RECOMBINANTLY MODIFIED PLASMIN

• US 20110003332A1
• Filed: 11/25/2008
• Published: 01/06/2011
• Est. Priority Date: 11/29/2007
• Status: Active Grant

• Current Assignee: Grifols Therapeutics LLC (Grifols SA)
• Original Assignee: Talecris Biotherapeutics Incorporated (Grifols SA)
• Inventors: Novokhatny, Valery
• Granted Patent: US 8,101,394 B2
• Time in Patent Office: Days
• 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

1-27. -27. (canceled)

28. A polynucleotide comprising a nucleotide sequence encoding a polypeptide having:

a) a single N-terminal kringle domain homologous to a kringle domain of native human plasminogen, wherein the last four amino acid residues within the single N-terminal kringle domain are VPQC; and

b) 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.

36. A vector comprising the polynucleotide of claim 28.

37. A cell comprising the vector of claim 36.

38. A polypeptide comprising:

a) a single N-terminal kringle domain homologous to a kringle domain of native human plasminogen, wherein the last four amino acid residues within the single N-terminal kringle domain are VPQC; and

b) 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.

39. The polypeptide of claim 38, wherein the single N-terminal kringle domain is homologous to kringle 1 or kringle 4 of native human plasminogen.

40. The polypeptide of claim 38, 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.

41. The polypeptide of claim 38, wherein the polypeptide is at least 90% identical to the sequence shown in SEQ ID NO:

2.

42. The polypeptide of claim 38, wherein the single N-terminal 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 N-terminal kringle domain 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.

43. The polypeptide of claim 38, wherein the polypeptide has an amino acid sequence as shown in SEQ ID NO:

2, and conservative substitutions thereof.

44. The polypeptide of claim 38, wherein the polypeptide has an arginine residue at a relative position analogous to that of position 85 of the amino acid sequence shown in SEQ ID NO:

2.

45. The polypeptide of claim 38, 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 is not VPQC.

46. A method for making one or more recombinant plasmin polypeptides having different N-termini, the method comprising:

contacting a polypeptide with a protease under conditions sufficient to cleave one or more peptide bonds thereby forming one or more recombinant plasmin polypeptides having different N-termini, wherein the polypeptide has;

(a) a single N-terminal kringle domain homologous to a kringle domain of native human plasminogen, wherein the last four amino acid residues within the single N-terminal kringle domain are VPQC; and

(b) 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.

47. The method of claim 46, wherein the polypeptide has an amino acid sequence shown in SEQ ID NO:

2.