Light-emitting fusion proteins and diagnostic and therapeutic methods therefor

• US 20030022198A1
• Filed: 03/19/2002
• Published: 01/30/2003
• Est. Priority Date: 03/20/2001
• Status: Active Grant

• Current Assignee: Dana-Farber Cancer Institute, Howard Hughes Medical Institute
• Original Assignee: Dana-Farber Cancer Institute
• Inventors: Kim, Tae-You, Kaelin, William G. Jr., Livingston, David M.
• Granted Patent: US 7,919,274 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: A61K 49/0045 the fluorescent agent being a peptide or protein used for imaging or diagnosis in vivo
• CPC Class Codes: A61K 49/0047 Green fluorescent protein [GFP]
• CPC Class Codes: C12Q 1/26 involving oxidoreductase
• CPC Class Codes: G01N 2333/90245 acting on paired donors with incorporation of molecular oxygen (1.14)
• CPC Class Codes: G01N 2500/02 Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)
• CPC Class Codes: G01N 33/573 for enzymes or isoenzymes

Abstract

Light-generating fusion proteins having a ligand binding site and a light-generating polypeptide moiety and their use as diagnostics, in drug screening and discovery, and as therapeutics, are disclosed. The light-generating fusion protein has a feature where the bioluminescence of the polypeptide moiety changes upon binding of a ligand at the ligand binding site. The ligand may be, for example, an enzyme present in an environment only under certain conditions, e.g., ubiquitin ligase in a hypoxic state, such that the light-generating fusion protein is “turned on” only under such conditions.

Claims

1. A non-invasive method for detecting the localization of an entity in a subject, comprising:

a) administering to said subject a light-generating fusion protein, or a cell expressing said light-generating fusion protein, said light-generating fusion protein comprising

i) a ligand binding site and

ii) a light-generating polypeptide moiety, wherein the light generation of said light-generating fusion protein changes upon binding of a ligand at said ligand binding site, said ligand binding site recognizing a ligand which is on said entity or a produced by said entity;

b) allowing for co-localization of said light-generating fusion protein or cell and an entity, wherein contact between said ligand binding site and said ligand causes a modification of a colinear effector site which alters the light generation of said light-generating polypeptide moiety; and

c) imaging said localized light-generating fusion protein, thereby localizing an entity in a subject.

2. The method of claim 1, wherein said entity is selected from the group consisting of a molecule;

a macromolecule;

a polymer;

a protein;

an antibody;

a protein complex;

a polysaccharide;

a nucleic acid;

a particle;

an inert material;

an organelle;

a cell;

an embryo;

a microorganism;

a bacteria;

a virus;

a fungus;

a prion;

a tumor;

a tissue;

a cellular environment comprising damaged tissue;

a cellular environment comprising diseased tissue;

a wound; and

an organ.

3. The method of claim 1, wherein said entity is selected from the group consisting of a tumor, a proliferating cell, a pathogen, and a cellular environment comprising hypoxic tissue.

4. The method of claim 1, wherein said ligand is an enzyme.

5. The method of claim 1, wherein said ligand is pVHL.

6. The method of claim 1, wherein said ligand is an enzyme selected from the group consisting of a kinase, a ligase, and a protease.

7. The method of claim 1, wherein said ligand is VBC.

8. The method of claim 4, wherein said ligand binding site does not comprise a kemptide or a malantide.

9. The method of claim 1, wherein said colinear effector site is congruent with said ligand binding site.

10. The method of claim 1, wherein said colinear effector site is in cis with said ligand binding site.

11. The method of claim 1, wherein said colinear effector site is in trans with said ligand binding site.

12. The method of claim 1, wherein said modification of said colinear effector site is a phosphorylation event.

13. The method of claim 1, wherein said modification of said colinear effector site is a cleavage event.

14. The method of claim 1, wherein said modification of said colinear effector site is a ubiquitination event.

15. The method of claim 1, wherein said light-generating polypeptide moiety is selected from the group consisting of bioluminescent and fluorescent polypeptide moieties.

16. The method of claim 1, wherein said light-generating polypeptide moiety is selected from the group consisting of ferredoxin IV, green fluorescent protein, red fluorescent protein, yellow fluorescent protein, blue fluorescent protein, the luciferase family and the aequorin family.

17. The method of claim 1, wherein said light-generating fusion protein further comprises a targeting moiety.

18. The method of claim 1, wherein said ligand binding site comprises a protein dimerization domain wherein binding of said ligand to said ligand binding site is effected via a secondary protein which mediates said ligand binding.

19. The method of claim 1, wherein the light generation of said light-generating fusion protein changes without altering the phosphorylational state of said light-generating fusion protein.

20. A method of imaging hypoxic tissue in a mammalian subject, comprising:

a) administering to a subject a light-generating fusion protein, or a cell expressing said light-generating fusion protein, comprising an ubiquitin ligase binding site and a light-generating polypeptide moiety, wherein the light generation of said light-generating fusion protein changes upon binding of a ubiquitin ligase at said ubiquitin ligase binding site, said ubiquitin ligase binding site recognizing a ubiquitin ligase present in hypoxic conditions in hypoxic tissue;

b) allowing for localization of said light-generating fusion protein or cell in hypoxic tissue in said subject, wherein contact between said ubiquitin ligase binding site and a ubiquitin ligase causes a modification of a colinear effector site which alters the light generation of said light-generating polypeptide moiety; and

;

c) measuring luminescence of said localized light-generating fusion protein and imaging said localized light-generating fusion protein, thereby imaging said hypoxic tissue.

