Targeted therapeutic proteins

• US 7,560,424 B2
• Filed: 10/16/2002
• Issued: 07/14/2009
• 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: LeBowitz, Jonathan H., Beverley, Stephen M.
• Primary Examiner(s): Romeo; David S
• Assistant Examiner(s): Gamett; Daniel C
• Application Number:
  US10/272,483
• Publication Number:
  US 20040006008A1
• Time in Patent Office: 2,463 Days
• Field of Search: None
• US Class Current: 514/1.1
• CPC Class Codes: A61K 2039/505 comprising antibodies
• CPC Class Codes: A61K 2039/515 Animal cells
• 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 35/12 Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells vaccines or medicinal preparations containing antigens or antibodies A61K39/00
• CPC Class Codes: A61K 35/54 Ovaries; Ova; Ovules; Embryos; Foetal cells; Germ cells
• 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 38/44 Oxidoreductases (1)
• CPC Class Codes: A61K 38/47 acting on glycosyl compounds (3.2), e.g. cellulases, lactases
• CPC Class Codes: A61K 39/3955 against proteinaceous materials, e.g. enzymes, hormones, lymphokines
• 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: 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: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• 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 16/28 against receptors, cell surface antigens or cell surface determinants
• CPC Class Codes: C07K 2317/77 Internalization into the cell
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/01 containing a localisation/targetting motif
• 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 2319/33 : fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
• 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 2319/74 : 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
• CPC Class Codes: C12N 9/0051 : acting on a sulfur group of donors (1.8)
• CPC Class Codes: C12N 9/2402 : hydrolysing O- and S- glycosyl compounds (3.2.1)
• CPC Class Codes: C12Y 302/01022 : Alpha-galactosidase (3.2.1.22)

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 targeted therapeutic fusion protein comprising:

a lysosomal enzyme;

a mutein of mature human IGF-II having an amino acid sequence at least 80% identical to mature human IGF-II, wherein the mutein binds to the human cation-independent mannose-6-phosphate receptor in a mannose-6-phosphate-independent manner with a dissociation constant of 10<sup>−

7</sup> M or less at pH 7.4, and has diminished binding affinity for the IGF-I receptor relative to the affinity of naturally-occurring human IGF-II for the IGF-I receptor;

wherein the lysosomal enzyme is targeted to a mammalian lysosome in a mannose-6-phosphate-independent manner and is therapeutically active in vivo.

2. The targeted therapeutic fusion protein of claim 1, wherein the mutein of mature human IGF-II comprises amino acids 48-55 of mature human IGF-II.

3. The targeted therapeutic fusion protein of claim 1, wherein the mutein of mature human IGF-II comprises, at corresponding positions, 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.

4. The targeted therapeutic fusion protein of claim 1, wherein the amino acid sequence 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.

5. A targeted therapeutic fusion protein of claim 1, wherein the mutein of mature human IGF-II comprises the amino acid sequence phenylalanine-arginine-serine.

6. A targeted therapeutic fusion protein of claim 1, wherein the mutein of mature human IGF-II comprises amino acids 8-28 and 41-61 of mature human IGF-II.

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

8. The targeted therapeutic fusion protein of claim 7, wherein the human disease is a lysosomal storage disease.

9. A nucleic acid encoding the therapeutic fusion protein of claim 1.

10. A cell comprising the nucleic acid of claim 9.

11. A method of producing a therapeutic fusion protein, the method comprising the step of providing to the cell of claim 10 conditions permitting expression of the therapeutic fusion protein.

12. The method of claim 11, comprising culturing the cell in vitro.

13. The method of claim 11, further comprising harvesting the therapeutic fusion protein.

14. A method of treating a patient with a lysosomal storage disease, the method comprising administering to the patient the therapeutic fusion protein of claim 1.

15. The targeted therapeutic fusion protein of claim 1, wherein the mutein differs from mature human IGF-II at a position selected from the group consisting of amino acid 9, amino acid 19, amino acid 26, and amino acid 27.

16. The targeted therapeutic fusion protein of claim 1, wherein the mutein is a fragment of mature human IGF-II.

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

18. The targeted therapeutic fusion protein of claim 1, wherein the mutein comprises a deletion or a replacement of amino acids 62-67 of mature human IGF-II.

19. The targeted therapeutic fusion protein of claim 1, wherein the mutein comprises a deletion or a replacement of amino acids 29-40 of mature human IGF-II.

20. The targeted therapeutic fusion protein of claim 1, wherein the mutein comprises at least an amino acid substitution selected from the group consisting of Tyr27Leu, Leu43Val, and Ser26Phe.

21. The targeted therapeutic fusion protein of claim 8, wherein the lysosomal storage disease is Pompe Disease.

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

23. The targeted therapeutic fusion protein of claim 8, wherein the lysosomal storage disease is Gaucher Disease.

24. A 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 and has diminished binding affinity for the IGF-I receptor relative to the affinity of naturally-occurring human IGF-II for the IGF-I receptor;

wherein the mutein differs from mature human IGF-II only by a deletion or a replacement of amino acids 1-7,

wherein the lysosomal enzyme is targeted to a mammalian lysosome in a mannose-6-phosphate-independent manner and is therapeutically active in vivo.

25. The targeted therapeutic fusion protein of claim 24, wherein a cellular or subcellular deficiency in the lysosomal enzyme is associated with a lysosomal storage disease.

26. The targeted therapeutic fusion protein of claim 25, wherein the lysosomal storage disease is Pompe Disease.

27. The targeted therapeutic fusion protein of claim 25, wherein the lysosomal storage disease is Fabry Disease.

28. The targeted therapeutic fusion protein of claim 25, wherein the lysosomal storage disease is Gaucher Disease.

29. A 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 and has diminished binding affinity for the IGF-I receptor relative to the affinity of naturally-occurring human IGF-II for the IGF-I receptor,

wherein the mutein differs from mature human IGF-II only by a deletion or a replacement of amino acids 62-67,

wherein the lysosomal enzyme is targeted to a mammalian lysosome in a mannose-6-phosphate-independent manner and is therapeutically active in vivo.

30. A 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 and has diminished binding affinity for the IGF-I receptor relative to the affinity of naturally-occurring human IGF-II for the IGF-I receptor;

wherein the mutein differs from mature human IGF-II only by a deletion or a replacement of amino acids 29-40,

wherein the lysosomal enzyme is targeted to a mammalian lysosome in a mannose-6-phosphate-independent manner and is therapeutically active in vivo.

31. A 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 and has diminished binding affinity for the IGF-I receptor relative to the affinity of naturally-occurring human IGF-II for the IGF-I receptor;

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 lysosomal enzyme is targeted to a mammalian lysosome in a mannose-6-phosphate-independent manner and is therapeutically active in vivo.

32. A 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 and has diminished binding affinity for the IGF-I receptor relative to the affinity of naturally-occurring human IGF-II for the IGF-I receptor;

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 lysosomal enzyme is targeted to a mammalian lysosome in a mannose-6-phosphate-independent manner and is therapeutically active in vivo.

33. A targeted therapeutic fission 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 and has diminished binding affinity for the IGF-I receptor relative to the affinity of naturally-occurring human IGF-II for the IGF-I receptor;

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 lysosomal enzyme is targeted to a mammalian lysosome in a mannose-6-phosphate-independent manner and is therapeutically active in vivo.