Angiostatin fragments and method of use

• US 6,024,688 A
• Filed: 04/24/1998
• Issued: 02/15/2000
• Est. Priority Date: 03/08/1996
• Status: Expired due to Term

• Current Assignee: Children's Medical Center Corporation
• Original Assignee: Children's Medical Center Corporation
• Inventors: O'Reilly, Michael S., Folkman, M. Judah, Cao, Yihai
• Primary Examiner(s): Carlson, Karen Cochrane
• Application Number:
  US09/066,028
• Time in Patent Office: 662 Days
• Field of Search: 514/12, 424/94.64, 530/350, 530/380
• US Class Current: 514/13.3
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• 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
• CPC Class Codes: G01N 33/574 for cancer

Abstract

Fragments of an endothelial cell proliferation inhibitor and method of use therefor are provided. The endothelial proliferation inhibitor is a protein derived from plasminogen, or more specifically is an angiostatin fragment. The angiostatin fragments generally correspond to kringle structures occurring within the endothelial cell proliferation inhibitor. The endothelial cell inhibiting activity of these fragments provides a means for inhibiting angiogenesis of tumors and for treating angiogenic-mediated disease.

Claims

1. A method of inhibiting angiogenesis in a mammal comprising administering to the mammal an angiogenesis inhibiting amount of a kringle region fragment of a angiostation molecule that has anti-angiogenic activity in vivo.

2. The method of claim 1, wherein the kringle region fragment has an ED₅₀ of less than about 500 nM.

3. The method of claim 1, wherein the angiostation fragment is derived from murine plasminogen, human plasminogen, Rhesus plasminogen, porcine plasminogen or bovine plasminogen.

4. The method of claim 1, wherein the kringle region fragment comprising an amino acid sequence selected from the group consisting of SEQ ID NO:

7;

SEQ ID NO;

8;

SEQ ID NO;

9;

SEQ ID NO;

10;

SEQ ID NO;

11;

SEQ ID NO;

12;

SEQ ID NO;

13;

SEQ ID NO;

14;

SEQ ID NO;

15;

SEQ ID NO;

16;

SEQ ID NO;

17;

SEQ ID NO;

18;

SEQ ID NO;

19;

SEQ ID NO;

20;

SEQ ID NO;

21;

SEQ ID NO;

22;

SEQ ID NO;

23;

SEQ ID NO;

24;

SEQ ID NO;

25;

SEQ ID NO;

26;

SEQ ID NO;

27;

SEQ ID NO;

28;

SEQ ID NO;

29;

SEQ ID NO;

30;

SEQ ID NO;

31;

SEQ ID NO;

32;

SEQ ID NO;

33;

SEQ ID NO;

34;

SEQ ID NO;

35;

SEQ ID NO;

36;

SEQ ID NO;

37;

SEQ ID NO;

38;

SEQ ID NO;

39;

SEQ ID NO;

40;

SEQ ID NO;

41 and SEQ ID NO;

42 or a combination thereof.

5. The method of claim 1, wherein the kringle region fragment is a kringle 1.

6. The method of claim 5, wherein the kringle 1 has an ED₅₀ of less than about 350 nM.

7. The method of claim 6, wherein the kringle 1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:

7;

SEQ ID NO;

8;

SEQ ID NO;

9;

SEQ ID NO;

10 and SEQ ID NO;

11.

8. The method of claim 5, wherein the kringle 1 comprising an amino acid sequence of SEQ ID NO:

8.

9. The method of claim 1, wherein the kringle region fragment is a kringle 2.

10. The method of claim 9, wherein the kringle 2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:

12;

SEQ ID NO;

13;

SEQ ID NO;

14;

SEQ ID NO;

15 and SEQ ID NO;

16.

11. The method of claim 9, wherein the kringle 2 comprising an amino acid sequence of SEQ ID NO:

13.

12. The method of claim 1, wherein the kringle region fragment is a kringle 3.

13. The method of claim 12, wherein the kringle 3 has an ED₅₀ of less than about 475 nM.

14. The method of claim 12, wherein the kringle 3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:

17;

SEQ ID NO;

18;

SEQ ID NO;

19;

SEQ ID NO;

20 and SEQ ID NO;

21.

15. The method of claim 12, wherein the kringle 3 comprising an amino acid sequence of SEQ ID NO:

18.

16. The method of claim 1, wherein the kringle region fragment is a kringle 2-3.

17. The method of claim 16, wherein the kringle 2-3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:

24;

SEQ ID NO;

25;

SEQ ID NO;

26;

SEQ ID NO;

27 and SEQ ID NO;

28.

