Recombinantly modified plasmin

• US 8,512,980 B2
• Filed: 05/03/2012
• Issued: 08/20/2013
• Est. Priority Date: 11/29/2007
• Status: Expired due to Term

• Current Assignee: Grifols Therapeutics LLC (Grifols SA)
• Original Assignee: Grifols Therapeutics Incorporated (Grifols SA)
• Inventors: Novokhatny, Valery
• Primary Examiner(s): NASHED, NASHAAT T
• Application Number:
  US13/463,437
• Publication Number:
  US 20120276611A1
• Time in Patent Office: 474 Days
• Field of Search: None
• US Class Current: 435/68.1
• CPC Class Codes: A61P 7/02 Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C12N 11/04 entrapped within the carrier, e.g. gel or hollow fibres
• CPC Class Codes: C12N 9/6435 Plasmin (3.4.21.7), i.e. fibrinolysin
• CPC Class Codes: C12Y 304/21007 Plasmin (3.4.21.7), i.e. fibrinolysin

Abstract

Polynucleotides and polypeptides relating to a recombinantly modified plasmin(ogen) molecule are provided. The plasmin(ogen) molecule has a single kringle domain N-terminal to the activation site present in the native human plasminogen molecule, combined such that no foreign sequences are present, and exhibits lysine-binding and significant enzymatic characteristics associated with the native enzyme.

Claims

1. A method for making a recombinant plasmin polypeptide, the method comprising:

(a) providing a polypeptide having a single N-terminal kringle domain wherein the last four amino acid residues within the kringle domain are identical to residues at positions 91-94 of SEQ ID NO;

2; and

a C-terminal domain activation site and serine protease domain;

wherein the polypeptide binds to immobilized lysine; and

wherein the polypeptide is at least 95% identical to the sequence shown in SEQ ID NO;

2;

(b) contacting the polypeptide provided in step (a) with a protease under conditions sufficient to cleave one or more peptide bonds thereby forming an active recombinant plasmin polypeptide.

2. The method of claim 1, wherein the polypeptide has an amino acid sequence shown in SEQ ID NO:

2.

3. The method of claim 1, wherein providing comprises expressing a DNA sequence shown in SEQ ID NO:

1, or a degenerate variant thereof, in E. coli.

4. The method of claim 1, wherein the polypeptide is at least 98% identical to the sequence shown in SEQ ID NO:

2.

5. The method of claim 1, wherein amino acid residues 2-9 of the polypeptide are identical to residues at positions 2-9 of SEQ ID:

NO;

2.

6. The method of claim 1, wherein the polypeptide has the amino acid sequence shown in SEQ ID NO:

2 with no more than 15 amino acid substitutions.

7. The method of claim 1, wherein the polypeptide has the amino acid sequence shown in SEQ ID NO:

2 with no more than 10 amino acid substitutions.

8. The method of claim 1 wherein the polypeptide has the amino acid sequence shown in SEQ ID NO:

2 with no more than 8 amino acid substitutions.

9. The method of claim 1, wherein the polypeptide has the amino acid sequence shown in SEQ ID NO:

2 with no more than 5 amino acid substitutions.

10. The method of claim 1, wherein the polypeptide has the amino acid sequence shown in SEQ ID NO:

2 with no more than 3 amino acid substitutions.

11. The method of claim 1, wherein the polypeptide exhibits a lower binding affinity for fibrinogen than the binding affinity for fibrinogen of mini-plasmin.

12. The method of claim 1, wherein the polypeptide exhibits higher binding affinity for partially cleaved fibrin than the binding affinity for partially cleaved fibrin of mini-plasmin.

13. The method of claim 1, wherein the polypeptide exhibits a fibrinolytic activity that is inhibited by α

2-antiplasmin at a rate of inhibition that is at least about 5-fold faster than the rate of inhibition of the fibrinolytic activity of mini-plasmin by α

2-antiplasmin.

14. The method of claim 1, wherein the rate of inhibition is at least 10-fold to 40-fold faster than the rate of inhibition of miniplasmin.

15. The method of claim 1, wherein the immobilized lysine is lysine bound to a solid support matrix selected from the group consisting of lysine-agarose, lysine-hydrogel, and lysine-cross-linked agarose.

16. The method of claim 14, wherein the immobilized lysine is lysine-cross-linked agarose.

17. The method of claim 1, wherein the polypeptide has reduced immunogenicity as compared to a reference polypeptide, wherein the reference polypeptide has a primary amino acid sequence identical to the primary amino acid sequence of the polypeptide;

with the proviso that the last four amino acid residues of the single N-terminal kringle domain of the reference polypeptide are not identical to residues at positions 91-94 of SEQ ID NO;

2.

18. The method of claim 1, wherein the protease is streptokinase, urokinase, or tissue plasminogen activator (tPA).

19. The method of claim 1, wherein the protease is streptokinase.