Nucleic acid molecules encoding transmembrane serine proteases, the encoded proteins and methods based thereon

• US 7,700,341 B2
• Filed: 02/02/2001
• Issued: 04/20/2010
• Est. Priority Date: 02/03/2000
• Status: Expired due to Fees

• Current Assignee: Dendreon Pharmaceuticals, Inc. (Ya Fei Yuan,)
• Original Assignee: Dendreon Corporation (Bausch Health Cos., Inc.)
• Inventors: Madison, Edwin L., Yeh, Jiunn-Chern, Ong, Edgar O.
• Primary Examiner(s): Pak; Yong D
• Application Number:
  US09/776,191
• Publication Number:
  US 20030119168A1
• Time in Patent Office: 3,364 Days
• Field of Search: 435/212, 435/440, 435/69.1, 435/71.1, 435/252.3, 435/320.1, 536/23.2, 536/23.5, 536/23.7
• US Class Current: 435/212
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 35/04 specific for metastasis
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 9/14 Vasoprotectives; Antihaemorrhoidals; Drugs for varicose therapy; Capillary stabilisers
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C12N 9/6424 Serine endopeptidases (3.4.21)

Abstract

Provided herein is are polypeptides that include the protease domain of a type II transmembrane serine protease (MTSP) as a single chain. Methods using the polypeptides to identify compounds that modulate the protease activity of an MTSP are provided. Also provided are MTSPs designated MTSP3 and MTSP4 and a form of an MTSP designated MTSP6.

Claims

1. An isolated, substantially purified single-chain polypeptide, consisting only of a protease domain of a type-II membrane-type serine protease (MTSP) or a proteolytically active fragment thereof as a single chain, wherein:

a free Cys, which is in the protease domain and which is normally disulfide bonded to a Cys in the pro domain of the full length MTSP, is replaced with another amino acid; and

the MTSP protease domain or the proteolytically active fragment thereof has serine protease activity as a single chain.

2. The substantially purified polypeptide of claim 1, wherein the MTSP portion has an N-terminus that comprises IVNG, ILGG, VGLL or ILGG.

3. The substantially purified polypeptide of claim 1, wherein the MTSP is selected from among MTSP1, MTSP3, MTSP4 and MTSP6.

4. The substantially purified polypeptide of claim 1, wherein the MTSP protease domain consists of a sequence of amino acid residues selected from among amino acids 615-855 of SEQ ID No. 2, amino acids 205-437 of SEQ ID NO. 4, the amino acid residues set forth as SEQ ID No. 6 or as amino acids 217-443 in SEQ ID No. 12.

5. The substantially purified polypeptide of claim 1 that has at least about 95% sequence identity with a protease domain consisting of a sequence of amino acid residues selected from among amino acids 615-855 of SEQ ID No. 2, amino acids 205-437 of SEQ ID NO. 4, the amino acids set forth as SEQ ID No. 6, and amino acids 217-443 in SEQ ID No. 12.

6. The polypeptide of claim 1, wherein a free Cys in the protease domain is replaced with a serine.

7. The polypeptide of claim 1, wherein the MTSP is selected from among corin, MTSP 1, enteropeptidase, human airway trypsin-like protease (HAT), TMPRSS2, and TMPRSS4.

8. A conjugate, comprising:

a) a polypeptide of claim 1, and

b) a targeting agent linked to the protein directly or via a linker, wherein the conjugate has serine protease activity.

9. The conjugate of claim 8, wherein the targeting agent permits

i) affinity isolation or purification of the conjugate;

ii) attachment of the conjugate to a surface;

iii) detection of the conjugate;

or

iv) targeted delivery to a selected tissue or cell.

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

11. The support of claim 10, wherein the polypeptides comprise an array.

12. The support of claim 11, wherein the array comprises polypeptides having different MTSP protease domains.

13. A method for identifying candidate anti-tumor compounds that inhibit the protease activity of an MTSP, comprising:

contacting a polypeptide of claim 1 with a substrate proteolytically cleaved by the MTSP, 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 change the amount cleaved compared to a control, whereby compounds that modulate the activity of the MTSP are identified.

14. The method of claim 13, wherein the test compounds are small molecules, peptides, peptidomimetics, natural products, antibodies or fragments thereof.

15. The method of claim 13, wherein a plurality of the test compounds are screened simultaneously.

16. The method of claim 13, wherein the change in the amount cleaved is assessed by comparing the amount cleaved in the presence of the test compound with the amount in the absence of the test compound.

17. The method of claim 13, wherein a plurality of the polypeptides are linked to a solid support, either directly or via a linker.

18. The method of claim 13, wherein the polypeptides comprise an array.

19. The method of claim 13, wherein the polypeptides comprise a plurality of different MTSP proteases.

20. A method of identifying a compound that specifically binds to a single chain protease domain of an MTSP, comprising:

contacting a polypeptide of claim 1 with a test compound or plurality thereof under conditions conducive to binding thereof; and

identifying compounds that specifically bind to the MTSP single chain protease domain or compounds that inhibit binding of a compound known to bind to the MTSP single chain protease domain, wherein the known compound is contacted with the polypeptide before, simultaneously with or after the test compound.

21. The method of claims 20, wherein the polypeptide is linked either directly or indirectly via a linker to a solid support.

22. The method of claim 20, wherein the test compounds are small molecules, peptides, peptidomimetics, natural products, antibodies or fragments thereof.

23. The method of claim 20, wherein a plurality of the test substances are screened for simultaneously.

24. The method of claim 21, wherein a plurality of the polypeptides are linked to a solid support.

25. A conjugate, comprising:

a) an MTSP3 or an MTSP4 or the MTSP6 of claim 4; and

b) a targeting agent linked to the protein directly or via a linker.

26. The conjugate of claim 25, wherein the targeting agent permits

i) affinity isolation or purification of the conjugate;

ii) attachment of the conjugate to a surface;

iii) detection of the conjugate;

or

iv) targeted delivery to a selected tissue or cell.

27. A solid support comprising two or more polypeptides of claim 4 linked thereto either directly or via a linker.

28. The support of claim 27, wherein the polypeptides comprise an array.

29. A method for identifying compounds that modulate the protease activity of an MTSP of claim 1, comprising:

contacting the MTSP of claim 1 with a substrate proteolytically cleaved by the MTSP, 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 change the amount cleaved compared to a control, whereby compounds that modulate the activity of the MTSP are identified.

30. The method of claim 29, wherein the test compounds are small molecules, peptides, peptidomimetics, natural products, antibodies or fragments thereof.

31. The method of claim 29, wherein the change in the amount cleaved is assessed by comparing the amount cleaved in the presence of the test compound with the amount in the absence of the test compound.

32. The method of claim 29, wherein a plurality of the test substances are screened for simultaneously.

33. The method of claim 32, wherein a plurality of the polypeptides are linked to a solid support.

34. A method of identifying a compound that specifically binds to an MTSP protease domain, comprising:

contacting an MTSP protease domain of claim 4 with a test compound or plurality thereof under conditions conducive to binding thereof; and

identifying compounds that specifically bind to the MTSP.

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

36. The method of claim 34, wherein the test compounds are small molecules, peptides, peptidomimetics, natural products, antibodies or fragments thereof.

37. The method of claim 34, wherein a plurality of the test substances are screened for simultaneously.

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