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

• US 7,888,092 B2
• Filed: 10/30/2009
• Issued: 02/15/2011
• 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:
  US12/590,011
• Publication Number:
  US 20100105121A1
• Time in Patent Office: 473 Days
• Field of Search: None
• 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. A nucleic acid molecule, comprising a sequence of nucleic acids encoding a single chain polypeptide that consists 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 in the protease domain, which is normally disulfide bonded to a Cys in the pro-domain of the full length MTSP, is replaced with another amino acid;

the nucleic acid molecule contains a stop codon so that only the single chain MTSP protease domain, or proteolytically active fragment thereof, can be expressed; and

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

2. The nucleic acid molecule of claim 1, wherein the MTSP portion has an N-terminus that comprises IVNG, ILGG, VGLL or ILGG.

3. The nucleic acid molecule of claim 1, wherein the MTSP is selected from among MTSP1, MTSP3, MTSP4 and MTSP6.

4. The nucleic acid molecule 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 acids set forth as SEQ ID NO;

6 or as amino acids 217-443 in SEQ ID NO;

12.

5. The nucleic acid molecule of claim 1, wherein the encoded polypeptide 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 nucleic acid molecule of claim 1, wherein the MTSP protease domain is encoded by a sequence of nucleic acid residues selected from among nucleotides 1865-2585 of SEQ ID NO:

1, nucleotides 873-1571 of SEQ ID NO;

3, the nucleotides set forth as SEQ ID NO;

5 and nucleotides 916-1596 of SEQ ID NO;

11.

7. The nucleic acid molecule of claim 1, wherein a free Cys in the protease domain of the polypeptide is replaced with a serine.

8. The nucleic acid molecule of claim 1, wherein the MTSP is selected from among corin, MTSP1, enteropeptidase, human airway trypsin-like protease (HAT), TMPRSS2, and TMPRSS4.

9. The nucleic acid molecule of claim 1, wherein:

the encoded MTSP protease domain is linked directly or via a polypeptide linker to a targeting agent; and

the encoded conjugate has serine protease activity.

10. The nucleic acid molecule of claim 1, wherein the nucleic acid encoding the MTSP protease domain is operatively linked to a nucleic acid encoding a signal sequence.

11. The nucleic acid molecule of claim 9, 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.

12. A vector, comprising the nucleic acid molecule of claim 1.

13. A cell, comprising the vector of claim 12.

14. The cell of claim 13 that is a eukaryotic cell.

15. The cell of claim 13 that is a yeast cell.

16. The cell of claim 15 that is a Pichia species cell.

17. A method for production of a single chain polypeptide that consists only of a protease domain of a type-II membrane-type serine protease (MTSP), or a proteolytically active fragment thereof, as a single chain, the method comprising:

growing a cell of claim 13 under conditions, whereby the single chain MTSP polypeptide is expressed; and

isolating the single chain MTSP polypeptide.

18. The method of claim 17, wherein the nucleic acid encoding the MTSP polypeptide is operatively linked to a nucleic acid encoding a signal sequence, whereby the encoded MTSP polypeptide is secreted into the cell culture medium.

19. The method of claim 17, wherein the cell is a eukaryotic cell.

20. The method of claim 17, wherein the cell is a yeast cell.

21. The method of claim 20, wherein the cell is a Pichia species cell.