Targeted therapeutic proteins

• US 20040005309A1
• Filed: 10/16/2002
• Published: 01/08/2004
• Est. Priority Date: 05/29/2002
• Status: Abandoned Application

• Current Assignee: Biomarin Pharmaceutical Incorporated (Amgen Inc.)
• Original Assignee: Symbiontics, Inc. (Amgen Inc.)
• Inventors: Sly, William S., Beverley, Stephen M., LeBowitz, Jonathan H.
• Application Number:
  US10/272,531
• Publication Number:
  US 20040005309A1
• Time in Patent Office: Days
• US Class Current: 424/94.61
• 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 47/6425 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: 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

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. An underglycosylated targeted therapeutic comprising:

a therapeutic agent that is therapeutically active in a human lysosome; and

a lysosomal targeting domain that binds an extracellular domain of human cation-independent mannose-6-phosphate receptor and

(i) does not bind a mutein in which amino acid 1572 of the human cation-independent mannose-6-phosphate receptor is changed from isoleucine to threonine;

or

(ii) binds the mutein with a dissociation constant at least ten times the dissociation constant for binding the extracellular domain of human cation-independent mannose-6-phosphate receptor.

12. The underglycosylated targeted therapeutic of claim 1, wherein the portion of IGF-II comprises amino acids 48-55 of human IGF-II.

2. An underglycosylated targeted therapeutic comprising:

a therapeutic agent that is therapeutically active in a human lysosome; and

a lysosomal targeting domain that is capable of binding a receptor domain consisting essentially of repeats 10-15 of the human cation-independent mannose-6-phosphate receptor.

3. The underglycosylated targeted therapeutic of claim 2, wherein the lysosomal targeting domain is capable of binding a receptor domain consisting essentially of repeats 10-13 of the human cation-independent mannose-6-phosphate receptor.

4. The underglycosylated targeted therapeutic of claim 3, wherein the lysosomal targeting domain binds a receptor domain consisting essentially of repeats 11-12 of the human cation-independent mannose-6-phosphate receptor.

5. The underglycosylated targeted therapeutic of claim 4, wherein the lysosomal targeting domain binds a receptor domain consisting essentially of repeat 11 of the human cation-independent mannose-6-phosphate receptor.

6. The underglycosylated targeted therapeutic of claim 5, wherein the lysosomal targeting domain binds a receptor domain consisting essentially of amino acids 1508-1566 of the human cation-independent mannose-6-phosphate receptor.

7. The underglycosylated targeted therapeutic of claim 2, wherein the lysosomal targeting domain binds the receptor domain with a submicromolar dissociation constant at pH 7.4.

8. The underglycosylated targeted therapeutic of claim 7, wherein the dissociation constant is about 10⁻

7 M.

9. The underglycosylated targeted therapeutic of claim 7, wherein the dissociation constant is less than about 10⁻

7 M.

10. An underglycosylated targeted therapeutic comprising:

a therapeutic agent that is therapeutically active in a human lysosome; and

a binding moiety sufficiently duplicative of human IGF-II such that the binding moiety binds the human cation-independent mannose-6-phosphate receptor.

11. The underglycosylated targeted therapeutic of claim 10, wherein the binding moiety is an organic molecule having a three-dimensional shape representative of at least a portion of IGF-II.

13. The underglycosylated targeted therapeutic of claim 11, wherein the portion of IGF-II comprises at least three amino acids selected from the group consisting of amino acids 8, 48, 49, 50, 54, and 55 of human IGF-II.

14. The underglycosylated targeted therapeutic of claim 11, wherein the organic molecule has a hydrophobic moiety at a position representative of amino acid 48 of human IGF-II and has a positive charge at about pH 7.4 at a position representative of amino acid 49 of human IGF-II.

15. The underglycosylated targeted therapeutic of claim 10, wherein the binding moiety comprises a polypeptide comprising the 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.

16. The underglycosylated targeted therapeutic of claim 10, wherein the binding moiety comprises a polypeptide comprising an amino acid sequence at least 60% identical to human IGF-II.

