Nucleic acid molecules encoding a transmembrane serine protease 9, the encoded polypeptides and methods based thereon

• US 7,105,333 B2
• Filed: 03/27/2002
• Issued: 09/12/2006
• Est. Priority Date: 03/27/2001
• Status: Expired due to Fees

• Current Assignee: Dendreon Pharmaceuticals, Inc. (Ya Fei Yuan,)
• Original Assignee: Deadreon Corporation
• Inventors: Madison, Edwin L., Ong, Edgar O.
• Primary Examiner(s): Nashed, Nashaat T.
• Assistant Examiner(s): Moore, William W.
• Application Number:
  US10/112,221
• Publication Number:
  US 20030166851A1
• Time in Patent Office: 1,630 Days
• Field of Search: 435/226, 435/69.1, 435/471, 536/23.2, 530/350
• US Class Current: 435/226
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C12N 9/6424 Serine endopeptidases (3.4.21)
• CPC Class Codes: Y10S 436/809 Multifield plates or multicontainer arrays

Abstract

Provided herein are type II transmembrane serine protease 9 (MTSP9) polypeptides. Zymogen and activated forms of these polypeptides as well as single and two chain forms of the protease domain are also provided. Methods using the polypeptides to identify compounds that modulate the protease activity of an MTSP9 are provided.

Claims

1. A substantially purified single or two chain protease that either comprises an amino acid sequence having at least 97% identity to the amino acid sequence of the MTSP9 protease set forth in SEQ ID No. 18, or comprises an amino acid sequence having at least 97% identity to the sequence of amino acid residues encoded by the sequence of nucleotides set forth in SEQ ID No. 17.

2. The protease of claim 1 that is an activated two chain protease.

3. A substantially purified single or two chain protease of claim 1, comprising an MTSP9 protease domain

having the sequence of amino acids set forth at positions 11–

242 in SEQ ID No. 6.

4. The substantially purified protease of claim 1, wherein the protease is a human protease.

5. The protease of claim 1, wherein a free Cysteine in the protease domain is replaced with another amino acid.

6. The protease of claim 5, wherein the replacing amino acid is a serine.

7. The protease of claim 1, wherein the protease comprises at least amino acids 85–

87 and/or 160–

165 of SEQ ID No. 18.

8. The substantially purified single or two chain protease of claim 3 that comprises the sequence of amino acids set forth in SEQ ID No. 18.

9. The substantially purified protease of claim 3 that consists of the sequence of amino acids set forth in SEQ ID No. 18.

10. The substantially purified protease of claim 3 that is an activated two chain protease.

11. The protease of claim 3, wherein a free Cysteine in the protease domain is replaced with another amino acid.

12. The protease of claim 11, wherein the replacing amino acid is a serine.

15. A conjugate comprising the protease of claim 1 or claim 13 and a targeting agent linked to the protease directly or via a linker.

16. The conjugate of claim 15, wherein the targeting agent permits

affinity isolation or purification of the conjugate;

attachment of the conjugate to a surface;

detection of the conjugate;

or

targeted delivery of the conjugate to a selected tissue or cell.

17. A conjugate comprising the protease of claim 3 or claim 8 and a targeting agent linked to the protease directly or via a linker.

18. The conjugate of claim 17, wherein the targeting agent permits

affinity isolation or purification of the conjugate;

attachment of the conjugate to a surface;

detection of the conjugate;

or

targeted delivery of the conjugate to a selected tissue or cell.

21. A solid support comprising two or more proteases of claim 1 linked thereto either directly or via a linker.

22. The support of claim 21, wherein the proteases comprise an array.

23. The support of claim 22, wherein the array further comprises a plurality of different protease domains.

24. A method for identifying compounds that inhibit the protease activity of the MTSP9 protease of claim 1 or claim 13, comprising:

contacting the MTSP9 protease of claim 1 or claim 13 with a substrate that is proteolytically cleaved by the protease, and, either simultaneously, before or after, adding a test compound or plurality thereof;

measuring the amount of substrate cleaved in the presence of the test compound; and

selecting compounds that decrease the amount of substrate cleaved compared to a control,

thereby identifying compounds that inhibit the activity of the protease.

25. The method of claim 24, wherein the test compounds are small molecules, peptides, peptidomimetics, natural products, antibodies or fragments thereof that inhibit the activity of the protease.

26. The method of claim 24, wherein the control measures the amount of substrate cleaved in absence of the test compound.

27. The method of claim 24, wherein a plurality of the test compounds are screened simultaneously.

28. The method of claim 27, wherein a plurality of the proteases are linked to a solid support, either directly or via a linker.