21. The method of claim 20, wherein said ligand binding site comprises the amino acid sequence Y-X₁-Leu-X₂-Pro_h-X₃-X4-X5-X₆-Y′

, wherein

a) Pro_h is hydroxylated proline;

b) X₁, X₂, X₄, X₅, and X₆ are independently Gly, Ala, Val, Leu, Ile, Pro, Met, Phe, or Trp;

c) X₃ is Ser, Thr, or Tyr; and

d) Y and Y′

are independently present or absent and, if present, independently comprise a peptide having from 1 to 600 amino acids.

22. The method of claim 20, wherein said ligand binding site comprises the amino acid sequence corresponding to the N-terminal residues 1-600 of HIF1α

, numbered in accordance with wild-type HIF1α

, wherein residue 564 is hydroxylated proline.

23. The method of claim 20, wherein said ligand binding site comprises the amino acid sequence corresponding to the N-terminal residues 1-600 of HIF1α

, numbered in accordance with wild-type HIF1α

, wherein residue 402 is hydroxylated proline.

24. The method of claim 20, wherein said ligand binding site comprises the amino acid sequence corresponding to the N-terminal residues 1-600 of HIF1α

, numbered in accordance with wild-type HIF1α

, wherein either or both of residues 402 and 564 are hydroxylated proline.

25. The method of claim 20, wherein said ligand binding site comprises a 4 to 12 amino acid sequence corresponding to the residues adjacent to and/or surrounding residue 564, inclusive, of HIF1α

, numbered in accordance with wild-type HIF1α

, wherein residue 564 is hydroxylated proline.

26. The method of claim 20, wherein said ligand binding site comprises a 12 to 14 amino acid sequence corresponding to the residues adjacent to and/or surrounding residue 564, inclusive, of HIF1α

, numbered in accordance with wild-type HIF1α

, wherein residue 564 is hydroxylated proline.

27. The method of claim 20, wherein said ligand binding site comprises a 20 to 30 amino acid sequence corresponding to the residues adjacent to and/or surrounding residue 564, inclusive, of HIF1α

, numbered in accordance with wild-type HIF1α

, wherein residue 564 is hydroxylated proline.

28. The method of claim 20, wherein said ligand binding site comprises a 80 to 120 amino acid sequence corresponding to the residues adjacent to and/or surrounding residue 564, inclusive, of HIF1α

, numbered in accordance with wild-type HIF1α

, wherein residue 564 is hydroxylated proline.

29. A method of measuring prolyl hydroxylase activity, comprising the steps of:

a) contacting a test sample with a light-generating fusion protein comprising an HIF1α

polypeptide moiety having binding character for prolyl hydroxylase and a light-generating polypeptide moiety, wherein the light generation of said light-generating polypeptide moiety changes upon binding of a prolyl hydroxylase to said HIF1α

polypeptide moiety; and

b) determining prolyl hydroxylase activity by measuring the luminescence of said test sample.

30. A method of monitoring the activity of an enzyme of interest, comprising:

a) exposing, to a fusion protein, a genetically manipulated cell or transgenic non-human mammal containing a recombinant DNA molecule comprising a binding site for a heterologous DNA binding domain upstream of a reporter gene, said reporter gene encoding a light emitting protein;

said fusion protein comprising an enzyme fused to a heterologous DNA binding domain; and

b) monitoring the production of the light emitting protein in the presence of substrate for the enzyme.

31. The method of claim 30, wherein said cell or transgenic non-human mammal is exposed to said fusion protein by transfecting said cell or mammal with a second recombinant DNA molecule encoding said fusion protein.

32. The method of claim 30, wherein said light emitting protein comprises luciferase or GFP.

33. The method of claim 30, wherein said heterologous DNA binding domain of said fusion protein comprises TETr.

34. The method of claim 33, wherein said binding site for said heterologous DNA binding domain is TETo.

35. The method of claim 30, wherein said enzyme of said fusion protein comprises cdk2 or cyclin E.

36. A light-generating fusion protein comprising a ligand binding site and a light-generating polypeptide moiety, whereupon ligand binding to said ligand binding site alters the luminescence of said light-generating polypeptide moiety without altering the phosphorylational state of said fusion protein.

37. The light-generating fusion protein of claim 36, wherein said light-generating fusion protein does not comprise a kemptide or a malantide.

38. The light-generating fusion protein of claim 36, wherein said ligand binding site is selected from the group consisting of a ligase binding site, a protease binding site, a ubiquitin ligase binding site, and a HIF1α

polypeptide moiety.

39. The light-generating fusion protein of claim 37, wherein said ligand binding site recognizes a ligand on an entity or a product of said entity.