18. The method of claim 16, wherein the kringle 2-3 comprising an amino acid sequence of SEQ ID NO:

25.

19. The method of claim 1, wherein the kringle region fragment is a kringle 1-3 protein.

20. The method of claim 19, wherein the kringle 1-3 has and ED₅₀ of less than about 100 nM.

21. The method of claim 19, wherein the kringle 1-3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:

29;

SEQ ID NO;

30;

SEQ ID NO;

31;

SEQ ID NO;

32 and SEQ ID NO;

33.

22. The method of claim 19, wherein the kringle 1-3 comprising an amino acid sequence of SEQ ID NO:

30.

23. The method of claim 1, wherein the kringle region fragment is a kringle 1-2.

24. The method of claim 23, wherein the kringle 1-2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:

34;

SEQ ID NO;

35;

SEQ ID NO;

36;

SEQ ID NO;

37 and SEQ ID NO;

38.

25. The method of claim 23, wherein the kringle 1-2 comprises an amino acid sequence of SEQ ID NO:

35.

26. The method of claim 1, wherein the administering step comprises administering a combination of two or more kringle region fragments.

27. The method of claim 26, wherein the combination of kringle region fragments comprises a kringle 1 and a kringle 2.

28. The method of claim 27, wherein the kringle 1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:

7;

SEQ ID NO;

8;

SEQ ID NO;

9;

SEQ ID NO;

10 and SEQ ID NO;

11, and the kringle 2, comprising an amino acid sequence selected from the group consisting of SEQ ID NO;

12;

SEQ ID NO;

13;

SEQ ID NO;

14;

SEQ ID NO;

15 and SEQ ID NO;

16.

29. The method of claim 27, wherein the kringle 1 consisting of an amino acid sequence of SEQ ID NO:

8 and the kringle 2 protein consisting of an amino acid sequence of SEQ ID NO;

13.

30. The method of claim 26, wherein the combination of kringle region fragments comprises a kringle 1 and a kringle 2-3.

31. The method of claim 30, wherein the kringle 1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:

7;

SEQ ID NO;

8;

SEQ ID NO;

9;

SEQ ID NO;

10 and SEQ ID NO;

11, and the kringle 2-3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO;

24;

SEQ ID NO;

25;

SEQ ID NO;

26;

SEQ ID NO;

27 and SEQ ID NO;

28.

32. The method of claim 30, wherein the kringle 1 comprising an amino acid sequence of SEQ ID NO:

8 and the kringle 2-3 comprising an amino acid sequence of SEQ ID NO;

25.

33. The method of claim 26, wherein the combination of kringle region fragments comprises a kringle 1, a kringle 2 and a kringle 3.

34. The method of claim 33, wherein the kringle 1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:

7;

SEQ ID NO;

8;

SEQ ID NO;

9;

SEQ ID NO;

10 and SEQ ID NO;

11, the kringle 2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO;

12;

SEQ ID NO;

13;

SEQ ID NO;

14;

SEQ ID NO;

15 and SEQ ID NO;

16, and the kringle 3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO;

17;

SEQ ID NO;

18;

SEQ ID NO;

19;

SEQ ID NO;

20 and SEQ ID NO;

21.

35. The method of claim 33, wherein the kringle 1 comprising an amino acid sequence of SEQ ID NO:

8, the kringle 2 comprising an amino acid sequence of SEQ ID NO;

13 and the kringle 3 comprising an amino acid sequence of SEQ ID NO;

18.

36. The method of claim 26, wherein the combination of kringle region fragments comprises a kringle 2 and a kringle 3.

37. The method of claim 36, wherein the kringle 2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:

12;

SEQ ID NO;

13;

SEQ ID NO;

14;

SEQ ID NO;

15 and SEQ ID NO;

16, and the kringle 3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO;

17;

SEQ ID NO;

18;

SEQ ID NO;

19;

SEQ ID NO;

20 and SEQ ID NO;

21.

38. The method of claim 36, wherein the kringle 2 consists essentially of an amino acid sequence of SEQ ID NO:

13 and the kringle 3 consists essentially of an amino acid sequence of SEQ ID NO;

18.

39. The method of claim 1, wherein the mammal is a human.

40. The method of claim 1, wherein the angiogenesis-mediated disease is a cancer.

41. A method of inhibiting endothelial cell proliferation comprising administering to an endothelial cell a proliferation inhibiting amount of a kringle region fragment of a plasminogen molecule that has endothelial cell proliferation inhibiting activity in vitro.