29. The method of claim 28, wherein the proteases comprise an array.

30. A method for identifying compounds that inhibit the protease activity of the MTSP9 protease of claim 3, comprising:

contacting the MTSP9 protease of claim 3 with a substrate that is proteolytically cleaved by the protease, and, either simultaneously, before or after, adding a test compound or plurality thereof;

measuring the amount of substrate cleaved in the presence of the test compound; and

selecting compounds that decrease the amount of substrate cleaved compared to a control,

thereby identifying compounds that inhibit the activity of the protease.

31. A method for identifying compounds that inhibit the protease activity of an MTSP9 protease of claim 8, comprising:

contacting the MTSP9 protease of claim 8 with a substrate that is proteolytically cleaved by the protease, and, either simultaneously, before or after, adding a test compound or plurality thereof;

measuring the amount of substrate cleaved in the presence of the test compound; and

selecting compounds that decrease the amount of substrate cleaved compared to a control,

thereby identifying compounds that inhibit the activity of the protease.

33. A method of identifying a compound that specifically binds to a single-chain and/or two-chain MTSP9 protease of claim 1 or claim 13 comprising:

contracting an MTSP9 protease of claim 1 or claim 13 with a test compound or plurality thereof under conditions conductive to binding of the test compound to the protease, and selecting test compounds that remain bound specifically to the protease compared to a control,

thereby identifying test compounds that specifically bind a single chain and/or two chain form of the protease.

34. The method of claim 33, wherein the protease is linked either directly or indirectly via a linker to a solid support.

35. The method of claim 33, wherein the test compounds are small molecules, peptides, peptidomimetics, natural products, antibodies or fragments thereof.

36. The method of claim 33, wherein a plurality of the test compounds are screened simultaneously.

37. The method of claim 33, wherein a plurality of the proteases are linked to a solid support.

38. A method of identifying a compound that specifically binds to a single-chain and/or two-chain MTSP9 protease of claim 3 comprising:

contacting an MTSP9 protease of claim 3 with a test compound or plurality thereof under conditions conductive to binding of the test compound to the protease and selecting test compounds that remain bound specifically to the protease compared to a control,

thereby identifying test compounds that specifically bind a single chain and/or two chain form of the protease.

39. A method of identifying a compound that specifically binds to the single-chain and/or two-chain form of an MTSP9 protease of claim 8 comprising:

contacting the MTSP9 protease of claim 6 with a test compound or plurality thereof under conditions conductive to binding of the test compound to the protease; and

selecting test compounds that remain bound specifically to the protease compared to a control,

thereby identifying test compounds that specifically bind to the protease.

41. A method for identifying activators of the zymogen form of the MTSP9 protease of claim 1 or claim 13, comprising:

contacting a zymogen form of the MTSP9 protease of claim 1 or claim 5 with a substrate of the activated form of the protease;

adding a test compound, wherein the test compound is added before, after or simultaneously with the addition of the substrate; and

detecting cleavage of the substrate,

thereby identifying compounds that activate the zymogen.

42. The method of claim 41, wherein the substrate is a chromogenic substrate.

43. The method of claim 41, wherein the substrate is a L-pyroglutamyl-L-prolyl-L-arginine-p-nitroaniline hydrochloride.

44. The method of claim 41, wherein the test compound is a small molecule, a nucleic acid or a polypeptide.

13. A substantially purified single or two chain MTSP9 protease that comprises one or more amino acid substitutions at positions selected from the group of positions consisting of 185–

189 and 292 in the sequence set forth in SEQ ID No. 16.

14. A substantially purified MTSP9 protease that consists of the sequence of amino acids set forth at positions 11–

242 in SEQ ID No. 6.

19. A conjugate comprising the protease of claim 14 and a targeting agent linked to the protease directly or via a linker.

20. The conjugate of claim 19, wherein the targeting agent permits

affinity isolation or purification of the conjugate;

attachment of the conjugate to a surface;

detection of the conjugate;

or

targeted delivery of the conjugate to a selected tissue or cell.

32. A method for identifying compounds that inhibit the protease activity of the MTSP9 protease of claim 14 comprising:

contacting the MTSP9 protease of claim 14 with a substrate that is proteolytically cleaved by the protease, and, either simultaneously, before or after, adding a test compound or plurality thereof;

measuring the amount of substrate cleaved in the presence of the test compound; and

selecting compounds that decrease the amount of substrate cleaved compared to a control,

thereby identifying compounds that inhibit the activity of the protease.

40. A method of identifying a compound that specifically binds to the MTSP9 protease of claim 14 comprising:

contacting the MTSP9 protease of claim 14 with a test compound or plurality thereof under conditions conductive to binding of the test compound to the protease; and

selecting test compounds that remain bound specifically to the protease compared to a control,

thereby identifying test compounds that specifically bind to the protease.