40. The light-generating fusion protein of claim 36, further comprising a targeting moiety.

41. The light-generating fusion protein of claim 36, whereupon contact of said ligand binding site with a ligand causes a modification of a colinear effector site which alters the light generation of said light-generating polypeptide moiety.

42. A light-generating fusion protein comprising a ligand binding site and a light-generating polypeptide moiety, whereupon ligand binding to said ligand binding site alters the luminescence and phosphorylational state of said light-generating polypeptide moiety, whereby said alteration in the phosphorylational state of said light-generating fusion protein does not alter the light generation of said light-generating protein.

43. A light-generating fusion protein comprising a ubiquitin ligase binding site and a light-generating polypeptide moiety, wherein the light generation of said light-generating fusion protein changes upon binding of a ubiquitin ligase at said ubiquitin ligase binding site.

44. The light-generating fusion protein of claim 43, whereupon ligand binding to said ligand binding site alters the light generation of said light-generating fusion protein without altering the phosphorylational state of said light-generating fusion protein.

45. A light-generating fusion protein comprising a HIF1α

polypeptide moiety and a light-generating polypeptide moiety wherein the light generation of said light-generating fusion protein changes upon binding of prolyl hydroxylase to said HIF1α

polypeptide moiety.

46. The light-generating fusion protein of claim 45, whereupon ligand binding to said ligand binding site alters the light generation of said light-generating fusion protein without altering the phosphorylational state of said light-generating fusion protein.

47. A fusion protein comprising a HIF1α

polypeptide moiety and a suicide protein polypeptide moiety.

48. A fusion protein comprising a HIF1α

polypeptide moiety, a suicide protein polypeptide moiety, and a light-generating polypeptide moiety, wherein the light generation of said light-generating fusion protein changes upon binding of prolyl hydroxylase to said HIF1α

polypeptide moiety.

49. A fusion protein comprising a protease binding site and a light-generating polypeptide moiety wherein the light generation of said light-generating fusion protein changes upon binding of a protease to said protease binding site.

50. A light-generating fusion protein comprising a protein dimerization domain and a light-generating polypeptide moiety, wherein the light generation of said light-generating fusion protein changes upon ligand binding to said ligand binding site effected via a secondary protein which mediates said ligand binding.

51. A non-invasive method for detecting cancerous tissue in a subject, comprising:

a) administering to a subject a light-generating fusion protein, or a cell expressing said light-generating fusion protein, comprising a cyclin/cdk binding site and a light-generating polypeptide moiety, wherein the light generation of said light-generating fusion protein changes upon binding of a cyclin at said cyclin binding site, said cyclin binding indicative of cancerous tissue;

b) allowing for localization of said light-generating fusion protein or cell in cancerous tissue in said subject, wherein contact between said cyclin binding site and a cyclin causes a modification of a colinear effector site which alters the light generation of said light-generating polypeptide moiety; and

;

c) measuring luminescence from said localized light-generating fusion protein and imaging said localized light-generating fusion protein, thereby detecting cancerous tissue in said subject.

52. The method of claim 51, wherein said ligand binding site comprises the amino acid sequence Y-Lys-X₁-Leu-K-X₂-Leu-Y′

, wherein

a) X₁, X₂, are independently any one or more amino acids; and

b) Y and Y′

are independently present or absent and, if present, independently comprise a peptide having from 1 to 600 amino acids.

53. The method of claim 52, wherein said ligand binding site comprises the amino acid sequence PKPLKKLRFD (SEQ ID NO:

1).

54. The method of claim 51, wherein said ligand binding site comprises the amino acid sequence Y-X₁-Arg-Arg-Leu-Y′

, wherein

a) X₁ is Lys or Cys; and

b) Y and Y′

are independently present or absent and, if present, independently comprise a peptide having from 1 to 600 amino acids.

55. The method of claim 54, wherein said ligand binding site comprises the amino acid sequence GRPPVKRRLDLE (SEQ ID NO:

2).

56. The method of claim 54, wherein said ligand binding site comprises the amino acid sequence CCSKACRRLFGP (SEQ ID NO:

3).

57. The method of claim 51, wherein said ligand binding site does not comprise a kemptide or a malantide.

58. The method of claim 51, wherein said colinear effector site is congruent with said ligand binding site.

59. The method of claim 51, wherein said colinear effector site is in cis with said ligand binding site.

60. The method of claim 51, wherein said colinear effector site is in trans with said ligand binding site.

61. The method of claim 51, wherein said light-generating polypeptide moiety is selected from the group consisting of bioluminescent and fluorescent polypeptide moieties.

62. The method of claim 51, wherein said light-generating polypeptide moiety is selected from the group consisting of ferredoxin IV, green fluorescent protein, red fluorescent protein, yellow fluorescent protein, blue fluorescent protein, the luciferase family and the aequorin family.

63. The method of claim 51, wherein said light-generating fusion protein further comprises a targeting moiety.

64. The method of claim 51, wherein said ligand binding site comprises a protein dimerization domain wherein binding of said ligand to said ligand binding site is effected via a secondary protein which mediates said ligand binding.

65. The method of claim 51, wherein the light generation of said light-generating fusion protein changes without altering the phosphorylational state of said light-generating fusion protein.