42. The method of claim 41, wherein the kringle region fragment is selected from the group consisting of a kringle 1, a kringle 2, a kringle 3, kringle 2-3, kringle 1-3, kringle 1-2, or a combination thereof.

43. The method of claim 41, wherein the kringle region fragment comprising an amino acid sequence selected from the group consisting of SEQ ID NO:

7;

SEQ ID NO;

8;

SEQ ID NO;

9;

SEQ ID NO;

10;

SEQ ID NO;

11;

SEQ ID NO;

12;

SEQ ID NO;

13;

SEQ ID NO;

14;

SEQ ID NO;

15;

SEQ ID NO;

16;

SEQ ID NO;

17;

SEQ ID NO;

18;

SEQ ID NO;

19;

SEQ ID NO;

20;

SEQ ID NO;

21;

SEQ ID NO;

24;

SEQ ID NO;

25;

SEQ ID NO;

26;

SEQ ID NO;

27;

SEQ ID NO;

28;

SEQ ID NO;

29;

SEQ ID NO;

30;

SEQ ID NO;

31;

SEQ ID NO;

32;

SEQ ID NO;

33;

SEQ ID NO;

34;

SEQ ID NO;

35;

SEQ ID NO;

36;

SEQ ID NO;

37;

SEQ ID NO;

38;

or a combination thereof.

44. The method of claim 41, wherein the kringle region fragment has an endothelial cell ED₅₀ of less than about 500 nM.

45. A method of inhibiting angiogenesis in a mammal comprising administering to the mammal an angiogenesis inhibiting amount of a kringle region fragment of an angiostatin molecule that has anti-angiogenic activity in vivo, wherein said fragment contains amino acid substitutions, deletions, and additions, wherein said fragment retains anti-angiogenic actitvity.

46. The method of claim 45, wherein said fragment is derived from the amino acid sequence selected from the group consisting of SEQ ID NO:

7;

SEQ ID NO;

8;

SEQ ID NO;

9;

SEQ ID NO;

10;

SEQ ID NO;

11;

SEQ ID NO;

12;

SEQ ID NO;

13;

SEQ ID NO;

14;

SEQ ID NO;

15;

SEQ ID NO;

16;

SEQ ID NO;

17;

SEQ ID NO;

18;

SEQ ID NO;

19;

SEQ ID NO;

20;

SEQ ID NO;

21;

SEQ ID NO;

22;

SEQ ID NO;

23;

SEQ ID NO;

24;

SEQ ID NO;

25;

SEQ ID NO;

26;

SEQ ID NO;

27;

SEQ ID NO;

28;

SEQ ID NO;

29;

SEQ ID NO;

30;

SEQ ID NO;

31;

SEQ ID NO;

32;

SEQ ID NO;

33;

SEQ ID NO;

34;

SEQ ID NO;

35;

SEQ ID NO;

36;

SEQ ID NO;

37;

SEQ ID NO;

38;

SEQ ID NO;

39;

SEQ ID NO;

40;

SEQ ID NO;

41; and

SEQ ID NO;

42 or a combination thereof.

47. A method of inhibiting endothelial cell preliferation comprising administering to an endothelial cell an endothelial cell proliferation inhibiting amount of a kringle region fragment of an angiostatin molecule that has anti-angiogenic activity in vitro, wherein said fragment contains amino acid substitutions, deletions, and additions, wherein said fragment retains anti-angiogenic actitvity.

48. The method of claim 47, wherein said fragment is derived from the amino acid sequence selected from the group consisting of SEQ ID NO:

7;

SEQ ID NO;

8;

SEQ ID NO;

9;

SEQ ID NO;

10;

SEQ ID NO;

11;

SEQ ID NO;

12;

SEQ ID NO;

13;

SEQ ID NO;

14;

SEQ ID NO;

15;

SEQ ID NO;

16;

SEQ ID NO;

17;

SEQ ID NO;

18;

SEQ ID NO;

19;

SEQ ID NO;

20;

SEQ ID NO;

21;

SEQ ID NO;

22;

SEQ ID NO;

23;

SEQ ID NO;

24;

SEQ ID NO;

25;

SEQ ID NO;

26;

SEQ ID NO;

27;

SEQ ID NO;

28;

SEQ ID NO;

29;

SEQ ID NO;

30;

SEQ ID NO;

31;

SEQ ID NO;

32;

SEQ ID NO;

33;

SEQ ID NO;

34;

SEQ ID NO;

35;

SEQ ID NO;

36;

SEQ ID NO;

37; and

SEQ ID NO;

38 or a combination thereof.