17. The underglycosylated targeted therapeutic of claim 16, wherein the amino acid sequence comprises, at positions corresponding to positions 54 and 55 of human IGF-II, amino acids each of which are uncharged or negatively charged at pH 7.4.

18. The underglycosylated targeted therapeutic of claim 10 wherein the binding moiety comprises a polypeptide having antiparallel alpha-helices separated by not more than five amino acids.

19. An underglycosylated targeted therapeutic comprising:

a therapeutic agent that is therapeutically active in a human lysosome; and

a polypeptide comprising the amino acid sequence phenylalanine-arginine-serine.

20. An underglycosylated targeted therapeutic comprising:

a therapeutic agent that is therapeutically active in a human lysosome; and

a polypeptide comprising an amino acid sequence at least 75% homologous to amino acids 48-55 of human IGF-II.

21. An underglycosylated targeted therapeutic comprising:

a therapeutic agent that is therapeutically active in a human lysosome;

amino acids 8-28 of human IGF-II; and

amino acids 41-61 of human IGF-II.

22. The underglycosylated targeted therapeutic of claim 21, wherein amino acids 8-28 and 41-61 are present in a single polypeptide.

23. An underglycosylated targeted therapeutic comprising:

a therapeutic agent that is therapeutically active in a human lysosome;

amino acids 41-61 of human IGF-II; and

a mutein of amino acids 8-28 of human IGF-II, the mutein differing from 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.

24. An underglycosylated therapeutic fusion protein comprising:

a therapeutic domain and

a subcellular targeting domain that binds to an extracellular domain of a receptor on an exterior surface of a cell and, upon internalization of the receptor, permits localization of the therapeutic domain to a subcellular compartment where the therapeutic domain is therapeutically active.

25. The underglycosylated therapeutic fusion protein of claim 24, wherein the subcellular compartment is selected from the group consisting of a lysosome, an endosome, endoplasmic reticulum, and golgi complex.

26. The underglycosylated therapeutic fusion protein of claim 25, wherein the subcellular compartment is a lysosome.

27. The underglycosylated therapeutic fusion protein of claim 24, wherein the receptor undergoes continuous endocytosis.

28. The underglycosylated therapeutic fusion protein of claim 24, wherein the therapeutic domain has a therapeutic enzymatic activity.

29. The underglycosylated therapeutic fusion protein of claim 28, wherein a cellular or subcellular deficiency in the enzymatic activity is associated with a human disease.

30. The underglycosylated therapeutic fusion protein of claim 29, wherein the human disease is a lysosomal storage disease.

31. A method of treating a patient, the method comprising administering to the patient the underglycosylated therapeutic fusion protein of claim 24.

32. A method of treating a patient, the method comprising:

(i) synthesizing an underglycosylated targeted therapeutic comprising a therapeutic agent that is therapeutically active in a mammalian lysosome and a targeting moiety that binds human cation-independent mannose-6-phosphate receptor in a mannose-6-phosphate-independent manner; and

(ii) administering the underglycosylated targeted therapeutic to the patient.

33. The method of claim 32, further comprising, prior to step (i),

identifying a targeting moiety that binds human cation-independent mannose-6-phosphate receptor in a mannose-6-phosphate-independent manner.

34. The method of claim 33, wherein the targeting moiety is identified by screening a nucleic acid or peptide library.

35. A method of producing a targeted therapeutic, the method comprising the steps of:

(a) providing a molecular model defining a three-dimensional shape representative of at least a portion of human IGF-II;

(b) identifying a candidate IGF-II analog having a three-dimensional shape corresponding to the three-dimensional shape representative of at least a portion of human IGF-II; and

(c) producing an underglycosylated therapeutic agent directly or indirectly bound to the candidate IGF-II analog, wherein the therapeutic agent is therapeutically active in a mammalian lysosome.

36. The method of claim 35, further comprising determining whether the compound produced in step c binds to the human cation-independent mannose-6-phosphate receptor.