Recombinantly Modified Plasmin

• US 20100304465A1
• Filed: 04/21/2005
• Published: 12/02/2010
• Est. Priority Date: 04/22/2004
• Status: Active Grant

• Current Assignee: Grifols Therapeutics LLC (Grifols SA)
• Original Assignee: Talecris Biotherapeutics Incorporated (Grifols SA)
• Inventors: Novokhatny, Valery, Hunt, Jennifer A.
• Granted Patent: US 8,034,913 B2
• Time in Patent Office: Days
• US Class Current: 435/219
• 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, and exhibits lysine-binding and significant enzymatic characteristics associated with the native enzyme.

Claims

1. A polynucleotide comprising a nucleotide sequence encoding a polypeptide having a single N-terminal kringle domain homologous to a kringle domain of native human plasminogen;

and a C-terminal domain activation site and serine protease domain homologous to the corresponding domains in human plasminogen;

wherein the polypeptide binds to immobilized lysine.

2. The polynucleotide of claim 1, wherein the N-terminal kringle domain is homologous to kringle 1 or kringle 4 of native human plasminogen.

3. The polynucleotide of claim 1, wherein the encoded polypeptide is at least 90% or 95%, or 98% identical to the sequence shown in SEQ ID NO:

2.

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

2.

5. The polynucleotide of claim 1, wherein the encoded polypeptide is the sequence shown in SEQ ID NO:

2.

6. The polynucleotide of claim 1, wherein the nucleotide sequence of the polynucleotide is the sequence shown in SEQ ID NO:

1 or a degenerate variant thereof.

7. The polynucleotide of claim 1, wherein the nucleotide sequence encodes a polypeptide having an N-terminal kringle domain homologous to the kringle 1 or kringle 4 domain of native human plasminogen;

and a C-terminal domain activation site and serine protease domain homologous to the corresponding domains in human plasminogen.

8. The polynucleotide of claim 1, wherein the nucleotide sequence encodes a polypeptide having a single N-terminal kringle domain at least 90% identical to the kringle 1 or kringle 4 domain of native human plasminogen;

and a C-terminal domain at least 90% identical to the activation site and serine protease domain of human plasminogen.

13. The polypeptide 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, lysine-cross-linked agarose.

14. The polypeptide of claim 13, wherein the immobilized lysine is lysine-cross-linked agarose.

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

16. The polypeptide 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.

20. An expression vector comprising a polynucleotide of claim 1.

21. A cultured cell comprising the expression vector of claim 20.

9. A polypeptide comprising a single N-terminal kringle domain homologous to a kringle domain of native human plasminogen;

and a C-terminal domain activation site and serine protease domain homologous to the corresponding domains in human plasminogen, wherein the polypeptide binds to immobilized lysine.

10. The polypeptide of claim 9, wherein the N-terminal kringle domain is homologous to kringle 1 or kringle 4 of native human plasminogen.

11. The polypeptide of claim 9, 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.

12. The polypeptide of claim 11, wherein the rate of inhibition is at least about 10-fold, 20-fold, 30-fold, or 40-fold faster than the rate of inhibition of mini-plasmin.

17. A polypeptide comprising a single kringle domain located N-terminal to a plasminogen activation site and plasminogen serine protease domain, wherein the kringle domain has at least one residue greater amino acid sequence identity with kringle 1 or kringle 4 of native human plasminogen than with kringle 5 of native human plasminogen, and wherein conservative substitutions of the single kringle region relative to the native sequences of kringles 1 and 4 of human plasminogen are not considered as differing from the native sequences for purposes of the identity comparison with kringle 5.

18. The polypeptide of claim 17, wherein the polypeptide has an amino acid sequence as shown in SEQ ID NO:

2, and conservative substitutions thereof.

19. The polypeptide of claim 17, wherein the polypeptide has an arginine residue at a relative position analogous to that of position 76 of the amino acid sequence shown in SEQ ID NO:

2.