Modified and stabilized GDF propeptides and uses thereof

• US 8,222,384 B2
• Filed: 05/04/2009
• Issued: 07/17/2012
• Est. Priority Date: 02/08/2001
• Status: Expired due to Fees

• Current Assignee: Wyeth LLC (Pfizer Inc.)
• Original Assignee: Wyeth LLC (Pfizer Inc.)
• Inventors: Wolfman, Neil M., Khor, Soo-Peang, Tomkinson, Kathleen N.
• Primary Examiner(s): WOODWARD, CHERIE MICHELLE
• Application Number:
  US12/434,758
• Publication Number:
  US 20100305194A1
• Time in Patent Office: 1,170 Days
• Field of Search: None
• US Class Current: 536/23.5
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 19/00 Drugs for skeletal disorders
• CPC Class Codes: A61P 19/02 for joint disorders, e.g. arthritis, arthrosis
• CPC Class Codes: A61P 19/08 for bone diseases, e.g. rachitism, Paget's disease
• CPC Class Codes: A61P 19/10 for osteoporosis
• CPC Class Codes: A61P 21/00 Drugs for disorders of the muscular or neuromuscular system
• CPC Class Codes: A61P 21/04 for myasthenia gravis
• CPC Class Codes: A61P 21/06 Anabolic agents androgens A61P5/26
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 3/00 Drugs for disorders of the metabolism of the blood or the extracellular fluid A61P7/00
• CPC Class Codes: A61P 3/04 Anorexiants; Antiobesity agents
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: C07K 14/475 Growth factors; Growth regulators
• CPC Class Codes: C07K 14/51 Bone morphogenetic factor; Osteogenins; Osteogenic factor; Bone-inducing factor
• CPC Class Codes: C07K 16/18 against material from animals or humans
• CPC Class Codes: C07K 2319/00 Fusion polypeptide

Abstract

Modified and stabilized propeptides of Growth Differentiation Factor proteins, such as GDF-8 and Bone Morphogenetic Protein-11, are disclosed. Also disclosed are methods for making and using the modified propeptides to prevent or treat human or animal disorders in which an increase in muscle tissue would be therapeutically beneficial. Such disorders include muscle or neuromuscular disorders (such as amyotrophic lateral sclerosis, muscular dystrophy, muscle atrophy, congestive obstructive pulmonary disease, muscle wasting syndrome, sarcopenia, or cachexia), metabolic diseases or disorders (such as such as type 2 diabetes, noninsulin-dependent diabetes mellitus, hyperglycemia, or obesity), adipose tissue disorders (such as obesity), and bone degenerative diseases (such as osteoporosis).

Claims

1. An isolated nucleic acid molecule encoding a GDF-8 propeptide comprising a nucleic acid sequence that encodes a GDF-8 propeptide that is at least 95% identical to SEQ ID NO:

5 and that is modified at the aspartate residue corresponding to aspartate 76 of SEQ ID NO;

5.

2. The nucleic acid molecule of claim 1, wherein the GDF-8 propeptide has an increased in vivo or in vitro half life relative to a corresponding unmodified GDF-8 propeptide.

3. The nucleic acid molecule of claim 1, wherein the GDF-8 propeptide inhibits one or more GDF-8 activities chosen from negative regulation of skeletal muscle mass, modulation of preadipocyte differentiation, inhibition of muscle formation, inhibition of muscle cell growth, inhibition of muscle development, regulation of muscle-specific enzymes, inhibition of myoblast cell proliferation, modulation of preadipocyte differentiation to adipocytes, increasing sensitivity to insulin, regulation of glucose uptake, glucose hemostasis, and modulation of neuronal cell development and maintenance.

4. The nucleic acid molecule of claim 1, wherein the nucleic acid encodes an alanine residue at the position corresponding to aspartate 76 of SEQ ID NO:

5.

5. The nucleic molecule acid of claim 1 further comprising a nucleic acid sequence encoding an Fc region of an IgG molecule.

6. The nucleic acid molecule of claim 5, wherein the IgG molecule is IgG1 or IgG4.

7. The nucleic acid molecule of claim 5, wherein the nucleic acid sequence encoding an Fc region of an IgG molecule encodes an amino acid sequence that is at least 95% identical to SEQ ID NO:

15 or SEQ ID NO;

16.

8. The nucleic acid molecule of claim 1, further comprising a nucleic acid sequence encoding albumin or an albumin derivative.

10. A vector comprising the nucleic acid molecule of claim 1.

11. The vector of claim 10 further comprising a nucleic acid molecule that encodes an Fc region of an IgG molecule.

12. The vector of claim 11 further comprising a linker peptide between the nucleotides encoding the GDF-8 propeptide and the IgG Fc region.

13. The vector of claim 12, wherein the linker peptide comprises nucleotides encoding for the amino acid sequence consisting of glycine-serine-glycine-serine (GSGS)(SEQ ID NO:

17).

14. A host cell comprising the vector of any one of claims 10, 11, 12, and 13.

15. The vector of claim 10, wherein the vector is a viral vector.

9. A nucleic acid molecule encoding a GDF-8 propeptide comprising a nucleic acid sequence that is at least 95% identical to SEQ ID NO:

6 and encodes a propeptide that is modified at the aspartate residue corresponding to aspartate 76 of SEQ ID NO;

5.

16. A nucleic acid molecule encoding a GDF-8 propeptide comprising a nucleic acid sequence that encodes a GDF-8 propeptide that is at least 95% identical to SEQ ID NO:

5 and that is modified at the aspartate residue corresponding to aspartate 76 OF SEQ ID NO;

5, wherein the encoded propeptide inhibits at least one GDF-8 biological activity.

18. The nucleic acid molecule of claim 16, wherein aspartate 76 is changed to alanine, and said nucleic acid molecule further comprises a nucleic acid sequence encoding an Fc region of an IgG molecule fused carboxy terminal to the GDF-8 propeptide.

19. The nucleic acid molecule of claim 18, wherein said IgG molecule is an IgG1 or IgG4 molecule.

20. The nucleic acid molecule of claim 18, wherein said IgG Fc region is 95% identical to SEQ ID NO:

15 or SEQ ID NO;

16.

21. The nucleic acid molecule of claim 18, further comprising a nucleic acid sequence encoding a signal sequence located amino terminal to the GDF-8 propeptide.

22. The nucleic acid molecule of claim 18, further comprising a nucleic acid sequence encoding a peptide linker between the GDF-8 propeptide and the IgG Fc region.

23. The nucleic acid molecule of claim 18, wherein the GDF-8 propeptide and IgG Fc fusion protein encoded by the nucleic acid sequence is at least 95% identical to the mature GDF-8 propeptide and IgG Fc fusion protein comprised by the amino acid sequence of SEQ ID NO:

22.

24. A vector comprising the nucleic acid molecule of claim 23.

25. A host cell comprising the nucleic acid molecule of claim 23 operatively linked to a regulatory sequence.

26. The nucleic acid molecule of claim 18, wherein said GDF-8 propeptide is at least 240 amino acids in length.

17. A nucleic acid molecule encoding a GDF-8 propeptide comprising a nucleic acid sequence that encodes a GDF-8 propeptide that is at least 95% identical to SEQ ID NO:

6 and that is modified at the aspartate residue corresponding to aspartate 76 OF SEQ ID NO;

5, wherein the encoded propeptide inhibits at least one GDF-8 biological activity.