomal storage disease is Pompe Disease.

13. The therapeutic fusion protein of claim 11, wherein the lysosomal storage disease is Fabry Disease.

14. The therapeutic fusion protein of claim 11, wherein the lysosomal storage disease is Gaucher Disease.