Modified ADAMTS4 molecules and method of use thereof

• US 20040142863A1
• Filed: 07/29/2003
• Published: 07/22/2004
• Est. Priority Date: 07/29/2002
• Status: Active Grant

• Current Assignee: Wyeth (Pfizer Inc.)
• Original Assignee: Wyeth (Pfizer Inc.)
• Inventors: Corcoran, Christopher John, LaVallie, Edward R., Zeng, Weilan, McDonagh, Thomas, Freeman, Bethany A., Georgiadis, Katy E., Racie, Lisa A., Flannery, Carl R.
• Granted Patent: US 7,118,902 B2
• Time in Patent Office: Days
• US Class Current: 514/12
• CPC Class Codes: A61K 2039/505 comprising antibodies
• 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 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C12N 9/6489 Metalloendopeptidases (3.4.24)

Abstract

The present invention relates to modified ADAMTS4 proteins having improved stability comparing to the corresponding native, unmodified proteins. The modified ADAMTS4 proteins can be expressed and isolated in large quantities, thus allowing further characterization of the proteins, such as crystallographic and enzyme kinetic studies. The purified, stable proteins would also facilitate the production of anti-ADAMTS antibodies and the development of inhibitors to ADAMTS enzymes.

Claims

1. An isolated, modified ADAMTS4 protein with improved stability compared to a naturally-occurring, full-length ADAMTS4 protein, said modified ADAMTS4 protein differing from the naturally-occurring, full-length ADAMTS4 protein by at least one amino acid.

2. The modified ADAMTS4 protein of claim 1, wherein the naturally-occurring, full-length ADAMTS4 protein comprises the amino acid sequence recited in SEQ ID NO:

15.

3. The modified ADAMTS4 protein of claim 1, wherein a difference in amino acid sequences between the modified ADAMTS4 protein and the naturally-occurring, full-length ADAMTS4 protein is introduced by altering at least one amino acid in the naturally-occurring, full-length ADAMTS4 protein using a method selected from the group consisting of substituting, deleting, inserting, and chemically modifying amino acid residues.

4. The isolated, modified ADAMTS4 protein of claim 1, comprising a deletion of all or a portion of an ADAMTS4 spacer domain, wherein the modified ADAMTS4 protein has aggrecanase activity.

5. The isolated, modified ADAMTS4 protein of claim 1, wherein said protein does not have auto-catalytic activity.

6. The isolated, modified ADAMTS4 protein of claim 1, wherein said protein comprises a peptide tag.

7. The isolated, modified ADAMTS4 protein of claim 1, wherein said protein comprises a mutation in ADAMTS4 catalytic domain that abolishes the aggrecanase activity of said modified ADAMTS4 protein.

8. The isolated, modified ADAMTS4 protein of claim 7, further comprising a deletion of all or a portion of ADAMTS4 spacer domain.

10. An isolated polynucleotide, said polynucleotide comprising a nucleotide sequence encoding the modified ADAMTS4 protein of claim 1.

12. A vector comprising the polynucleotide of claim 10 in operative association with an expression control sequence.

14. A method for producing a modified ADAMTS4 protein, said method comprising:

introducing a polynucleotide comprising a nucleotide sequence encoding the modified ADAMTS4 protein of claim 1 into a host cell;

incubating said host cell under conditions that allow expression of the modified ADAMTS4 protein from the polynucleotide; and

purifying the modified ADAMTS4 protein from the host cell.

16. A method of identifying an inhibitor of the modified ADAMTS4 protein of claim 1, said method comprising the steps of:

determining the aggrecanase activity of the modified ADAMTS4 protein;

contacting the modified ADAMTS4 protein with a candidate agent;

determining the aggrecanase activity of the modified ADAMTS4 protein in the presence of said candidate agent; and

determining whether said candidate agent affects the activity of the modified ADAMTS4 protein.

17. A pharmaceutical composition for treating an aggrecanase-related disease, comprising:

(a) an inhibitor of the modified ADAMTS4 protein of claim 1 or an antibody that binds specifically of the modified ADAMTS4 protein of claim 1;

and

(b) a pharmaceutically acceptable carrier.

18. The pharmaceutical composition of claim 17, wherein the antibody inhibits the aggrecanase activity of the modified ADAMTS4 protein.

19. A method for treating an aggrecanase-related disease in a mammal, said method comprising the step of:

introducing into the mammal an effective amount of the pharmaceutical composition of claim 17.

20. The method of claim 19, wherein the aggrecanase-related disease is osteoarthritis.

9. An isolated, modified ADAMTS4 protein with improved stability compared to an ADAMTS4 protein having an amino acid sequence recited in SEQ ID NO:

15, said modified ADAMTS4 protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOS;

17, 19, 22, 24, 26, 27, 29, 31, 32, 40, and 46-53.

11. An isolated polynucleotide, said polynucleotide comprising a nucleotide sequence encoding the modified ADAMTS4 protein of claim 9.

13. A vector comprising the polynucleotide of claim 11 in operative association with an expression control sequence.

15. A method for producing a modified ADAMTS4 protein, said method comprising:

introducing a polynucleotide comprising a nucleotide sequence encoding the modified ADAMTS4 protein of claim 9 into a host cell;

incubating said host cell under conditions that allow expression of the modified ADAMTS4 protein from the polynucleotide; and

purifying the modified ADAMTS4 protein from the host cell.